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Microbial Cell Factories

Volume 5 Suppl 1, 2006Meeting abstracts

The 4th Recombinant Protein Production Meeting:a comparative view on host physiologyBarcelona, Spain21–23 September 2006

Published: 10 October 2005

ORAL PRESENTATIONS

S1The moss bioreactor offers best of both worldsfor biopharmaceutical productionEva L Decker and Ralf ReskiPlant Biotechnology, Faculty of Biology, University of Freiburg,79104 Freiburg, Germany

Microbial Cell Factories 2006, 5(Suppl 1):S1

Transgenic plants are promising alternatives for the productionof recombinant pharmaceutical proteins (plant molecular farm-ing). Plants as higher eukaryotes perform posttranslationalmodifications similar to those of mammalian cell lines. Low-costcultivation and safe pathogen-free production are furtheradvantages. However, field cultivation of transgenic plants raisessocial, environmental and regulatory challenges that need to beaddressed when considering how plant-made pharmaceuticalsmight be successfully commercialized. Especially GMP conditionsare hard to achieve for the field production of pharmaceuticals.Moreover, plant-specific protein N-glycosylation was shown tobe immunogenic, a fact that represents a drawback for many plantsystems as biofactories for a broad spectrum of biopharmaceu-ticals. The moss Physcomitrella patens offers unique properties as asafe contained system for protein production [1]. It is grown inthe dominant haploid gametophytic stage as tissue suspensioncultures in photobioreactors. Photo-autotrophic growth enablescultivation in a simple and cheap mineral medium. The generationof stable transgenic lines is easy and a relatively short-termprocedure. The moss is genetically well characterized anddisplays no special codon usage preferences allowing the high-level expression of human cDNAs without prior codonoptimization. Strong promoters for foreign gene expressionhave been characterized from both, heterologous as well asendogenous genes. Efficient secretory signals and a transienttransfection system allow the secretion of freshly synthesizedproteins to the surrounding medium. The secretory systemoffers continuous harvesting of the product without the necessityto lyse the producing cells and eased downstream isolation andpurification steps. The key advantage of Physcomitrella comparedto other plant systems is its high degree of nuclear homologousrecombination enabling targeted gene replacements. By thismeans, plant-specific glycosyltransferase genes, i.e. beta1,2-xylosyltransferase and alpha1,3-fucosyltransferase were specifi-cally knocked out and human-type beta1,4-galactosyltransferasewas introduced. The resultant moss strains provide proteins withnon-immunogenic humanized glycan patterns.

Here we present an overview of the relevant aspects forestablishing moss as a production system for recombinantbiopharmaceuticals.AcknowledgementsFinancial support by the German Federal Ministry of Educationand Research (BMBF) as well as the Fonds der ChemischenIndustrie is gratefully acknowledged.Reference1. Decker EL and Reski R: The moss bioreactor. Curr Opin

Plant Biol 2004, 7:166–170.

S2Enhanced production of recombinant proteinsby systems biotechnological approachesSang Yup LeeMetabolic Engineering National Research Laboratory,Department of Chemical and Biomolecular Engineering,Department of BioSystems, BioProcess Engineering ResearchCenter, and Bioinformatics Research Center, Korea AdvancedInstitute of Science and Technology (KAIST), 373-1Guseong-dong, Yuseong-gu, Daejeon 305-701, Korea


The recent development of high-throughput experimentaltechniques has resulted in rapid accumulation of a wide rangeof biological data and information at different levels: genome,transcriptome, proteome, metabolome, and fluxome data. Thistechnology-driven discovery science is now allowing theidentification of unprecedentedly large numbers of individualcomponents and molecules of a biological system, thus providinga foundation for a profound understanding of biologicalprocesses. It is currently true that the identification of thesecomponents and molecules alone is not sufficient to characterizetheir functions and interactions in a global scale. However, evennot truly global scale information and knowledge newly foundfrom such omics studies can be successfully employed for straindevelopment. Also, it is increasingly accepted that in silico analysisof the cellular network is promising to discover a knowledge mapfor deciphering the functions and characteristics of the biologicalsystems. In silico genome-scale metabolic models can be used tounderstand the status of the complex cellular system andinvestigate inherent cellular properties. As such, hypothesis-driven in silico experiments can be invaluable to improve ourability to predict the cellular behavior of microorganisms undervarious genetic and environmental conditions. In this lecture, Iwill show some successful examples of "local engineering of

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cellular components based on global omics data" towardenhanced production of recombinant proteins. Also, systemsbiotechnological research cycle that allows efficient straindevelopment by combining in silico and wet experiments will bediscussed.AcknowledgementsThis work was supported by the Korean Systems BiologyProgram from the MOST, LG Chem Chair Professorship, IBM,Microsoft and BK21 project.

S3Industrial aspects of protein productionby filamentous fungiMichael WardGenencor International, 925 Page Mill Rd, Palo Alto,CA 94304, USA


Aspergillus niger and Trichoderma reesei are filamentous fungi thatare used extensively for large-scale industrial production ofsecreted proteins. Recombinant DNA technology has beenemployed to create strains that over-produce native proteins orthat produce foreign proteins. Standard methods of strainconstruction involve use of strong promoters, insertion ofmultiple copies of expression cassettes into the genome, and, insome cases, secretion of a foreign protein as a fusion with anative, secreted protein. However, for manufacturing atindustrial scale it is sometimes necessary to improve theproductivity of the strains further. Despite the fact that ourknowledge of the molecular genetics of filamentous fungi hasimproved dramatically over the last few years it has been difficultto use this knowledge for rational strain improvement.Consequently, classical strain improvement methods involvingmutagenesis and screening are still important and continue to beimproved. Aspects of strain and fermentation process improve-ment for industrial enzyme production will be discussed.

S4The effects of expressing anti-apoptotic genes onmammalian cell survival, physiology, and proteinproductionMichael J. BetenbaughDepartment of Chemical and Biomolecular Engineering, JohnsHopkins University, Baltimore, Maryland, USA 21218 E-mail:[email protected]


Mammalian cell culture is used for the production of proteinsfor numerous therapeutics, diagnostics, vaccines, and thegeneration of cells used in biomedical devices. During cellculture, the cells are exposed to numerous external and internalinsults such as nutrient depletion, toxin accumulation, viralinfections, and external shear stress. These events can some-times cause the cells to trigger a biochemical cascade calledapoptosis or programmed cell death in which the cells activelyparticipate in their own demise. The programmed cell death(PCD) cascade decreases the number of viable cells in abioreactor and leads to the premature termination of cellculture runs. Cell death may also lower productivity sincebioreactor resources must be utilized to replace dead or dying

cells. As a result, methodologies that limit the activation of thiscascade are desirable and represent one of the most significantapplications of metabolic engineering in cell culture. In this way,it may be possible to extend mammalian cell lifetimes andfunction for multiple biotechnology and bioengineering applica-tions. The engineering of production cell lines to express anti-apoptotic genes may have numerous potential process benefits,including enhanced cell survival, increased protein expressionand improved product quality. A number of natural anti-apoptosis genes have been identified in both eucaryotes andviruses and these anti-apoptosis proteins are being used to blockor limit the cell death cascade. Unfortunately, these anti-apoptosis proteins can be degraded in culture or otherwiseeliminated. In order to increase their activity, some of the anti-apoptosis proteins can be modified to limit degradation in cellsystems. In addition, apoptosis pathways include feedback andfeedforward loops that lead to amplification of the apoptoticresponse. Strategies that block cell death at multiple pointsalong the cascade may limit the amplification of these apoptoticsignals. As a result, the expression of multiple anti-apoptosisgenes has been explored in order to extend cell survival andimprove protein production. More recently, the role ofbioprocess environments is being examined by investigatingsurvival of cells engineered to include anti-apoptosis genes indifferent bioreactors with various operating conditions. The useof different bioreactors allows cell culture engineers to examinethe relationship between anti-apoptosis engineering and cellphysiology and productivity for mammalian cell systems.

S5The making and breaking of an efficient antibodyfactoryRoberto SitiaUniversita Vita-Salute San Raffaele Scientific Institute, 20132Milano, Italy E-mail: [email protected]


Upon encounter with antigen, long-lived B lymphocytesdifferentiate into short-lived plasma cells, the terminal effectorsof the humoral immune response. Plasma cells are specialized inimmunoglobulin (Ig) secretion, each of them being capable ofreleasing thousands of molecules per second. We haveperformed proteomics analyses to unravel mechanistically themassive de novo ER biogenesis during terminal plasma celldifferentiation, with emphasis on how cells adapt ER qualitycontrol to exuberant Ig production and secretion. These studiesrevealed that waves of functionally related proteins areproduced to increase the capacity of the antibody factory, andled to the identification of novel ER chaperones. As to themechanisms that lead to plasma cell death, we show that in thelate phases of plasmacytic differentiation, when antibodyproduction becomes maximal, proteasomal activity unexpect-edly decreases. The excessive load for the reduced proteolyticcapacity correlates with accumulation of polyubiquitinatedproteins, stabilization of endogenous proteasomal substrates(including Xbp1s, Ik-Ba and Bax), onset of apoptosis, andsensitization to proteasome inhibitors. A developmental pro-gram seems therefore to link plasma cell death to proteinproduction, explaining the peculiar sensitivity of normal andmalignant plasma cells to proteasome inhibitors.

Microbial Cell Factories 2006, 5(Suppl 1) http://www.microbialcellfactories.com/supplements/5/S1


S6Monitoring the dynamics of transcriptionand translation within the time course ofrecombinant e. coli cultivationsKarin Durrschmid1, Wolfgang Schmidt-Heck2,Thomas Hrebicek3, Helga Reischer1, Norbert Auer1,Gerd Margreiter1, Reinhard Guthke2, Andreas Rizzi3

and Karl Bayer11Department of Biotechnology, University of NaturalResources and Applied Life Sciences, 1190 Vienna, Austria2Leibniz Institute for Natural Products research and InfectionBiology-Hans Knoell Institute (HKI), 07745 Jena, Germany3Institute of Analytical Chemistry, University of Vienna, 1090Vienna, Austria E-mail: [email protected]


Background: Among the key objectives for the optimization ofrecombinant protein production on industrial scale high yield ofthe product is one of the most important issues. To achieve highquantities of recombinant protein strong host/vector systems areutilized, generally leading to an overburden of the host cellmetabolism. To cope with different types of stress cells haveevolved complex regulatory entities acting on the highest level ofmetabolic regulation, such as the stringent response network.Highly specific signal molecules of these regulatory networks, likesigma factors and ppGpp, as well as global regulators like ArcA,Crp and Fis enable accurate up-and downregulation of specificgenes [1, 2]. The there by altered expression profile confersincreased resistance to adverse conditions. As the exploitation ofthe cell factory will always approach the physiological limits of thecellular machinery the short- as well as the long-term impact ofhigh recombinant gene expression rates have to be investigated.Therefore key variables of metabolic stress to define and thusquantify the actual metabolic load of host cell metabolism can beinferred from variations of genome and proteome patterns. Dueto the availability of whole genome E. coli microarrays thistechnology provides an efficient tool for screening overalltranscriptional changes in the course of fermentation processes.2D-Fluorescence Differencel Gel Electrophoresis (DIGE, Amer-sham Biosciences) and MALDI-MS are established to identifychanges on proteome level. A combination of these threemethods (microarrays, DIGE and MS) paired with the applicationof time series experiments during a recombinant fermentationprocess provide an insight into the black box of cell metabolismand improve the understanding of cellular physiology.Results: The design of expression profiling experiments,measuring the rate of transcription of mRNA and expressionof proteins, is a very important issue in bioprocessing. Most ofthese studies have been carried out in flask cultures, wherebynon defined conditions were applied [3]. In accordance withgood experimental design it is important to ensure that thechanges in expression profiles are due to the perturbing event.To fulfil these requirements chemostat cultivation is performedin our study to monitor the behavior of the production strainE. coli HMS 174(DE3)(pET11a) under stress conditions. Thushomogeneous samples and steady state conditions can beguaranteed and aimed perturbation is applied. Different induc-tion strategies are used to exert different metabolic loads on theproduction organism and two soluble proteins are expressed inorder to detect the impact of the recombinant protein on theproduction organism.

Microarray and DIGE data show a significant increase in thenumber of altered genes and respectively proteins, whereof adramatic change in the cell during this kind of stress conditioncould be derived. Evaluation of these data was performedaccording to their assignment to metabolic pathways as well asto regulatory profiles.Conclusion: Microarray analysis as well as DIGE combinedwith MS proved to be well suited tools for monitoring changeson transcriptome and proteome level of time-series experi-ments during recombinant cultivations. The acquired dataprovide the basis to identify interactions and bottlenecksbetween metabolic pathways and regulatory circuits of theproduction organism and should enable reverse engineering ofhost cell for the optimal exploitation for recombinant proteinproduction.AcknowledgementsThis work is supported by the Austrian Center of Biopharma-ceutical Technology.References1. Cashel M, Gentry DR, Hernandez VJ and Vinella D, et al:

The stringent response Escherichia coli and Salmo-nella typhimurium. Cell Mol Biol Washington DC: Amer-ican Society for Microbiology Press: Neidhardt FC, et al1986, 1410–1438.

2. Martinez-Antonio A and Collado Vides J: Identifyingglobal regulators in transcriptional regulatory net-works in bacteria. Curr Opin Microbiol 2003, 6:482–489.

3. Yoon SH, Han MJ, Lee SY, Jeong KJ and Yoo J: CombinedTranscriptome and Proteome Analysis of Escher-ichia Coli During High Cell Density Culture. BiotechnBioeng 2002, 81(7):753–767.

S7Targeting expression of expanded polyglutamineproteins to the endoplasmic reticulum ormitochondria prevents their aggregationErwann Rousseau1, Benjamin Dehay1, Lea Ben-Haıem2,Yvon Trottier2, Michel Morange1 and Anne Bertolotti11Laboratoire de Genetique Moleculaire, CNRS UMR8541,Ecole Normale Superieure, 46 rue d’Ulm, 75230 ParisCedex 05, France2Institut de Genetique et de Biologie Moleculaire et Cellulaire,INSERM-CNRS-Universite Louis Pasteur, BP 10142, 67404Illkirch Cedex, France


Aggregation of misfolded proteins is a characteristic of severalneurodegenerative diseases. The huntingtin amino-terminalfragment with extended polyglutamine repeat forms aggregatesclosely associated with chaperones both in the cytoplasm andthe nucleus. As each cellular compartment contains distinctchaperones and because the molecular mechanisms controllingpolyglutamine aggregation are largely unknown, we decided toinvestigate the influence of different cellular environments onthe aggregation of this pathological protein. We found thataggregation of a protein containing a polyglutamine stretch ofpathological length is abolished when its expression is targetedto the endoplasmic reticulum. Once retrogradely transportedoutside of the endoplasmic reticulum, the aggregation-pronepolyglutamine containing protein recovers its ability to aggre-gate. When expressed in the mitochondria, a protein containing



73 glutamines is entirely soluble while the nucleo-cytosolicequivalent has an extremely high tendency to aggregate. Ourdata imply that polyglutamine aggregation is a propertyrestricted to the nucleo-cytosolic compartment and suggestthe existence of compartment-specific co-factors promoting orpreventing aggregation of pathological proteins. Implications forrecombinant proteins production will be discussed.

S8Protein quality control systems: Mechanismsand applicationsAxel Mogk and Bernd BukauZentrum fur Molekulare Biologie Heidelberg, Im NeuenheimerFeld 282, 69120 Heidelberg, Germany


A protein quality control system, consisting of molecularchaperones and proteases, controls the folding status of proteinsand prevents the aggregation of misfolded proteins by eitherrefolding or degrading aggregation-prone species. During severestress conditions or upon protein overproduction this protec-tion system can be overwhelmed by high substrate load, resultingin the formation of protein aggregates. In such emergencysituations, ClpB/Hsp104, a ring-forming AAA+ chaperone, canbecome a key player for cell survival, as it has the extraordinarycapacity to rescue proteins from an aggregated state incooperation with an Hsp70 chaperone system.We could recently demonstrate that ClpB/Hsp104 extractsunfolded polypeptides from an aggregate by threading themthrough its central pore. This translocation activity is necessarybut not sufficient for aggregate solubilization. In addition, themiddle (M) domain of ClpB and the Hsp70 system have essentialroles, potentially by providing an additional unfolding force,which facilitates the extraction of misfolded proteins fromaggregates. Here we report on both, novel mechanistic aspectsof the disaggregation machinery and its application to increasethe solubility of model substrates.

S9Protein aggregation into bacterial inclusion bodiesis a specific kinetically driven processNatalia S de Groot1, Montse Morell2, Josep Vendrell1,2,Francesc X Aviles1,2 and Salvador Ventura1,21Departament de Bioquimica I Biologia Molecular2Institut de Biotecnologia I de Biomedicina UniversitatAutonoma de Barcelona, 08193 Bellaterra (Barcelona), Spain


Background: Bacterial inclusion bodies are major bottlenecksin protein production and are hampering the development oftop priority research areas such as structural genomics.Inclusion body formation was formerly considered to occurvia non-specific association of hydrophobic surfaces in foldingintermediates rendering biologically inactive protein. Increasingevidence, however, indicates that protein aggregation in bacteriais a rather specific event which might result in active inclusionbodies [1, 2].Results: Here, first we have used fluorescence resonanceenergy transfer and microscopy to investigate the degree towhich unrelated proteins expressed in the same cells coaggre-gate with one another. Our data reveal that in bacteria, protein

aggregation is a specific event even among highly aggregation-prone polypeptides expressed at high levels.Second, we have investigated the effect of a large set of single-point mutants of one of these proteins on its specific activityonce deposited in inclusion bodies. We find that the activity ofsuch aggregates significantly correlates with the predictedaggregation rates for each mutant.Conclusion: Overall the data in this study confirms that in vivoprotein aggregation depends on molecular recognition and suggeststhat rationally tuning the kinetic competition between folding andaggregation might result in highly active, inclusion bodies. Theexploration of this technology during recombinant protein produc-tion would have a significant biotechnological value.References1. Carrio M, Gonzalez-Montalban N, Vera A, Villaverde A and

Ventura S: Amyloid-like properties of bacterial inclu-sion bodies. J Mol Biol 2005, 347:1025–1037.

2. Garcia-Fruitos E, Gonzalez-Montalban N, Morell M, Vera A,Ferraz RM, Aris A, Ventura S and Villaverde A: Aggrega-tion as bacterial inclusion bodies does not implyinactivation of enzymes and fluorescent proteins.Microb Cell Fact 2005, 4:27.

S10Kinetics of aggregation and structural propertiesof proteins in inclusion bodies studied by Fouriertransform infrared spectroscopyAntonino Natalello1, Diletta Ami1,Pietro Gatti-Lafranconi1, Ario de Marco2,Marina Lotti1 and Silvia Maria Doglia11Department of Biotechnology and Biosciences, University ofMilano-Bicocca, Piazza della Scienza 2, 20126 Milano, Italy2EMBL Scientific Core Facilities, Mayerhofstr. 1, D-69117,Heidelberg, Germany


Background: Protein aggregation plays a crucial role inmedical sciences and in biotechnology, as it occurs in severaldiseases and in the expression of recombinant proteins inbacterial cells in the form of inclusion bodies (IBs). Interestingly,it has been suggested that the presence of native-like structureswithin IBs [1, 2, 3, 4] improves the efficiency of refoldingprotocols that employ mild solubilization methods [5]. Thisproperty could also explain the residual enzymatic activity ofrecombinant proteins in IBs, with possible applications inbiocatalysis [6].As recombinant protein production in bacteria is a central issuein biotechnology, it would be instructive to monitor the kineticsof protein aggregation and the extent of native-like secondarystructures within IBs.Results: We will present a new Fourier transform infraredspectroscopy (FT-IR) approach to study the aggregation ofrecombinant proteins in E. coli in the form of aggregates ofincreasing complexity. The method enables us to monitor thekinetics of aggregate formation within intact cells in a rapid andnon invasive way and to obtain structural information onproteins within IBs. We will report results on four recombinantproteins: human growth hormone (h-GH), human interferon-alpha-2b (IFN-alpha-2b) [4, 7], Pseudomonas fragi lipase [3], andgreen fluorescent protein – glutathione S-transferase fusionprotein (GFP-GST) [8].



Kinetics of aggregate formation was investigated at differentproduction temperatures. The rate of protein aggregation,monitored by the marker band of aggregation in the FT-IRabsorption spectrum (Amide I band), was found to increase withthe raising of production temperature. Furthermore, the proteinexpression in its soluble and insoluble fraction was alsoevaluated by the analysis of the FT-IR spectrum, in excellentagreement with SDS-PAGE analysis.To obtain structural information on protein aggregates,extracted IBs were also studied in the Amide I absorptionregion. Two structural features were observed, namely thepresence of native-like residual structures and the intermole-cular �-sheet interaction of proteins within IBs.Interestingly, for the same protein the residual native-likestructures in IBs were found to change with the level ofexpression. Therefore, by modulating the culture conditions,the extent of native-like structures in IBs can be optimised withuseful applications in biotechnology.Furthermore, additional structural features were obtained bythe comparison of the FT-IR spectra of the native form, IBs andthermal aggregates for the same protein.Conclusion: This FT-IR analysis offers a simple and rapidmethod to monitor in vivo the development of aggregatesformed by heterologous proteins and the effect of culturecondition modification on the process. Furthermore, themethod indicates that aggregating proteins modify at differentextent their secondary structures from native a-helices andintramolecular �-sheets to intermolecular �-sheets typical ofamorphous aggregates and fibrils.AcknowledgementsThis work was supported by INFM (Istituto Nazionale Fisica dellaMateria) grant to S.M.D. The support of F.A.R. (Fondo di Ateneoper la Ricerca) grants to S.M.D. and M.L. is also acknowledged.References1. Oberg K, Chrunyk BA, Wetzel R and Fink AL: Native-like

secondary structure in interleukin-1 beta inclusionbodies by attenuated total reflectance FTIR. Biochem-istry 1994, 33:2628–2634.

2. Przybycien TM, Dunn JP, Valax P and Georgiou G:Secondary structure characterization of beta-lacta-mase inclusion bodies. Protein Eng 1994, 7:131–136.

3. Ami D, Natalello A, Gatti-Lafranconi P, Lotti M andDoglia SM: Kinetics of inclusion body formationstudied in intact cells by FT-IR spectroscopy. FEBSLett 2005, 579:3433–3436.

4. Ami D, Natalello A, Taylor G, Tonon G and Doglia SM:Structural analysis of protein inclusion bodies byFourier transform infrared microspectroscopy. Bio-chim Biophys Acta 2006 in press.

5. Patra AK, Mukhopadhyay R, Mukhija R, Krishnan A, Garg LCand Panda AK: Optimization of inclusion body solubi-lization and renaturation of recombinant humangrowth hormone from Escherichia coli. Protein ExprPurif 2000, 18:182–192.


7. Ami D, Bonecchi L, Calı S, Orsini G, Tonon G andDoglia SM: FT-IR study of heterologous protein

expression in recombinant Escherichia coli stains.Biochim Biophys Acta 2003, 1624:6–10.

8. Schrodel A and de Marco A: Characterization of theaggregates formed during recombinant proteinexpression in bacteria. BMC Biochemistry 2005, 6:10.

S11Investigation of the inclusion body formationprocess by FTIR spectroscopyGerd Margreiter1, Manfred Schwanninger2,Christian Obinger2 and Karl Bayer11Department of Biotechnology, BOKU – University of NaturalResources and Applied Life Sciences, Vienna, Austria2Department of Chemistry, BOKU – University of NaturalResources and Applied Life Sciences, Vienna, Austria E-mail:[email protected]


Background: The great demand on high amounts of pureprotein for pharmaceutical applications and for research madeEschericha coli one of the most important cell factories forrecombinant protein production. Although it is a well studiedorganism showing high productivity, the recombinant proteinfrequently aggregates and forms the so called inclusion bodies(IBs). The entire aggregation process is still poorly understood.Recent research showed that proteins expressed as inclusionbodies have extensive native-like secondary structures and thatthe formation of IBs is a result of specific aggregation betweenfolding intermediates of protein molecules. However, solubilisa-tion of inclusion bodies by application of high concentrations ofchaotropic reagents the secondary structure is destroyed,leading to a random coil formation of the protein structureand exposure of hydrophobic surfaces. The loss of secondarystructure due to solubilisation and the interaction of therebyexposed protein domains lead to undesired aggregation andmisfolding of the target protein. These reactions are consideredto be responsible for poor recovery of bioactive proteins fromIBs [1, 2, 3, 4]. Therefore, solubilisation processes which providethe conservation of the existing secondary structure of the IBswill probably lead to higher yields of bioactive protein.Consequently it is important to evaluate to which extent thestructural properties of the IBs can be influenced by cultivationconditions and induction strategy [5].For structural analysis the method of choice is FTIR (FourierTransformed Infrared) spectroscopy, because of its possibleapplication to study proteins regardless their physical form both insolution and in solid precipitates. FTIR is very sensitive for analysis ofthe secondary structure and for detecting conformational changes.Results: Solubilisation of IBs with different buffers andrefolding: To find an alternative solubilisation method, a testseries with buffers of different pH and urea concentrations wasperformed. Using Tris/Cl pH11.4 containing 3–5 M urea, as wellas Tris/Cl pH11 containing 4 and 5 M urea the IBs of the modelprotein (autoprotease-GFP fusion protein) were dissolvedcompletely (also with 8 M urea pH8 as a standard method).Refolding was performed by dilution and showed a higher yieldfor IBs which were dissolved by buffer with high pH and lessurea compared to IBs dissolved by 8 M urea pH8. FTIRspectroscopy of the protein dissolved in the different buffers(8 M urea vs. 4 M urea pH11.4) showed shifts of the amide Iband, indicating differences of the secondary structure.



Impact of cultivation conditions on IB structure:Harvested inclusion bodies of four fed-batch cultivationsdiffering in cultivation temperature (30˚C and 37˚C) andinduction strategy (partial and full induction with IPTG) havebeen analysed by ATR-FTIR-spectroscopy. IBs of the cultivationat 30˚C and full induction showed a shift of 2 wavenumberscompared to the others, what could be an evidence fordifferences in the structure, and the refolding experimentresulted in a higher yield. Furthermore different ratios of solubleand insoluble recombinant protein fractions were obtained atthe different cultivation conditions.Conclusion: Experiments with alternative dissolving condi-tions compared to the standard method (complete unfoldingwith high concentrations of chaotrops) demonstrated the highpotential of conserving existing protein structures in inclusionbodies to obtain higher refolding yields. In addition, dataobtained by FTIR spectroscopy and the different ratios ofsoluble to aggregated fraction confirm the impact of differentcultivation conditions on the folding process of the hetero-logous expressed proteins. Furthermore, the recent observa-tions of the heterogeneity of folding states in inclusion bodiespoints out the demand of a new view of those aggregates.Monitoring the entire folding process of the recombinantprotein combined with improved downstream strategies has ahigh potential to increase renaturation efficiency and to improveprotein quality. Further research on diverse proteins isnecessary to get deeper insight into inclusion body formation.References1. Khan R, Appa Rao K, Eshwari A, Totey S and Panda A:

Solubilization of recombinant ovine growth hor-mone with retention of native-like secondary struc-ture and its refolding from the inclusion bodies ofEscherichia coli. Biotechnol Prog 1998, 14:722–728.

2. Patra A, Mukhopadhyay R, Mukhija R, Krishnan A, Garg Land Panda A: Optimization of inclusion body solubi-lization and renaturation of recombinant humangrowth hormone from Escherichia coli. Protein ExprPurif 2000, 18:182–192.

3. Oberg K, Chrunyk BA, Wetzel R and Fink AL: Nativelikesecondary structure in interleukin-1 beta inclusionbodies by attenuated total reflectance FTIR. Biochem-istry 1994, 33:2628–2634.

4. Umetsu M, Tsumoto K, Ashish K, Nitta S, Tanaka Y,Adschiri T and Kumagai I: Structural characteristics andrefolding of in vivo aggregated hyperthermophilicarchaeon proteins. FEBS Lett 2004, 557:49–56.

5. Georgiou G and Valax P: Expression of correctly foldedproteins in Escherichia coli . Curr Opin Biotechnol 1996,7:190–197.

S12Environment matters – An E. coli case study. Howprocess technology affects cell physiology andproduct quality/quantityUrsula RinasGBF – German Research Centre for BiotechnologyMascheroder Weg 1, D-38124 Braunschweig, Germany


What matters most?? Genes or environment? Of course, bothmatter, but environment matters more. "Bad genes" can be

overcome in an adequate environment and "good genes" can besuppressed under inappropriate conditions.The impact of process technology on cell physiology andproduct quality/quantity will be discussed using a comprehensivesystems biotechnology approach. Production of a recombinanthuman growth factor in small-scale shaker flasks, as well as inbatch and industrial relevant fed-batch cultures using differentinduction strategies will be evaluated using proteome, tran-scriptome, metabolome and fluxome profiling techniques. Theresults clearly show that the commonly observed responsestowards induced recombinant protein production such asgrowth rate reduction, changes in cell morphology, inductionof stress responses and corresponding alterations in geneexpression profiles, as well as alterations of central catabolic andanabolic activities strongly depend on, and thus can be effectivelymanipulated by the environmental conditions to achieve the goalof product formation as desired.

S13Substrate feeding strategies in Pichia pastorisfed-batch cultivation processes: Analysis of keyparameters influencing recombinant proteinproductionRamon Ramon, Oriol Cos, Pau Ferrer,Jose Luis Montesinos and Francisco ValeroDepartament d’Enginyeria Quımica, E.T.S.E., UniversitatAutonoma de Barcelona, 08193-Bellaterra (Cerdanyola delValles), Spain


Background: An important number of heterologous proteinshave been produced in the methylotrophic yeast Pichia pastorisusing the methanol-inducible alcohol oxidase promoter [1].Cultivation conditions and host physiology have an importantimpact on the final yields and productivities for heterologousprotein production [2]. Recently, the effect of the Mutphenotype and gene dosage on the heterologous productionof a Rhizopus oryzae lipase (ROL) in P. pastoris has been studied infed-batch bioprocesses with a manual (off-line) methanolconcentration control [4]. These studies demonstrated thatvariations of the residual methanol concentration influencedrastically in the specific consumption and production rates. Toavoid this problem a predictive control algorithm coupled with aPI feedback controller has been satisfactorily implemented [5].This set-up has allowed for further analysis of several keyparameters influencing heterologous protein production inP. pastoris fed-batch cultivation processes. In particular, theimpact of i) the residual methanol concentration present in theculture broth and ii) co-feeding of a multicarbon substrate andmethanol on process performance will be illustrated in a P.pastoris Muts phenotype strain secreting a Rhizopus oryzae lipase(ROL) as a reporter protein.Results: The effect of methanol concentration on hetero-logous ROL production during the fed-batch phase was analysedby performing cultivations at different methanol set points,ranging from 0.5 to 1.75 g· L-1. The maximal lipase activity (490UA· mL-1), specific yield (11236 UA· g-1biomass), productivity(4901 UA· L-1· h-1) and specific productivity (112 UA· g-1biomass ·h-1) were reached for a residual methanol concentration setpoint of 1 g· L-1. Notably, these parameters are almost 2-foldhigher than those obtained with a manual control at a similar



methanol set-point. The study of the consumption (qs) andproduction rates (qp) showed very different patterns for theserates depending on the methanol concentration set-point:In all cultivations maximal qs values were obtained at thebeginning of the induction phase; shortly after this point,qs started to exponentially decrease.The evolution of the extracellular lipolytic activity wascompletely different depending on the residual methanolconcentration at the fed-batch phase. In particular, when themethanol set point was set at 0.5 g· L-1, the qp reached amaximum of 340 UA· gX-1· h-1 at the beginning of the inductionphase, followed by a sharp decrease to almost zero values after20 h of induction. Since no proteolytic degradation of theproduct was observed, the exponential decrease in the productsecretion rate was probably indicative that ROL synthesis hadalso stopped. In the fed-batch cultivation carried out at a setpoint of 1 g· L-1, the lipolytic activity values remained very lowduring a considerable period (20 h of induction phase); after thislag phase, a maximum qp value of 440 UA· gX-1· h-1 is reachedafter 40 h of induction phase, which was followed by anexponential decrease to values below 100 UA· gX-1· h-1 after75 h of fed-batch phase. Again, neither extracellular proteaseactivity nor important levels of cellular lysis were detected. Theqp values during the fed-batch cultivation at a set point of 1.75 g·L-1 were kept rather constant throughout the bioprocess withsignificantly lower maximal (60 UA· gX-1· h-1) and mean qpvalues. In this cultivation, extracellular lipolytic activity increasedsteadily until 110 h. After this point, lipolytic activity slightlydecreased by the effect of cellular lysis. However, obtainedlevels (150 UA· mL-1) were similar to those obtained at thebeginning of the induction phase when a methanol set-point of0.5 g· L-1 was used (137 UA· mL-1) in only 4 hours.Overall, these results indicated that, although cell growth isgenetically limited (the host strain is Muts and, therefore, has avery limited methanol assimilation capacity), the synthesis andsecretion rates are still greatly influenced by the residualmethanol concentration (as expected from the observation thattranscription levels from the AOX1 promoter are highest atmethanol limiting concentrations [1]).Since ROL expression has been shown to trigger the unfoldedprotein response [6], cells growing under carbon/energylimitation may not be able to sustain highest ROL productionrates. To overcome this problem, we tested the effect of anadditional, non-repressing, carbon source (sorbitol) on cellperformance and heterologous protein expression and secretionduring the methanol-fed induction phase. Remarkably, growth ofMuts cells in a batch cultivation using sorbitol and methanol ascarbon sources showed that both substrates were co-assimilatedsimultaneously along the growth phase. Conversely, sorbitol andmethanol were assimilated sequentially by wild type cells.Hence, we performed replica fed-batch cultivations at amethanol residual concentration of 0.5 g L-1 and sorbitolbelow 2 g L-1 simultaneously fed. The maximum qp valuesreached at the beginning of the induction phase were similar(about 340 UA· gX-1· h-1). Although the qp decreased after thispoint, production rate stabilized at around 200 UA· gX-1· h-1

until the end of the bioprocess. By using this strategy, themaximal lipolytic activity was 3.5 fold higher than in the fed-batch at 0.5 g L-1 of methanol as a single carbon source. Thespecific yield, productivity and specific productivity were alsoimproved by 2.5, 2.6 and 2-fold, respectively.

Conclusion: The combined use of a P. pastoris Muts strain andthe control of the residual methanol concentration during thefed-batch phase allow for the modulation of the ROL productionrates. Since ROL expression triggers the UPR, cells growingunder carbon and energy source limitation appear particularlysensitive to ROL production rates (highest process productiv-ities are not achieved under methanol-limiting conditionsi.e. when AOX1 promoter transcription levels are probablyhighest).Notably, mixed carbon source co-assimilation seems to supportcell’s adaptation to the stress caused by ROL secretion,i.e. allowing for sustained specific secretion rates and boostingprocess productivities and yields.AcknowledgementsThis work was supported by a grant from the Spanish Programon Chemical Processes Technologies (CTQ2004-00300).References1. Lin-Cereghino J and Cregg JM: Heterologous protein

expression in the methylotrophic yeast Pichia pas-toris . FEMS Microbiol Rev 2000, 24:45–66.

2. Macauley-Patrick S, Fazenda ML, McNeil B and Harvey LM:Heterologous protein production using the Pichiapastoris expression system. Yeast 2005, 22:249–270.

3. Zhang W, Inan M and Meagher MM: Fermentationstrategies for recombinant protein expression inthe methylotrophic yeast Pichia pastoris. BiotechnolBioprocess Eng 2000, 5:275–287.

4. Cos O, Serrano A, Montesinos JL, Ferrer P, Cregg JM andValero F: Combined effect of methanol utilization(Mut) phenotype and gene dosage on recombinantprotein production in Pichia pastoris fed-batchcultures. J Biotechnol 2005, 116:321–335.

5. Cos O, Ramon R, Montesinos JL and Valero F: A simplemodel-based control for Pichia pastoris allows amore efficient heterologous protein productionbioprocess. in press.

6. Resina D, Cos O, Gasser B, Mauer M, Valero F,Mattanovich D and Ferrer P: Analysis and engineeringof bottlenecks in Rhizopus oryzae lipase productionin Pichia pastoris using the mitrogen source-regu-lated FLD1 promoter. in press.

S14A rational approach to optimize feed profiles forthe maximization of productivity of secretedproteins expressed in Pichia pastorisMichael Maurer1, Brigitte Gasser1, Manfred Kuhleitner2

and Diethard Mattanovich1,31University of Natural Resources and Applied Life SciencesVienna, Department of Biotechnology, Institute of AppliedMicrobiology, Vienna, Austria2University of Natural Resources and Applied Life SciencesVienna, Department of Integrative Biology, Institute ofMathematics, Vienna, Austria3School of Bioengineering, University of Applied SciencesFH-Campus Vienna, Austria


Background: Yeast processes for the production of secretedrecombinant proteins are usually performed in fed batch.Although it is established that the productivity of such processes



is defined by the space time yield, there is a lack of knowledgecorrelating physiological data to process related data defined bythe feed profile. Typically these fed-batches employ eitherexponential or linear feed rates. Based on our experience, bothprotocols are not optimal solutions for secreted proteins.Using a Pichia pastoris clone expressing the Fab fragment of thehuman monoclonal antibody 2F5 under the glyceraldehydephosphate dehydrogenase (GAP) promoter, we determined thecorrelation of specific product formation rates (qP) as well asbiomass yields (YX/S) in relation to a wide range of specificgrowth rates (�). Measuring transcript levels of the productspecific genes as well as 49 host genes related to growth, proteinsynthesis, oxygen and nutrient limitation responses, and proteinsecretion stress (UPR, ERAD, posttranslational processing),enabled the determination of net specific transcription rates.These parameters were determined in chemostat cultures in arange of dilution rates (=�) between 0.02 and 0.2 h-1.Results: qP showed an asymptotic relation to �, which couldbe modeled by a Monod-type equation. YX/S could be describedby approximation of the maintenance coefficient m and themaximum yield coefficient. The specific transcription rates ofthe product related genes (under control of the GAPpromoter), as well as those of glycolytic (e.g. PFK1) andribosomal genes correlated with �. TCA cycle related genes,and some stress genes were more independent of growth, whileUPR controlled genes were more strongly expressed at high �.The determination of a relation of qP, YX/S and � enabled thedevelopment of an optimization model for fed batch based onthe Microsoft Excel Solver. This optimization tool allows easilyto impose different constraints on the model, like minimumproduct concentration or maximum biomass concentration,both being key parameters for downstream processing.Depending on the constraints, different combinations ofexponentially and linearly increasing feed rates were calculatedas optimal feed protocols. Model predictions were comparedwith actual fed batch data.Conclusion: These data provide evidence that recombinantprotein secretion is decoupled from growth and specific mRNAsynthesis at higher specific growth rates, thus approaching asaturation level at higher �. Consequently the development of �over time is a critical parameter for an optimal fed batch process.

S15Expression of soluble and membraneproteins in E. coliA James Link, Ki-Jun Jeong and George GeorgiouInstitute for Molecular and Cell Biology and Department ofChemical Engineering, University of Texas, Austin, TX 78712,USA


Our lab has developed a number of tools for enhancing theexpression of soluble secreted protein as well as membraneproteins in bacteria. Specifically: (1) We have explored mutagen-esis/screening and also host cell engineering strategies for theexpression of mammalian and prokaryotic membrane proteins.This work has led to the development of simple approaches thatcan be used to obtain significantly increased yields of membraneproteins in E.coli. (2) Developed a high through strategy relying onflow cytometry for the isolation of mutant proteins exhibitingenhanced expression from combinatorial libraries. This latter

technology has been used for the expression maturation of scFvand FAB antibody fragments.Representative results illustrating the power of these methodswill be discussed.

S16Enhancing recombinant glycoprotein yield andquality using gene targeted CHO cells linesDanny Chee Furng Wong1,2, Yih Yean Lee1, Kathy TinKam Wong1, Peter Morin Nissom1, May May Lee1

and Miranda Gek Sim Yap11Bioprocessing Technology Institute, Biomedical SciencesInstitutes, 20 Biopolis Way, #06-01 Centros, Singapore1386682Department of Chemical & Biomolecular Engineering,National University of Singapore, 10 Kent Ridge Crescent,Singapore 119260


Background: It has been widely reported that CHO cellsundergo apoptosis in culture, despite nutrient supplementationthrough fed-batch strategies. An understanding of apoptosissignaling can thus enable the identification of key genetic targetsfor the engineering of cell lines that could prolong cultureviability and attain higher cell densities to effectively improverecombinant glycoprotein yield and quality.Results: Transcriptional profiling using microarray technology,which allowed for the expression profiling of thousands ofdistinct genes simultaneously, has been adopted for the analysis ofapoptosis signaling in a CHO cell fed-batch culture system. Thesubsequent analysis has allowed for the identification of earlyapoptosis signaling genes, which were significantly up- or down-regulated as the culture transited from the exponential to thestationary phase. Novel gene targeted CHO cell lines weredeveloped by targeting the following anti- and pro-apoptosisgenes, either individually or in combination: Fadd, Faim, Alg-2,Requiem and Molecular Chaperones. Comparison of theseengineered CHO cell lines with the parental CHO in terms ofrecombinant glycoprotein yield and glycosylation profiles will bepresented. As hypothesized, the gene targeted CHO cell linesexhibited improved resistance to apoptosis, resulting in pro-longed culture viability and more importantly concurrentimprovement in recombinant protein yield for fed-batch cultures.Conclusion: This study showed that by harnessing theinformation from gene expression analyses, a rational approachto the development of robust CHO cell lines possessing thedesired culture characteristics to enhance recombinant proteinyields and quality can be achieved.

S17A novel engineered insect cell line for pro-proteinprocessing and activationMaria Cristina Sidoli, Mariantonietta Rubino,Lucia Iuzzolino, Beatrice Bellanti, Angela Molteni,Patrizia Arioli, Loredana Redaelli and Daniele CarettoniBiochemistry, AXXAM srl, San Raffaele Biochemical SciencePark, Milan, Italy


Background: A large number of therapeutically relevantproteins, including secreted enzymes, hormones and neuropep-



tides, need post-translational proteolytic processing to becomemature and functional. A problem in their heterologousexpression is often represented by the limited ability of thehost cell to efficiently and accurately mature the recombinantlyexpressed zymogens and precursors. This hurdle often impairstheir expression level and solubility, and an in vitro maturationstep is always required to recover functionally active products.However, the latter procedure cannot be universally applied,since in many cases functional enzymes can be obtained onlywhen the post-translational processing is accomplished by thephysiological peptidase in the proper cell compartment.Results: To tackle this bottleneck in recombinant proteinproduction, we have developed an insect cell line able toprocess inactive zymogens. Stably transfected cells have beenisolated through serial limiting dilutions and clone pool analysishas been performed to ensure the expression of therecombinant gene. Functional screening has been applied tocharacterize the final clones, based on the ability of the cellsinfected with recombinant baculovirus to secrete properlyprocessed human enzymes. The selected clone was demon-strated to be competent for processing of different enzymeclasses, including lipase, serine endopeptidase and metallopro-tease. The processing extent of the secreted enzymes strictlycorrelated with an increase in their catalytic activity, confirmingthat cleavage occurred at the proper activation site. The finalvalidation was performed applying a high-throughput approach,whereby media of insect cells grown in 24-deep-well blockswere processed using a 96-well plate fully-automated roboticprocedure to purify the recombinant proteins, whose catalyticactivity was assayed in 384-well plate format with a homo-geneous fluorescence-based readout. Pro-protein processingstably transfected cells were easily adapted to grow inminiaturized format (2 ml in 24-deep-well block) and wereused for mid-scale production of a recombinant humanpeptidase, demonstrating good compliance with differentcultivation conditions.Conclusion: We have developed a novel stably transfectedinsect cell line able to effectively process recombinant pro-containing proteins.

S18Antisense RNA based control of detrimentalfactors for recombinant gene expression inEscherichia coli – down-regulation of RNase EChristian Kemmer and Peter NeubauerBioprocess Engineering Laboratory, Dept. Process & Environm.Engin. and Biocenter Oulu, P.O. Box 4300, University of Oulu,FIN-90014 Oulu, Finland E-mail: [email protected]


Background: Messenger RNA decay is an important mechan-ism for controlling gene expression in all organisms. The rate ofthe mRNA degradation directly affects the steady stateconcentration of mRNAs and therefore influences the proteinsynthesis. It is assumed that RNase E is the initial point forgeneral mRNA decay in E. coli. RNase E is starting the mRNAdecay by performing the initial cut. The resulting fragments arethen further degraded by endo- and exonucleolytic activity inthe cell. During recombinant protein production mRNAinstability may be the major bottleneck for a successful product

formation. While RNase E initiates the degradation of mostmRNAs in E coli, it is likely that the enzyme is also responsiblefor the degradation of recombinant RNAs. Therefore a systemwhich allows the controlled reduction of RNase E might lead toa higher product formation in recombinant production pro-cesses. As RNase E is essential for cell viability and knockoutmutants can be not cultured, we investigated the possibility fordown-regulation of the level of RNase E by antisense RNAs.Results: During this study the antisense RNA based approachcould be established and indicated that the reduction of theintracellular level of RNase E in E. coli is possible. The expressionof antisense RNAs showed no influence on the cell growth. In afluorescence based sandwich hybridisation assay the amount ofantisense RNAs was sequence specifically quantified. We couldprove that the induction of antisense RNAs was followed by a25-fold increase of the detectable antisense RNA molecules percell. The antisense RNA level was maintained above 400molecules per cell. When the cells passed into the stationaryphase the level of expressed antisense RNAs decreasedmarkedly. Western blot experiments revealed the strongestreduction in the RNase E protein level 90 min after theinduction of antisense RNAs. The RNase E level could bedecreased to a maximum of 35% of the wild type level. Whenthe growth entered the stationary phase the RNase E level wasmaintained at 50 to 60% of the wild type level.Conclusion: In contrast to eukaryotic cells, where the RNAitechnology is widely used, this technology is nearly unex-plored in bacteria, although different natural systems useantisense RNA based silencing and antisense RNA mechan-isms have bee used to control plasmid stability. Only recentlyfew studies have indicated that antisense based strategies maybe a way to regulate the biosynthesis of global regulatoryproteins and metabolites and may be beneficial for theexpression of recombinant target proteins as well [1, 2, 3, 4].Our initial results clearly indicate the possibility to controlthe expression of RNase E, a key enzyme for mRNAdegradation, by antisense RNAs in E. coli. This was challengingand surprising due to the feed-back control mechanism whichkeeps the RNase E concentration in the cell constantindependent on the growth stage. Investigations to studythe effect of the down-regulation of RNase E on the stabilityof heterologous genes are ongoing.AcknowledgementsThis study was supported by the TEKES "Neobio" programReferences1. Desai RP and Papoutsakis ET: Antisense RNA strategies

for metabolic engineering of Clostridium acetobuty-licum. Appl Environ Microbiol 1999, 65:936–945.

2. Srivastava R, Cha HJ, Peterson MS and Bentley WE:Antisense downregulation of sigma (32) as a tran-sient metabolic controller in Escherichia coli: Effectson yield of active organophosphorus hydrolase. ApplEnviron Microbiol 2000, 66:4366–4371.

3. Tchurikov NA, Chistyakova LG, Zavilgelsky GB,Manukhov IV, Chernov BK and Golova YB: Gene-specificsilencing by expression of parallel complementaryRNA in E. coli . J Biol Chem 2000, 275:26523–26529.

4. Kim JY and Cha HJ: Down-regulation of acetatepathway through antisense strategy in E. coli:improved foreign protein production. Biotechnol Bioeng2003, 83:841–853.



S19The first fruits of an HTP membrane platform:crystal structure of the CorA Mg2+ transporterVladimir V Lunin1, Elena Dobrovetsky2,Galina Khutoreskaya2, Rongguang Zhang3,Andrzej Joachimiak3, Declan A Doyle4,Alexey Bochkarev2,5,6, Michael E Maguire7,Aled M Edwards1,2,3,5,6 and Christopher M Koth2,81Department of Medical Biophysics, University of Toronto, 112College Street, Toronto, ON, Canada2Banting and Best Department of Medical Research, Universityof Toronto, 112 College Street, Toronto, ON, Canada3Structural Biology Center & Midwest Center for StructuralGenomics, Biosciences Division, Argonne National Laboratory,9700 S. Cass Av. Argonne, IL 60439, USA4Structural Genomics Consortium Botnar Research Centre,Oxford, Oxon OX3 7LD, UK5Department of Medical Genetics and Microbiology, Universityof Toronto, 112 College Street, Toronto, ON, Canada6Structural Genomics Consortium, Banting Institute, 100College Street, Toronto, ON, Canada7Department of Pharmacology, Case Western ReserveUniversity, Cleveland, OH 44106-4965, USA8Vertex Pharmaceuticals Incorporated, 130 Waverly St.,Cambridge, MA 02139 USA


Membrane proteins constitute 30% of prokaryotic and eukar-yotic genomes but comprise a small fraction of the entries inprotein structural databases. A number of features of membraneproteins render them challenging targets for the structuralbiologist, among which the most important is the difficulty inobtaining sufficient quantities of purified protein. We havedeveloped robust procedures to express and purify largenumbers of prokaryotic membrane proteins. Using a set ofstandard conditions, expression can be detected in the mem-brane fraction for approximately 30% of cloned targets. To date,over 30 membrane proteins have been purified in quantitiessufficient for structural studies, typically in just two chromato-graphic steps. Theses include several transporters/channels,sensor kinases, and rhomboid intramembrane proteases. Usingthis system, we have recently crystallized and solved thestructure of the CorA magnesium transporter, the primaryMg2+ uptake system of most prokaryotes. Crystal structures of thefull-length ThermotogamaritimaCorA in an apparent closed state andits isolated cytoplasmic domain were determined at 3.9A and 1.85Aresolution respectively. Our HTP strategy for membrane proteins,and the first structure from this effort, will be discussed.

S20Highly active membrane proteins produced in acell-free expression systemLavinia Liguori1, Bruno Marques1, Ana Villegas-Mendez1,Romy Rothe1, Norbert Rolland2, Francoise Morel3

and Jean-Luc Lenormand11European Laboratory HumProTher, MENRT EA 2938, DBPC/Enzymologie, Centre Hospitalier Universitaire de Grenoble,BP 217, 38043 Grenoble Cedex 9, France2Laboratoire de Physiologie Cellulaire Vegetale, UMR 5168(CNRS/UJF/INRA/CEA Grenoble), Grenoble, Cedex 9 F-38054,France

3Groupe de Recherche et d’Etude du Processus Inflammatoire(GREPI), MENRT EA 2938, DBPC/Enzymologie, CentreHospitalier Universitaire de Grenoble, BP 217, 38043Grenoble Cedex 9, France


Background: Dysregulation in membrane protein activity isoften a primary step in human diseases. One of the major difficultiesin the study of membrane proteins is to recover a large enoughamount of recombinant proteins from the classical overexpressiontechniques. We have recently demonstrated the effectiveness of acell-free expression system in production of membrane proteins.The membrane proteins are produced in a soluble formwithout therequirement of a denaturation step. Most of the membrane proteinstested in this expression system are with native-like conformationwhatever the presence or not of lipid vesicles. Here, we examplifyour protein expression system on membrane proteins fromdifferent origins: the mammalian gp91-phox protein, the largesubunit of the flavocytochrome b558, the mitochondrial voltage-dependent anion channel, VDAC, and the chloroplastic outerenvelope protein from pea, OEP24.Results: Gp91phox is an integrated transmembrane proteincontaining the catalytic activity of the reducing NADPH oxidasecomplex. Full length or truncated gp91-phox proteins wereexpressed in soluble forms in E. coli lysate in presence of non ionicdetergents and in an oxidizing environment. Activities of theresulting proteins were tested in an electron transfer assay. Some ofthese gp91-truncated forms contained an enzymatic activity alike tothe cellular counterpart. For these truncated proteins, scale-upexperiments have been performed in order to use the purifiedproteins in crystallographic studies and structural analysis. VDACand OEP24 membrane proteins were overexpressed at 230 �g/mland 360 �g/ml respectively in our optimized cell-free proteinsynthesis system. Proteins are integrated in lipid vesicles in presenceof detergents resulting in proteoliposomes. In presence ofliposomes, OEP24 protein was recovered as dimers suggestingthat OEP24 protein is functional. The OEP24 in proteoliposomesforms a cation-selective channel as measured by changes insuspension turbidity in presence of KCl or sucrose. Activity ofrecombinant VDAC proteoliposomes was directly tested onmammalian cells by adding the VDAC proteoliposomes on cellsand by measuring the cytochrome c release and activation ofapoptosis. Immunofluorescence studies using anti-VDAC and anti-His antibodies indicate that recombinant VDAC is targeted to themitochondria.Conclusion: In conclusion, our recombinant technology toproduce membrane proteins in their native conformationsrepresents a new strategy for providing insights into functionaland structural informations on membrane proteins.AcknowledgementsThis study is supported by a grant from the EuropeanCommission (Marie Curie Excellence Grant).

S21Production, purification and structural analysis ofa cation efflux membrane protein from ThermusthermophilusOlga Kolaj1, Hui Li1, Elizabeth Burrowes1,Jeremy Moore1,2,3, Martin Caffrey1 and J Gerard Wall1,21Department of Chemical and Environmental Sciences,University of Limerick, Limerick, Ireland



2Materials and Surface Science Institute, University ofLimerick, Limerick, Ireland3Chemistry Department, The Ohio State University, Columbus,Ohio, 43210, USA


Background: A metal efflux protein CzrB, for cadmium and zincresistance protein B, was isolated during phage display-basedscreening of a Thermus thermophilus genomic library in Escherichiacoli. E. coli cells containing the czrB gene expressed from its nativepromoter exhibit increased efflux of, and resistance to, zinc andcadmium ions. Of biotechnological interest, however, czrB+ cells alsodisplay delayed cell lysis upon recombinant protein production [1].We have undertaken the cloning, production and purification ofCzrB in order to determine its structure. In addition, the 92-aacytoplasmic tail of this 291-aa protein has been cloned andproduced in E. coli for structural analysis.Results: czrB has been cloned and expressed, with N-terminal,C-terminal and no hexahistidine tags, under the control of anumber of promoters and in a variety of E. coli host strains tooptimise production. Cellular fractionation and immunoblottingrevealed from <5% to ~50% of the recombinant polypeptide to beassociated with the cytoplasmic membrane, depending onproduction parameters. Purification of the protein has beencarried out from cellular membrane fractions and cytoplasmicinclusion bodies to generate sufficient yields for crystallisationstudies. In addition, co-production of rare tRNAs and engineeringof czrB to improve its expression using the ribonuclease MazFapproach [2] are also under investigation to increase yields.Production and purification of the soluble, cytoplasmic tail ofCzrB has been optimised in parallel with analysis of the full-length protein (Figure 1). Preliminary crystallisation conditionswere obtained using a sparse matrix screen (Hampton Research,Crystal Screen I and II) with a protein concentration of 20 mg/ml. Polyethylene glycol and ammonium sulfate concentrationswere optimised to produce crystals which diffracted to 2.8 A.Conclusion: We have produced and purified the cation effluxprotein CzrB from T. thermophilus in an E. coli expression system.Expression has been optimised and crystallisation and structuralanalysis of the protein and its cytoplasmic tail are underway.

References1. Spada S, Pembroke JT and Wall JG: Isolation of a novel

Thermus thermophilus metal efflux protein thatimproves E. coli growth under stress conditions.Extremophiles 2002, 6:301–8.

2. Suzuki M, Zhang J, Liu M, Woychik NA and Inouye M:Single protein production in living cells facilitated byan mRNA interferase. Mol Cell 2005, 18:253–261.

S22An optimized method to produce halophilicproteins in Escherichia coliJulia Esclapez, Ma Jose Bonete, Monica Camacho,Carmen Pire, Juan Ferrer, Vanesa Bautista, Rosa MaMartınez-Espinosa, Basilio Zafrilla,Francisco Perez-Pomares and Susana DıazDepartment of Biochemistry and Molecular Biology, Universityof Alicante, 03080 Alicante, Spain


Background: The homologous and heterologous expressionof genes is a prerequisite for most biochemical studies of proteinfunction. Many systems have been carried out for proteinproduction in members of the Bacteria and Eukarya, howevermembers of the Archaea are less amenable to genetic manipula-tion. Only a few systems for high-level gene expression have beendeveloped for halophilic microorganisms. Because of this,mesophilic hosts, in particular Escherichia coli, have been usedto produce halophilic proteins for biochemical characterizationand crystallographic studies. Expression in E. coli has theadvantage to be faster and it will easily allow production on acommercial scale. In contrast, difficulties are encountered sinceenzymes from extreme halophiles require the presence of highsalt concentration for activity and stability, and the over-expressed product will need either reactivation or refolding ina salt solution, and so the purification techniques should becompatible with the high salt concentration required.For the last years, we have developed and refined a system toproduce and purify large amounts of recombinants proteinsfrom Haloferax mediterranei and Haloferax volcanii in themesophilic host E. coli.Results: Halophilic proteins have been overexpressed usingthe pET3a vector in E. coli BL21(DE3), for instance glucosedehydrogenase, glutamate dehydrogenase, nitrite reductase,extracellular a-amylase and isocitrate lyase from H. mediterraneiand isocitrate dehydrogenase from H. volcanii. The recombinantproteins were always obtained as inclusion bodies (see Figure 1),which were solubilised in the presence of urea. In most cases,the proteins were refolded by rapid dilution in a high saltconcentration buffers. The purification procedure of therecombinant proteins was based on the halophilic propertiesof this kind of enzymes. On the whole, the method consists of aprecipitation using ammonium sulphate and a chromatographyon DEAE-cellulose in the presence of that salt. The elution withsodium/potassium chloride yielded proteins in a pure and highlyconcentrated form (see Figure 2 and Table 1). Halophilicrecombinant proteins have been characterized and shown thesame biochemical characteristics as the enzymes isolated fromH. mediterranei and H. volcanii [1, 2, 3]. The high proteinconcentrations obtained has allowed us to carry on crystal-lization assays. In particular, the glucose dehydrogenase from

Figure 1 (abstract S21)

Production of CzrB-6his in E. coli BL21 (DE3) cells. Lane 1.Molecular weight marker; lane 2. E. coli control; lanes 3,4. whole cellextracts after CzrB production at 25˚C and 37˚C; lanes 5,6. membranefractions (concentrated 3.5 relative to whole extracts) after CzrBproduction at 25˚C and 37˚C.



H. mediterranei has been crystallized by the hanging-dropmethod using sodium citrate as the precipitant [4, 5].Conclusion: The overexpression, refolding and purificationmethod of halophilic proteins developed provides a fast, simple andefficient process that yields enzymes of high purity in large amounts.References1. Pire C, Esclapez J, Ferrer J and Bonete MJ: Heterologous

overexpression of glucose dehydrogenase from thehalophilic archaeon Haloferax mediterranei, anenzyme of the medium chain dehydrogenase family.FEMS Microbiol Lett 2001, 200:221–227.

2. Camacho M, Rodrıguez-Arnedo A and Bonete MJ: NADP-dependent isocitrate dehydrogenase from the halo-philic archaeon Haloferax volcanii : cloning, sequencedetermination and overexpression in Escherichiacoli. FEMS Microbiol Lett 2002, 209:155–160.

3. Dıaz S, Perez-Pomares F, Pire C, Ferrer J and Bonete MJ:Gene cloning, heterologous overexpression and

optimized refolding of the NAD-glutamate dehy-drogenase from Haloferax mediterranei. Extremophiles2006, 10:105–115.

4. Ferrer J, Fisher M, Burke J, Sedelnikova SE, Baker PJ,Gilmour DJ, Bonete MJ, Pire C, Esclapez J and Rice DW:Crystallization and preliminary X-ray analysis ofglucose dehydrogenase from Haloferax mediterranei.Acta Crystallogr D Biol Crystallogr 2001, 57:1887–1889.

5. Esclapez J, Britton KL, Baker PJ, Fisher M, Pire C, Ferrer J,Bonete MJ and Rice DW: Crystallization and preliminaryX-ray analysis of binary and ternary complexes ofHaloferax mediterranei glucose dehydrogenase. ActaCrystallograph Sect F Struct Biol Cryst Commun 2005, 61:743–746.


Expression of recombinant isocitrate dehydrogenase from H. volcaniiunder different temperatures. Lane 1. Molecular weight standards. Lane2. Wild-type isocitrate dehydrogenase. Lanes 3. Uninduced insolublefraction. Lanes 4 and 5. Induced insoluble fraction at 37˚C. Lanes 6 and 7.Induced insoluble fraction at 25˚C.


Purification of recombinant glucose dehydrogenase from H. mediterranei.Lane 1: Molecular weight standards. Lane 2: Wild type glucose dehydrogen-ase. Lane 3: Inclusion body fraction. Lane 4: (NH4)2SO4 precipitationsupernatant. Lane 5: (NH4)2SO4 precipitation pellet. Lane 6: Active fractionsfrom DEAE-cellulose.

Table 1 (abstract S22) Purification of recombinant glucose dehydrogenase from H. mediterranei

Enzyme fraction Vol. (ml) UT mgT Specific activity(U/mg)

Enrichment(-fold)

Yield (%)

Inclusion bodies refolding in 20 mMTris-HCl pH 7.4, 2 M NaCl, 1 mM EDTA

500 2210 20.9 106 1.0 100

(NH4)2SO4

Precipitation

Supernatant 500 2180 18.7 117 1.1 99

Pellet 20 27 1.7 16 – –DEAE-cellulose 18 1666 8.9 187 1.8 75



S23Monitoring of stress responsesThomas Schweder1, Britta Jurgen1, Birgit Voigt2,Daniel Pioch1, Michael Hecker2, Stefan Evers3

and Karl-Heinz Maurer31Pharmaceutical Biotechnology, Institute of Pharmacy,Ernst-Moritz-Arndt-University, D-17487 Greifswald,Germany2Institute of Microbiology, Ernst-Moritz-Arndt-University,D-17487 Greifswald, Germany3VTB Enzyme Technology, Henkel KGaA, D-40191Dusseldorf, Germany


Background: The consideration of bacterial stress and starvationresponses is of great importance for the successful establishment ofan industrial large scale fermentation process. Suitable analysistechniques for stress and starvation specific genes are thereforeparticularly interesting for the monitoring and control of suchprocesses. The combined methods of transcriptome analysis, highresolution two-dimensional polyacrylamide gel electrophoresis(2D-PAGE) and mass spectrometry have been extensively appliedfor the description of general and specific stress and starvationresponses of industrial microorganisms.Results: By means of proteomics and transcriptome analyses weidentified marker genes of the gram-positive bacteria Bacillus subtilisand Bacillus licheniformis. The expression of such marker genes isspecifically regulated by distinct stress and starvation conditions. Forboth bacteria, which represent important industrial hosts with along history in industrial enzyme production, we have filtered a setof marker genes, which could be used as indicators for process-relevant stress situations during protein production fermentationprocesses. For example, in Figure 1 starvation specific markerproteins for nitrogen, phosphate and glucose limitation of B.licheniformis are summarised.Such process-critical genes/proteins can be used as biomarkersin order to control the fitness and productivity of theseindustrial bacterial hosts during fermentation processes. DNA-and protein-chips specific for such process-relevant markergenes would be valuable diagnostic tools for the monitoring ofcellular physiology. In this respect fast mRNA and proteinanalytical techniques for an at-line monitoring of gene expressionduring bioprocesses are required. The electric chip techniquefulfills these requirements [2]. This technique allows a fast andreproducible expression analysis of process-relevant markergenes (see Figure 2).Conclusion: It is demonstrated that electric chips loaded withmRNA specific DNA-probes or with marker protein specificantibodies represent a suitable alternative for gene expressionanalyses in competition with the real time RT-PCR duringfermentation processes. The electric chip technique is easy toautomate and could be cheaper in the handling than theestablished gene expression analysis techniques. The electricbiochip combined with an automated sample preparationestablishes a basis for continuous at-line monitoring of hostcell physiology during industrial bioprocesses.AcknowledgementsThis study was financially supported by the Ministry ofEducation, Science and Culture of Mecklenburg-Vorpommern(grant number: 0202120) and the Federal Ministry of Education,and Science (BMBF) (grant number: 031U213A).

References1. Voigt B, Schweder T, Sibbald MJ, Albrecht D, Ehrenreich A,

Bernhardt J, Feesche J, Maurer KH, Gottschalk G, VanDijl JM and Hecker M: The extracellular proteome ofBacillus licheniformis grown in different media andunder different nutrient starvation conditions.Proteomics 2006, 6:268–281.

2. Jurgen B, Barken KB, Tobisch S, Pioch D, Wumpelmann M,Hecker M and Schweder T: Application of an ElectricalDNA-Chip for the Expression Analysis of Bioprocess-Relevant Genes of Bacillus subtilis. Biotechnol Bioeng 2005,92:299–307.

S24Design of transcriptional fusions of stresssensitive promoters and GFP to monitor theoverburden of E.coli hosts during recombinantprotein productionSabine Nemecek, Karoline Marisch, Renata Juricand Karl BayerDepartment of Biotechnology, University of Natural Resourcesand Applied Life Sciences, Vienna, Austria


Background: Nowadays a high number of recombinantproteins for therapeutical purposes in human and animal healthcare are produced by microbial systems, mainly in E.coli [1].Basically E.coli is well studied, sequenced [2] and characterizedbut changes in the cell composition during heterologous proteinexpression are poorly understood. Due to the lack of appro-priate sensors for monitoring alterations of E.coli cells and thehuge complexity of cellular systems, many of the present proteinproduction processes are still far from optimal. Aiming at optimalexploitation of the host cell enhanced knowledge of cellularreactions related to recombinant protein expression is required.Using current methods like DNA microarrays and 2-D-electro-phoresis changes of transcriptional and translational activity instress situations like heat shock, general stress response, nutrientlimitation, and stress caused by overexpression of heterologuesproteins can be monitored. However, acquisition of these data istime consuming; therefore the goal is to create new on-linesystems to monitor metabolic shifts. The major advantage of on-line process monitoring derives from immediate intervention in arunning cultivation process. 2D-multi-wavelength fluorescencespectroscopy (DELTA, Bioview) represents a powerful, non-invasive measurement principle for on-line monitoring providingdirect acquisition of biologically active fluorophores e.g. NAD(P)H [3] and detection of the reporter protein GFP. Using GFP asreporter is superior in comparison to other reporters like luxAB-genes or CAT, because it does not need substrates, cofactors oradditional stabilization to yield a fluorescence signal [4, 5].Therefore, GFPmut3.1 [6] was used as appropriate reporter byfusing stress relevant promoters and acquisition of resultingfluorescence [7].Relevant promoters upregulated during the protein productionprocess were derived from microarray data (Durrschmid,Reischer unpublished data). For the construction of thepromoter-reporter fusions the chaperone dnaK and the generalregulator for stress response sigma32 (rpoH), which is feed backregulated by other stress genes, were used [8]. To gain anefficient monitoring system for metabolic load two different



approaches were established. On the one hand the fusion ofpromoter and GFPmut3.1 were inserted into a low copy plasmid(pMMB67HE), kindly provided by Karaimann, [9] which iscompatible to the expression plasmid pET30a containingrhSOD (superoxiddismutase). The resulting 2-plasmid E.coliHMS174(DE3) strains were tested in shake flask experimentsand fed batch cultivations. On the other hand promoters-GFPcartridges were integrated in the E.coli genome, as described

by Datsenko and Wanner [10]. The expression plasmid wastransformed into the resulting monitoring host and themonitoring system was evaluated under protein productionconditions.Results: Fed batch cultivations (20 l) of 2-plasmid hosts wereperformed (� = 0,15 h-1). Tuning of recombinant geneexpression was achieved by controlled feed of inducer, wherebythe first generation in the feed phase was non-induced, followed


Colour coding of extracellular marker proteins of B. licheniformis for phosphate, glucose and nitrogen starvation conditions. Colour coding was donewith the Delta 2D software http://www.decodon.com. (P: phosphate starvation, C: glucose starvation, N: nitrogen starvation). [1]



by 3 generations in induced state with an increasing amount ofIPTG (from 0,75 to 2,5 �mol/gBDM).In the induced cultivation of E. coliHMS174(DE3) containingpMMB67HE:dnaKp:GFPmut3.1 and pET30arhSOD 95 mg SOD/gBDM were produced and compared with the non inducedcultivation (see Figure 1). The off-line measured fluorescence wasincreased in comparison to the non-induced cultivation, which isin accordance with the amount of GFP determined by ELISA. In

addition, the on-line fluorescence was increased during recom-binant protein production and was compared with cultivationwithout monitoring plasmid to confirm the obtained fluorescenceresults from GFP (Figure 2). The system with the dnaK-promotermonitoring plasmid was able to show fluorescence due to thestress caused by recombinant protein production.Our second approach for stress monitoring (genome integratedmonitoring cartridges) shows lower fluorescence signals and


(A) Schematic presentation of the electric chip principle and (B) analysis of a B. subtilis glucose-starvation marker gene with an electric DNA-chipduring a glucose-limited fermentation process. EBC = electric DNA-chip, RT-PCR = real time RT-PCR



less amount of GFP (data not shown). All induced cultivationsshow an increase in the amount of the stress alarmon ppGpp,indicating overburden of the cells.Conclusion: The concept of fusing stress relevant promoterswith GFP for monitoring the overburden of the cell was proven.The adoption of host cells with an additional monitoring plasmidfor the evaluation of the stress caused by recombinant proteinproduction was very successful. Regrettably the genome-integratedmonitoring cartridges did not generate a significant fluorescencesignal. To cope with the low fluorescence transcriptional amplifica-tion of the GFP signal is planned.AcknowledgementsThe work is supported by the Austrian Center of Biopharma-ceutical Technology (ACBT, http://www.acbt.at).

References1. Demain AL: Microbial biotechnology. Trends Biotechnol

2000, 18:26–31.2. Blattner FR, et al: The complete genome sequence of

Escherichia coli K-12. Science 1997, 277(5331):1453–74.3. Marose S, Lindemann C and Scheper T: Two-dimensional

fluorescence spectroscopy: a new tool for on-linebioprocessmonitoring. Biotechnol Prog 1998, 14(1):63–74.

4. Chalfie M, Tu Y, Euskirchen G, Ward WW andPrasher DC: Green fluorescent protein as markerfor gene expression. Science 1994, 263(5148):802–805.

5. Cha HJ, Srivastava R, Vakhira VN, Rao G and Bentley WE:Green fluorescent protein as a noninvasive probe inresting Escherichia coli cells. Appl Environ Microbiol1999, 65(2):409–414.

6. Cormack BP, Valdivia RH and Falkow S: FACS-optimizedmutants of green fluorescent protein (GFP).Gene 1996,173(1):33–38.

7. Reischer H, Schotola I, Striedner G, Potschacher F andBayer K: Evaluation of the GFP signal and itsaptitude for novel on-line monitoring strategies ofrecombinant fermentation processes. J Biotechnol2004, 108:115–125.

8. Wick LM and Egli T: Molecular Components ofPhysiological Stress Responses in Escherichia coli.Adv Biochem Eng Biotechnol 2004, 89:1–45.

9. Furste JP, Pansegrau W, Frank R, Blocker H, Scholz P,Bagdasarian M and Lanka E: Molecular cloning of theplasmid RP4 primase region in a multi-host-rangetacP expression vector. Gene 1986, 48:119–131.

10. Datsenko KA and Wanner BL: One-step inactivation ofchromosomal genes in Escherichia coli K-12 usingPCR products. PNAS 2000, 97:6640–6645.

S25Dynamic optimisation of a recombinant BHK-21culture based on elementary flux analysis andhybrid parametric/nonparametric modelingAna Teixeira1, Carlos Alves1, Paula Alves2,Manuel Carrondo1,2 and Rui Oliveira11REQUIMTE, Departamento de Quımica, Faculdade deCiencias e Tecnologia, Universidade Nova de Lisboa,P-2829-516 Caparica, Portugal2IBET/ITQB Instituto de Biologia Experimental e Tecnologia/Instituto de Tecnologia Quımica e Biologica, Apartado 12,P-2781-901 Oeiras, Portugal


Background: Metabolic flux analysis (MFA) and metabolicpathway analysis (MPA) are today fundamental tools to studycellular metabolism. Such tools can assist the generation ofpotential modifications that can alter the cell metabolic activitytoward bioprocess optimisation.Although MFA and MPA techniques have been mainly used formetabolic engineering [1], they may also be useful in otherphases of the bioprocess development cycle, namely foradvanced bioreactor monitoring and control [2, 3]. A numberof methods have been developed to study the structure ofbiochemical networks. From a process optimisation and controlpoint of view, the elementary flux modes (EFMs) method is


Comparison of E.coliHMS174(DE3) cultivations containing pMMB67HE:dnaKp:GFP and pET30arhSOD; amounts of SOD and GFP weredetermined by ELISA, spec. Fc = specific cellular off-line fluorescenceby SPECTRAmax GeminiXS at ex488nm/em530 cut off filter 515 nm.


On-line fluorescence data of a E.coliHMS174(DE3) cultivation containingpMMB67HE:dnaKp:GFP and pET30arhSOD(SRK8) compared with acultivation without monitoring plasmid (SRK10).



particularly attractive since it reduces network complexity to aminimal set of reactions. EFMs are unique for a given networkand can be considered as nondecomposable steady state fluxdistributions using a minimal set of reactions.In previous studies [4], an iterative batch-to-batch optimizationscheme was developed and applied to the optimization ofrecombinant BHK-21 expressing the fusion glycoprotein IgG1-IL2 used in cancer therapy [5]. The main objective of the presentstudy is complementing the previous batch-to-batch schemewith knowledge of the metabolic network of the biologicalsystem under consideration. The incorporation of reliablemechanistic knowledge in the batch-to-batch optimisationscheme, namely of the metabolic network in the form ofEFMs, may increase the ’extrapolation’ capacity and thus maycontribute to increase the rate of success of the proposedtechnique.Results: The metabolic network adopted (Fig. 1) is firstdecomposed into EFMs using the FluxAnalyser program [6].The system has seven EFMs. The hypothesis of balanced growthallows the elimination of the intermediate metabolites resultingin a simplified set of reactions (Table 1) connecting extracellularsubstrates with end-products.

The resulting set of reactions is the basis for the formulation ofthe following hybrid model structure:

d

dt

X

Glc

G

Lac

Amm

Ala

IgG

v

ln

⎡

⎣

⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢

⎤

⎦

⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥

=

−1 0 0 0 0 0 0

0 1 −− −− − −

⎡

⎣

⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢

1 0 0 2 0

0 0 0 1 1 5 0

0 2 0 0 0 0 0

0 0 0 1 2 2 0

0 0 0 1 0 0 0

0 0 0 0 0 0 1

⎤⎤

⎦

⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥

× ( )× ( )× ( )× ( )×

ρρρρρ

1

2

3

4

5

X

X Glc

X Glc

X G

X G

V

V

V

V

V

ln

ln(( )× ( )

× ( )

⎡

⎣

⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢

⎤

⎦

⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥

−

ρρ

6

7

X GlcG

X

D

X

Glc

G

L

V

V

v

ln

ln

aac

Amm

Ala

IgG

F

FGlc

G

⎡

⎣

⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢

⎤

⎦

⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥

+

⎡

⎣

⎢⎢⎢⎢⎢⎢

ο

ln

0

0

0

0

⎢⎢⎢⎢⎢

⎤

⎦

⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥

( )1

An artificial neural network was used to identify the reactionkinetics from data: the apparent specific growth rate (�-kd), thespecific protein synthesis rate (qIgG), and the EFM kinetics (�ifunctions in eq. 1). Measured data of one batch and four fed-batch runs was used. Figure 2 presents the identifiedintracellular flux distribution for one of the fed-batch runs.Analyzing such patterns we can take some conclusions. The mostenergetic EFM involving glucose and glutamine are re2 and re5,respectively. Looking at these two EFMs in Figure 2 we can verifythat glutamine seems to be the major source of energy during thegrowth phase since re5 is almost constant, while the metabolism ofglucose gradually changes from a state where it is mostly convertedto lactate (re1, a poor energetic pathway), to a state of completeoxidation of glucose via TCA cycle (re2). Zielke et al. (1984) havealready reported that glutamine becomes the predominant sourceof energy at low glucose concentration. On the other hand, in thedeath phase (�-kd<0) there is a shut down in the most energeticEFMs (re2 and re5) and the overflow metabolism takes place i.e., theproduction of lactate (re1) and alanine (re3) starts to increase. Thesemetabolic particularities of animal cells were well captured by thehybrid model which confirms its potentialities.Using the developed hybrid model, the process performance(described as the glycoprotein titre at the end of thebioreaction, eq. 2) is optimized with respect to glucose andglutamine feeding using a micro-genetic algorithm [9].

maxu

IgG IL f fJ C t V t= ( ) ( ) ( )−1 2 2

The final optimization results are presented in Fig. 3. Theoptimized strategy suggests to control glucose and glutamine atlow levels while cells are growing (Fig. 3a). During this periodcells use both nutrients in an increasingly efficient way: completeoxidation of both glucose (re2) and glutamine (re5) increaseswhile glucose converted into lactate (re1) and glutamineconverted into alanine (re3) decreases. As shown in Figure 3b,the ratios between the respective EFM and total glucose andglutamine consumption rates corroborates this metabolicefficiency improvement. When cells start dying (probablybecause ammonia reached toxic levels) the best strategyseems to be to increase the glutamine concentration. By doingso, a redistribution in the intracellular fluxes occurs that favoursproduct formation. The process productivity may be consider-ably increased applying the proposed nutrients feeding strategy.The final product titre predicted by the model is 25 mg/l againstthe 15 mg/l that had been obtained in the fed-batch experiments.Conclusion: In this work we present a novel bioreactoroptimisation method that incorporates detailed metabolic knowl-edge of the biological system under consideration. The method wasapplied to a recombinant BHK-21 cell line expressing a fusionglycoprotein. The method allows to identify metabolic fluxes overthe runtime of a bioprocess. Such knowledge allows to betterunderstand metabolic structural changes by the analysis of the

Table 1 (abstract S25) Elementary flux modes of the metabolicnetwork considered.

re1: Glucose 2 Lactatere2: Glucose 6 CO2

re3: Glutamine 2 CO2 + Ammonia+ Alaninere4: Glutamine Lactate + 2 CO2 + 2 Ammoniare5: Glutamine 5 CO2 + 2 Ammoniare6: Glucose + 3 Glutamine Purine + 2 CO2 + Ammoniare7: Glucose + 2 Glutamine Pyrimidine + 2 CO2 + Ammonia


Animal cells metabolic network [2, 7] From the reactions of table 1, itwas further assumed that re6=re7 since DNA and RNA are made up ofequal parts of purine and pyrimidine. Therefore, these two reactionswere substituted by their sum:2Glc+5 GlnPur+Pyr+4CO2+2Amm-Furthermore, it was considered that the 4th elementary mode hasnegligible flux, since lactate is mainly produced from glucose.



relative importance of elementary flux modes. The final hybridmodel was used to optimise the flux distribution towardsmaximising the final product titre. It was concluded that the processproductivity can be substantially improved by increasing theglutamine concentration during the cells death phase

AcknowledgementsThe authors acknowledge the financial support provided bythe Fundacao para a Ciencia e Tecnologia throughproject POCTI/BIO/57927/2004 and PhD grant SFRH/BD/13712/2003.


Optimization results.


Apparent specific growth rate (-kd), specific protein synthesis rate (rIgG) and elementary flux modes kinetics identified by the hybrid model.



References1. Follstad BD, Balcarcel RR, Stephanopoulos G and Wang DI:

Metabolic flux analysis of hybridoma continuousculture steady state multiplicity. Biotechnol Bioeng1999, 63:675–683.

2. Provost A and Bastin G: Dynamic metabolic modelingunder balanced growth condition. J Process Control2004, 14:717–728.

3. Mahadevan R, Burgard A, Famili I, Van Dien S and Schilling C:Applications of metabolic modeling to drive biopro-cess development for the production of value-addedchemicals. Biotechnol Bioprocess 2005, 10:408–417.

4. Teixeira A, Cunha A, Clemente J, Moreira J, Cruz H,Alves P, Carrondo M and Oliveira R: Modelling andoptimisation of a recombinant BHK-21 cultivationprocess using hybrid grey-box systems. J Biotechnol2005, 118:290–303.

5. Cruz HJ, et al: Process development of a recombinantantibody/interleukin-2 fusion protein expressed inprotein-free medium by BHK cells. J Biotechnol 2002,96:169–183.

6. Godia F and Cairo J: Metabolic engineering of animalcells. Bioprocesses Biosyst Eng 2002, 24:289–298.

7. Klamt S, Stelling J, Ginkel M and Gilles E: FluxAnalyser:exploring structure, pathways, and flux distributionsin metabolic networks on interactive flux maps.Bioinformatics 2003, 19:261–269.

8. Zielke HR, Zielke C and Ozand PT: Glutamine: a majorenergy source for cultured mammalian cells. Fed Proc1984, 43:121–125.

9. Krishnakumar K: Micro-Genetic Algorithms for Sta-tionary and Non-Stationary Function Optimization.SPIE: Intelligent Control and Adaptive Systems Philadelphia, PA;1989, 1196.

S26Monitoring of transcript regulation and proteinproduction related stress responses in Pichiapastoris secreting Fab antibody fragmentsBrigitte Gasser1, Michael Maurer1, Michael Sauer1,2,Markku Saloheimo3, Jari Rautio3, Merja Penttila3

and Diethard Mattanovich1,21University of Natural Resources and Applied Life SciencesVienna, Department of Biotechnology, Institute of AppliedMicrobiology, Vienna, Austria2School of Bioengineering, University of Applied SciencesFH-Campus Vienna, Austria3VTT Biotechnology, P.O. Box 1500, 02044 VTT, Espoo,Finland


Background: Protein production processes having the methylo-trophic yeast Pichia pastoris as heterologous production hostbecame increasingly important in the last decade. Although Pichiapastoris is known as a highly efficient expression system, there isonly little knowledge about the physiology and the genetics lyingunderneath. During the recent years, it has become evident that avariety of intrinsic, metabolic and environmental stresses may havea strong impact on recombinant protein production.Especially the production of complex proteins has turned out tohave a very low success rate. Several physiological studies have

demonstrated that many physiological processes, includingstress responses to environmental factors, and protein folding/aggregation and secretion are highly interrelated. Among theenvironmental factors influencing protein expression andsecretion, pH, osmolarity, oxygen availability and temperatureappear to be particularly important.For the production of heterologous proteins it seems implau-sible to cultivate the host cells at higher temperatures than thegrowth optimum, as the products naturally are heat sensitive.Lower temperatures, however, are often applied, usually basedon empiric data based on improved product formation orstability.Therefore a deeper understanding of the physiological andmolecular links between protein folding and temperature(-adaption/-stress) appears useful. While a lot of data havebeen collected regarding the regulatory events as a reaction totemperature changes, there is not much information on the truephysiological reaction of cells particularly in context ofheterologous protein expression.Results: The rapid transcriptional profiling method VTT-TRAC has been applied to monitor the levels of a subset ofmRNAs coding for UPR-regulated and stress-connected genes inchemostat cultivations of a P. pastoris strain secreting the 2F5antibody fragment. Specific marker genes have been chosen todeliver insights into the general physiological status of the cellsunder production conditions (including growth, protein synth-esis, oxygen and nutrient limitation responses) with the mainfocus on secretion stress connected genes (UPR, ERAD,posttranslational processing).As product formation is known to be strongly dependent oncultivation conditions, the influence of different cultivationtemperatures has been analysed.Transcript formation rates of the two respective product genes(for Fab light chain and heavy chain mRNA) have been set incorrelation to the mRNA levels of folding related genes such asKAR2 and PDI1, and additionally to the specific productformation rate of secreted Fab.Interestingly, although the transcriptional levels of the productgenes were reduced at lower temperature, specific productivity ofthe 2F5 Fab protein was significantly increased. Thus it is temptingto speculate that at lower temperature a reduced amount offolding stress is imposed on the cells, consequently leading to ahigher rate of correctly folded product. Also the chaperone KAR2/BiP, which is commonly seen as a marker of unfolded protein stressappeared among the genes downregulated at lower temperature.Additionally, the levels of intracellularly retained antibody frag-ments and the UPR marker protein BiP (Kar2) were analyzed byimmunofluorescence and flow cytometry.Conclusions: The robust, sensitive and cost-efficient tran-scriptional profiling method VTT-TRAC could be set up forP. pastoris. Apart from being useful for bioprocess monitoringand control of fermentation conditions, it was possible to obtainnew and valuable information regarding the physiologicalregulation of protein production in P. pastoris under alteringculture conditions and to reveal key regulatory pathwaysdetermining the efficiency of protein production.The induction of UPR-target genes due to heterologous proteinproduction could be shown for the first time in P. pastoris. Fromthe technological point of view, connections between growthtemperature as an example for environmental conditions andspecific productivity could be revealed at a transcriptional level,



once again confirming that a release of folding stress can lead tohigher product secretion rates.

S27High level Aspergillus production of proteinsDominique Aubert, Jan Lehmbeck, Mogens Trier Hansenand Carsten HjortExpression Technology, Novozymes A/S, Brydelysvej 26, 2880Bagsvœrd, Denmark


Background: The Aspergillus oryzae expression system hasbeen developed by Novozymes and used for recombinantprotein production since 1988. Later on Aspergillus niger andFusarium venenatum expression systems have been alsodeveloped.The rationale for entering development of the Aspergillus oryzaeexpression system was that we needed an efficient expressionsystem that could secrete large amounts of protein at highproduct purity for production of industrial enzymes.The choice of Aspergillus oryzae as the preferred expressionsystem was made from several different criteria. First of allAspergillus oryzae is a well characterized organism that has beenused in the food and fermentation industry in Japan for severalhundred years. This means that the products of the fungus havebeen recognized as "Generally Regarded As Safe" (GRAS). Thesystem was also used by producers of industrial enzymes usingsubmerged fermentation for production for decades as Asper-gillus oryzae has been used for production of amylases andproteases since the dawn of modern enzyme manufacturing.Another important advantage of Aspergillus oryzae was that itcould be genetically manipulated. Finally there were no patentsblocking the use of Aspergillus oryzae at the time.Results: The early recombinant strains were essentially wildtype strains transformed with plasmids based on a promoter anda selection marker that was cloned directly from differentAspergillus strains. Several shortcomings of these strains werequickly realized and work to improve the system was undertaken.The host strain produced a lot of unwanted proteins such asamylases, amyloglycosidases and proteases.In addition to improving the host strain, the expression vectorshave also been improved. In particular the promoter has beenthe subject of optimization.The promoter used in the first recombinant Aspergillus oryzaeproduction strains was the TAKA promoter, the promoterdriving the expression of the abundant TAKA amylase. We havecharacterized this promoter and found a specific transcriptionfactor called amyR and binding sites for that transcription factorin the TAKA promoter [1]. The detailed understanding of thepromoter has enabled us to design new synthetic promotersthat are substantially improved compared to the wild typeTAKA promoter.The improvements of the Aspergillus oryzae expression systemhave resulted in a versatile, clean and high yielding expressionsystem for enzyme production [2]. With these improvementsthe system is suitable for other uses than just expression ofenzymes, including production of bioparmaceuticals. So recently wehave initiated a number of projects aiming at developing biophar-maceutical products such as monoclonal antibodies and antimicro-bial peptides [3] using Aspergillus oryzae as expression host.

References1. Petersen KL, Lehmbeck J and Christensen T: A new

transcriptional activator for amylase genes inAspergillus. Mol Gen Genet 1999, 262:668–676.

2. Christensen BE, Mollgaard H, Kaasgaard S and Lehmbeck J:Methods for producing polypeptides in aspergillusmutant cells. Official Gazette of the United States Patent andTrademark Office Patents 2004, 1289.

3. PH Mygind, et al: Plectasin is a peptide antibiotic withtherapeutic potential from a saprophytic fungus.Nature 2005, 437:975–980.

S28Pseudomonas fluorescens – a robust expressionplatform for pharmaceutical protein productionDiane Retallack, J Carrie Schneider, Lawrence Chew,Tom Ramseier, Jeffrey Allen, Anant Patkar,Charles Squires, Henry Talbot and Jon MitchellBiotechnology R&D, Dowpharma, The Dow ChemicalCompany, 5501 Oberlin Dr. San Diego, CA 92121, USA


Background: A bottleneck to protein pharmaceutical produc-tion can be efficient expression of the target protein. APseudomonas fluorescens-based manufacturing platform for highyield production of non-glycosylated protein pharmaceuticalshas been developed. This platform is derived from P. fluorescensbiovar I strain MB101 [1]. The system’s performance is due tothe combination of a robust host strain, the availability ofextensive molecular biology and bioinformatics tools, and a welloptimized high cell density fermentation process. The SystemsBiology tools include a genomics and functional genomicscapability, a range of stable plasmid vectors of various copynumbers, non-antibiotic-dependent plasmid maintenance [2],multiple expression cassettes [3] and engineered host strains forstringent control of gene expression, and the ability to exportproteins to the cell’s periplasmic space.Results: The ability to export proteins to the periplasmicspace enables the formation of a precise N-terminus andformation of disulfide bonds. Moreover, export to the periplasmcan simplify downstream processing. Multiple native P. fluor-escens secretion leader sequences have been evaluated for theability to direct heterologous proteins to the periplasm. Severalsecretion leaders were shown to effectively enable secretion ofrecombinant proteins with precise N-terminal processing at theexpected amino acid. Yields of up to 18 g/L of secreted proteinhave been observed at the 20 L fermentation scale.Conclusion: P. fluorescens is a robust expression host for highyield expression of secreted proteins. The identification ofmultiple, effective secretion leaders that can direct secretion ofhigh levels of protein to the periplasm offers flexibility to identifythe best secretion leader for each recombinant protein.References1. Landry TD, Chew L, Davis JW, Frawley N, Foley HH,

Stelman SJ, Thomas J, Wolt J and Hanselman DS: Safetyevaluation of an alpha-amylase enzyme preparationderived from the archaeal order Thermococcales asexpressed in Pseudomonas fluorescens biovar I. RegulToxicol Pharmacol 2003, 37(1):149–168.



2. Schneider JC, Jenings AF, Mun DM, McGovern PM andChew LC: Auxotrophic markers pyrF and proC canreplace antibiotic markers on protein productionplasmids in high-cell-density Pseudomonas fluores-cens fermentation. Biotechnol Prog 2005, 21

(2):343–348.3. Retallack DM, Thomas TC, Shao Y, Haney KL, Resnick SM,

Lee VD and Squires CH: Identification of anthranilateand benzoate metabolic operons of Pseudomonasfluorescens and functional characterization of theirpromoter regions. Microb Cell Fact 2006, 5(1):1.

S29Enhanced protein expression through strainselection, gene disruption, improved vector designand co-expression of endogenous chaperonesDarrell Sleep, Chris Finni and Leslie EvansNovozymes Delta Ltd, Castle Court, 59 Castle Boulevard,Nottingham, UK


Backgound: The use of Saccharomyces cerevisiae as ahost system has been limited by the perception of limitedsecretion capacity, unstable episomal vectors and aberrant

glycosylation. Solutions to all of these limitations are nowavailable.Results: An analysis of a series of haploid laboratory yeaststrains revealed significant intra-strain variability and unstableplasmid segregation. By combining classic chemical mutagenesisand selection a family of highly efficient Saccharomyces cerevisiaestrains has been developed for the commercial production ofbiopharmaceutical products. When combined with a stable [1],high copy number [2], episomal expression vector system anda strong constitutive promoter, secreted recombinantprotein expression titres in excess of 4 g/L were achieved(see Figure 1). Specific genetic modifications to the hostwere also introduced to increase product yield and controlpost-translational modifications, such as proteolysis andglycosylation.The expression vectors have been further enhanced to facilitatethe stable co-expression of multiple proteins. When one ofthese proteins is a chaperone, the titre of co-expressedrecombinant transferrin was increased 15-fold. The applicabilityof this system has been demonstrated with a wide range ofheterologous proteins and is scalable from 10 mL shake flask tocGMP manufacture at high cell density fermentation (8,000 L) ina defined synthetic medium; designed to be integrated with cost-efficient downstream processing.


Enhancement of protein production through chemical mutagenesis and specific gene disruptions.



Conclusion: Significant intra-strain variability and unstableepisomal plasmid systems have limited the usefulness ofSaccharomyces cerevisiae as an industrial host for the productionof biopharmaceuticals. However co-enhancement of the episo-mal vector system and the host strains is not only possible buthas led to significant improvements in recombinant proteinproduction.References1. Chinery SA and Hincliffe E: A novel class of vector for

yeast transformation. Curr Genet 1989, 16:21–25.2. Sleep D, Finnis C, Turner AJ and Evans LR: Yeast 2 mm

plasmid copy number is elevated by a mutation inthe nuclear gene UBC4 . Yeast 2001, 18:403–421.

S30Impact of high throughput technology onrecombinant protein productionJo J Jones1, Angela M Bridges1, Andrew P Fosberry1,Jonathan Bullock2, Ian Hudson2, Richard M Hall1,Owen Jenkins1 and Jeffrey A Cole31Gene Expression and Protein Biochemistry, GlaxoSmithKline,New Frontiers Science Park, Coldharbour Road, Harlow, Essex,CM19 5AD, UK2The Automation Partnership, York Way, Royston,Hertfordshire, SG8 5WY, UK3School of Biosciences, University of Birmingham, Edgbaston,Birmingham B15 2TT, UK


Background: There are many challenges still to be overcomein the efficient production of recombinant protein from botheukaryotic and prokaryotic organisms. Often the problemsfaced are protein specific, and can result in unacceptable timedelays between target screening and structural elucidation. Arequirement therefore exists for rapid, parallel approaches thatcan identify suitable clones under a preliminary set offermentation conditions in days rather than months.Piccoloâ is a fully automated, high throughput system,specifically developed for the rapid optimisation of proteinproduction from both insect and microbial cells. The system iscapable of expressing a maximum of 1152 cultures, at volumesbetween 10 ml and 100 mls, followed by protein purificationusing affinity chromatography. A total of four different culturemedia, two antibiotics and two inducers can be used, inconjunction with three post induction temperatures. The opticaldensity of cultures is measured robotically to monitor growth,and the growing cultures can be both aerated and agitatedcontinuously to prevent anaerobic growth. Following expres-sion, the cells are centrifuged, lysed and the lysate bound toaffinity resin. The resin is then washed, eluted and the partiallypurified protein stored at 6˚C ready for analysis.Results are presented that show the increased efficiency ofcombining the automated primary screening of Piccoloâ with on-lineGFP sensors to optimise the subsequent fermentation processes.The use of an on-line sensor to measure GFP-tagged proteinexpression in fermenters has been described previously by Jones etal. [1]. In the pharmaceutical industry, reducing the time taken forscreening recombinant protein is becoming more important and thisautomated approach will undoubtedly have an important role.Results: Early results from experiments completed usingPiccoloâ are encouraging. To illustrate the potential of the

technology, an experiment to investigate different host strainsof E. coli was undertaken. The expression conditions wereinduction at an optical density of 1.0 with 0.5 mM IPTG followedby incubation for 7 h at 30˚C and for 3 h at 37˚C. Six proteins allwith an N-terminal 6-histidine tag were evaluated in threedifferent host strains. The experiment used a total of 720individual cultures, with samples in quadruplicate. Cultures weregrown in special custom built culture vessel blocks (CVBs)containing 24 wells per block fitted with an aeration assemblythat allowed both addition of gases and agitation. On-linegrowth measurements using an optical sensor were alsopossible. The culture blocks were incubated before inductionat 37˚C, moved to a post-induction incubator at the desiredinduction statistic for 30 minutes. After 30 minutes the CVB wasremoved from the incubator, IPTG was added and the CVBreturned for incubation for 3 h at 37˚C or 7 h at 30˚C. TheCVBs were removed, decoupled from their aeration assembliesand moved to cold storage at 6˚C until ready for purification.The CVB containing culture was centrifuged, the supernatantremoved and the cells lysed with lysis buffer. The CVB was againcentrifuged and the lysate mixed with affinity resin to allow forbinding of the affinity tag of the recombinant protein to theresin. After binding, the resin was washed twice and then eluted.The purified protein was collected in a 24 well plate andreturned to cold storage at 6˚C awaiting assay. Figure 1 showssome of the results obtained for the samples analysed by SDS-PAGE after purification.Conclusion: The potential of a high throughput automatedsystem for industry to screen a large number of conditions isdemonstrated. From this experiment alone, the choice ofexpression host is seen to influence the amount of recombinantprotein produced. Piccoloâ offers the scientist the capability toscreen a large number of factors in one experiment. Theoptimum conditions can then be scaled up into a fermenter, andif a GFP fusion protein is used, the recombinant proteinexpression can be followed in the fermenter on-line in real-timeusing a fluorescence sensor.Reference1. Jones JJ, Bridges AM, Fosberry AP, Gardner S, Lowers RR,

Newby RR, James PJ, Hall RM and Jenkins O: Potential ofreal-time measurement of GFP-fusion proteins.J Biotechnol 2004, 109:201–211.

S31Industrial scale production of chymosinwith Aspergillus nigerKarsten HellmuthDepartment of Process Development, Ch. Hansen GmbH,Gr. Drakenburger Str. 93-97, D-31582 Nienburg, Germany


Background: Aspergillus niger has a long history as a producerof food-grade enzymes and is established as a GRAS (GenerallyRecognized As Safe) organism. As Chymosin has been createdby nature to coagulate cow’s milk, it is not surprising thatchymosin is the most commonly used enzyme for cheesemaking.Since more than 10 years Chr. Hansen is producing chymosin bya large scale fermentation process.Results: The genome of A. niger was modified by inserting aprochymosin c-DNA via an expression vector. Chymosin issecreted as a fusion protein with glucoamylase and processed to



the active protein during fermentation. After cultivation thebiomass is inactivated by lowering the pH-value and separatedby filtration. For concentration and purification of chymosin aone-step EBA (Expanded Bed Adsorption) chromatography isused. This new production process is illustrated in Figure 1.During the last years the fermentation process was optimised bychanging various parameters like feeding strategy and inocula-tion procedure of the Seed fermentor. The concentration stepwith an aqueous two-phase system with PEG (poly-ethylen-glycol) and the purification step with an ion-exchange chroma-tography step were replaced by one-step EBA chromatography.Conclusion: The simplified downstream process reduced thecosts for waste water treatment and increased the recoveryyield. Due to its high quality, the unlimited availability and itscompetitiveness to other milk clotting enzymes the marketshare of fermentation produced chymosin (FPC) increasedsignificantly over the last years.

S32Filamentous fungi as cell factories for proteinproductionPeter J PuntTNO Quality of Life, Microbiology, PObox 360, 3700AJ Zeist,The Netherlands


Filamentous fungi have been used as sources of metabolites andenzymes for centuries. In the last few decades molecular genetictools have enabled us to improve metabolite and proteinproduction in these organisms. The use of gene-transfer systemsand the development of efficient and versatile fungal expressionand secretion vectors has allowed the generation of proteinoverproducing host strains. Also in the field of strain develop-ment for fungal metabolite production (e.g. beta-lactamantibiotics) and bioconversion processes (e.g. P450 based


SDS-PAGE of six different proteins (1 to 6) analysed in quadruplicate (A to D). The proteins were expressed in E. coli host strains BL21(DE3)*, BL21(DE3)pLysS and Rosetta2, purified after NiNTA affinity chromatography on Piccolo.Lane A1 = protein 1, replicate A; A2 = protein 2, replicate A;A3 = protein 3, replicate A etc.; B1 = protein 1, replicate B etc.



reactions) molecular genetic tools have resulted in significantimprovements.For the production of secreted proteins further improvementsof the first generation strains have been obtained by rationalstrain design. The development of protease deficient hoststrains obtained by classical or molecular approaches has beenvery successful. The recent development of fungal HighThroughput Screening approaches will allow further improvedstrain development in this area. For the secretion of proteins ofnon-fungal origin specific carrier-protein strategies have beendeveloped.

In several cases strain design in combination with fermentationprocess development has resulted in achieving commerciallyrelevant quantities of protein.Also solid state fermentation strategies for filamentous fungi arebeing explored for protein production and as a source of newmetabolites and enzyme activities. Given the complex nature offungal solid state and submerged fermentation processes, due tothe metabolic versatility and hyphal growth phenotype of theseorganisms, the newly emerging ’genomics’ technologies areideally suited to reach a further understanding and subsequentimprovement of these processes.


Chymosin production process; fermentation and downstream processing.



S33Genome-wide analysis of protein productionphysiology in the filamentous fungus TrichodermareeseiMarkku Saloheimo, Tiina Pakula, Mikko Arvas, Jari Rautioand Merja PenttilaVTT Biotechnology, P.O. Box 1500, 02044 VTT, Espoo, Finland


Background: Trichoderma reesei is a filamentous fungus usedwidely as a protein production host by the enzyme industry. It is anexcellent protein secretor; the highest native enzyme productionlevels in the streamlined industrial process are above 100 g/l [1]. T.reesei is also used as a host for recombinant protein production. Theunfolded protein response (UPR) pathway has been characterizedfrom this fungus in our group and we have discovered a novelsecretion stress response, repression under secretion stress (RESS)affecting the genes encoding secreted proteins. The genome of T.reesei was sequenced recently by the Joint Genome Institute.Results: The methods for genome-wide analysis of T. reeseiphysiology have been set up in our laboratory. For transcrip-tomics we use the method TRAC and commercial genome-wideoligonucleotide arrays. As a rapid and semi-automated methodTRAC is very useful in focused transcript analysis of multiplesamples e.g. in assessment of the quality of chemostat cultures,in choosing the right samples for genome-wide analysis and intransient experiments with many sample time points. Inproteomics we use conventional 2D-electrophoresis techni-ques. The developed methodology has been used to address thephysiology of T. reesei that is relevant for industrial productionof recombinant and native proteins. A strain expressing theMelanocarpus albomyces laccase, a protein not inducing theunfolded protein response, was investigated by transcriptomicsand proteomics. Surprisingly few physiological changes werefound, and most of them were detected only at the proteomelevel. We have also addressed the effects of certain keybioprocess parameters on productivity and cell physiologywith our experimental platform, including O2 deficiency, growthrate and cell density. The results of these studies will bediscussed.Conclusion: An experimental platform for genome-wide physio-logical studies in Trichoderma reesei has been founded and used forelucidation of the effects of recombinant protein production andcertain key bioprocess parameters. This work has started toaccumulate useful data that can contribute to increasing theunderstanding on protein production in eukaryotic microbes.Reference1. Cherry JR and Fidanstef AL: Directed evolution of

industrial enzymes: an update. Curr Opin Biotechnol2003, 14:438–443.

S34Expression efficiency and sequence-based factors:a comparative view on 79 human genes in PichiapastorisChristine Lang1 and Mewes Bottner1,21Technical University of Berlin, Institute of Biotechnology,Department of Microbiology and Genetics, Gustav-Meyer-Allee25, 13355 Berlin, Germany2ORGANOBALANCE GmbH, Gustav-Meyer-Allee 25, 13355Berlin, Germany


High yield expression of heterologous proteins is usually amatter of "trial and error". In the search of parameters with amajor impact on expression, we have applied a comparativeanalysis to 79 homologous strains of Pichia pastoris harbouringdifferent human cDNAs. Recombinant protein expression wasmonitored in a standardized procedure and classified withrespect to the expression level. More than ten sequence-basedparameters with a possible influence on the expression levelwere analysed. Three factors proved to have a statisticallysignificant association with the expression level. Low abundanceof AT-rich regions in the cDNA associates with a highexpression level, indicating that correct transcript processingis a major determinant for the expression success in this yeast.A comparatively high isoelectric point of the recombinantprotein associates with failure of expression and, finally, theoccurrence of a protein homologue in yeast is associated withdetectable protein expression. Interestingly, some often dis-cussed factors like codon usage or GC content did not show asignificant impact on protein yield.These results could provide a basis for a knowledge-orientedoptimisation of gene sequences both to increase protein yieldsand to help target selection and the design of high-throughputexpression approaches.

S35A novel yeast expression system based on ahormone-induced transcriptional cascadeMarıa Jose Quintero1, Miguel Arevalo-Rodrıguez2,Angel Cebolla2 and Sebastian Chavez11Departamento de Genetica, Universidad de Sevilla, Avda.Reina Mercedes, 6 Sevilla E-41012, Spain2Biomedal S.L., Sevilla 41092, Spain


Background: The yeast Saccharomyces cerevisiae is widelyutilized in gene expression projects, both as a model eukaryoticorganism and as a host for heterologous protein production.Much of this research and biotechnological activity demands theuse of highly regulated systems, able to provide accurate controlof the expression in gene function analysis, and timelyrecombinant protein synthesis during fermentative production.Different yeast expression systems have been developed thatcan be controlled at the transcriptional level. Among thesesystems, those based on the potent, tightly regulated GAL1-10promoter and its cognate transcriptional activator Gal4 aremost commonly used [1]. However, induction of the GAL systemrequires the presence of galactose and the absence of glucose inthe culture media [2], a major disadvantage when the metabolicchanges associated to this switch in carbon source are relevantto the study. In addition, the high cost of the inducer canpreclude scaling up production of a commercially valuableprotein using this system. A good alternative to regulatetranscription driven by GAL promoters is the incorporation ofthe hybrid protein developed by D. Picard [3], which combinesfeatures of three different transcriptional activators, the DNAbinding domain of Gal4, the hormone response domain of thehuman estrogen receptor (ER), and the transcription activationdomain of herpes-virus VP16. This chimerical protein activatestranscription from GAL1-10 promoters in the presence of



estradiol at micromolar concentrations, even in the presence ofglucose. However, constitutive expression of this transactivatororiginates a high basal activity of the GAL promoters in theabsence of the hormone, therefore diminishing its efficiency as atranscriptional regulator.Results: In order to improve this expression tool, we haveplaced the coding sequence of the Gal4-ER-VP16 hybridactivator under the control of a GAL promoter. The combinationof these regulatory elements results in an amplification feedbackloop that is triggered by the hormone and ultimately leads toenhanced expression of recombinant genes fused to similar GALpromoters (see Figure 1). The basal expression level of thissystem is as low as that of GAL-driven genes in glucose-containing media. This platform is back compatible with pre-existent GAL expression constructs.

We have further expanded the versatility and capacity of thissystem, by adding new regulatory elements to those alreadydescribed. To this end, we have placed the complete human ERcoding sequence under the control of a GAL promoter, andcombined it with another construct, constitutively expressingthe Gal4-ER-VP16 hybrid activator. This configuration generateda hormone-dependent transcriptional cascade that allowedaccurate regulation of any promoter containing an estrogen-responsive element (ERE) (Figure 2). The basal activity of theERE-containing promoter was not influenced by expression ofGal4-ER-VP16 in the absence of ligand. The new combinedsystem improved an order of magnitude the expression levelwindows of the individual regulatory circuits. In addition, wehave found that certain mutations affecting GAL regulationfurther increase the level of induction of this system.


Estradiol-triggered feedback loop.


Hormone-dependent transcriptional cascade.



Conclusion: We have tested two novel gene/protein expres-sion systems derived from the combination of differenteukaryotic transcription elements. One of the systems activatedexpression of genes under the control of GAL promoters inS. cerevisiae, uncoupling it from the galactose/glucose signalingand keeping the low basal activity found in GAL promoters. Theother system consisted of a cascade of estrogen-dependentactivators able to stimulate transcription from an ERE-containingpromoter. This new CASCADE system could constitute asimple and cost-efficient way to control heterologous proteinexpression in yeast, especially in projects requiring tightlycontrolled protein production, including functional genomicsand industrial recombinant protein production.AcknowledgementsThis work was supported by PROFIT grants from the SpanishMinisterio de Educacion y Ciencia (FIT-01000-2003-110 and CIT-01000-2005-32) and by the Andalusian Government (CVI-271).References1. Schneider JC and Guarente L: Vectors for expression of

cloned genes in yeast: regulation, overproduction,and underproduction. Methods Enzymol 1991,194:373–388.

2. Johnston M and Carlson M: Regulation of carbon andphosphate utilization. The Molecular Biology of the YeastSaccharomyces Cold Spring Harbor Laboratory Press, ColdSpring Harbor, NY: Jones EW, Pringle JR, Broach JR,2:193–281.

3. Louvion JF, Havaux Copf-B and Picard D: Fusion of GAL4-VP16 to a steroid-binding domain provides a tool forgratuitous induction of galactose-responsive genesin yeast. Gene 1993, 131:129–134.

S36Use of a "universal" yeast vector (CoMedä)system for the production of proteins inHansenula polymorpha and Arxula adeninivoransGerd Gellissen1, Gerhard Steinborn2

and Gotthard Kunze21PharmedArtis GmbH, 52074 Aachen, Germany2IPK Gatersleben, 06466 Gatersleben, Germany


Background: A range of yeasts has been developed asattractive production systems for recombinant proteins. Somelike Hansenula polymorpha [1] are already distinguished by animpressive track record as producers of valuable proteins thathave already reached the market whereas other newly definedsystems like Arxula adeninivorans [2] have yet to establishthemselves but demonstrate a great potential for industrialapplications. All yeast systems have special favorable character-istics, but also limitations and drawbacks – as is the case with allexpression systems. As there is clearly no single system that isoptimal for all possible proteins, it is advisable to assess severalselected organisms in parallel for their capability to produce aparticular protein in desired amounts and quality to avoid costlytime- and resource-consuming failures. The availability of avector that can be targeted to the various platform candidateswould greatly facilitate such a comparison. As such a vectorsystem (CoMedä) has been designed that is built up in amodular way [3]. Certain combinations of elements result invectors that can be addressed to a wide range of yeast hosts.

Results: The basic design of the CoMed vector is depicted inFigure 1. Several ARS sequences are available, a range of differentA. adeninivorans and H. polymorpha-derived rDNA sequences, avariety of dominant and auxotrophic selection markers and ofexpression cassettes equipped with a great selection of yeastpromoter elements. The final constructs can be linearized in away that leaves behind all sequences of bacterial origin.The design of vectors suited for a wide range of fungal organismsmust meet several prerequisites. Such a plasmid must contain atargeting element suitable for all test species. The promoter thatdrives heterologous gene expression must be functional in allthese organisms and the vector/host system must employ adominant selection marker or a sequence that can complementthe auxotrophy in all selected organisms. Certain combinationsof vector elements presented before fulfill all requested criteria.The rDNA is highly conserved, the rDNA genes are present inhigh copy numbers and they are readily accessible for efficienttranscription. The A. adeninivorans-derived TEF1-promoter wasfound to function in all yeast systems tested so far, for selectionthe A. adeninivorans-derived LEU2 gene is available that cancomplement the leucine auxotrophy of all leu- yeast strainsassessed so far.A vector containing a combination of an rDNA integrationsequence, the LEU2 selection marker and an expresion cassetteharbouring the TEF1 promoter for expression control wastherefore selected to address a range of respective auxotrophicyeast strains, among others A. adeninivorans and H. polymorpha.In a first example we tested both species for the capability toproduce IL-6. We observed a different extent of correctlyprocessed precursor molecules.The rDNA integration sequence provides a tool that is not onlysuited to address several platform candidates in parallel but alsoto co-integrate several vectors at the same time. The option wasexecuted for the generation of recombinant IFN�-secretingH. polymorpha strains.In a rDNA co-integration approach several genes of thesecretory pathway were assessed for their impact on thesecretion of the cytokine. Upon co-integration and co-expres-sion of CNE1 secretion of the interferon was found beconsiderably improved, the glycosylated secretion product wasof distinct size corresponding to core-glycosylated moleculesinstead of hyperglycosylated proteins present without co-expression of CNE1 [4].Conclusion: A "universal" yeast vector system based onrDNA integration has been developed which on one hand isable to address in parallel a range of selected expressionorganisms and by which on the other hand several expressionplasmids can be co-integrated. The newly developed systemconstitutes an attractive novel tool for the application of yeastexpression platforms to heterologous protein production.References1. Kang HA and Gellissen G: Hansenula polymorpha. G

Gellissen (ed) Production of recombinant proteins – novelmicrobial and eukaryotic expression systems Wiley-VCH,Weinheim; 2005, 111–142.

2. Boer E, Gellissen G and Kunze G: Arxula adeninivorans.In G Gellissen (ed) Production of recombinantproteins – novel microbial and eukaryotic expres-sion systems. Wiley-VCH, Weinheim 2005, 89–110.

3. Gellissen G, Kunze G, Gaillardin C, Cregg JM, Berardi E,Veenhuis M and van der Klei IJ: New yeast expression



platforms based on methylotrophic Hansenula poly-morpha and Pichiapastoris and dimorphic Arxulaadeninivorans and Yarrowia lipolytica- a comparison.FEMS Yeast Res 2005, 5:1079–1096.

4. Degelmann A, Muller F, Sieber H, Jenzelewski V, Suckow M,Strasser AWM and Gellissen G: Strain and processdevelopment for the production of cytokines inHansenula polymorpha. FEMS Yeast Res 2002, 2:349–361.

S37Recombinant protein expression system in coldloving microorganismsRosanna Papa1, Valentina Rippa1, Giovanni Sannia1,Gennaro Marino1,2 and Angela Duilio11Department of Organic Chemistry and Biochemistry,University Federico II, Naples, Italy2School of Biotechnological Sciences, University Federico II,Naples, Italy


Background: Soluble and functional proteins are of highdemand in modern biotechnology. Although many recombinantproteins have been successfully obtained from common prokar-yotic and eukaryotic hosts, these systems result to be oftenunproductive due to the peculiar properties of the protein to be

produced. Incorrect folding of the nascent polypeptide chains isone of the main problems occurring during heterologous proteinproduction in bacteria. Since formation of inclusion bodies oftenimpairs the recombinant production of valuable proteins, manyexperimental approaches have been explored to minimize thisundesirable effect [1, 2]. Expression of "difficult" proteins has alsobeen carried out by lowering the temperature at the physiologicallimit allowed for the growth of mesophilic host organisms(between 15 and 18˚C for Escherichia coli). Lowering thetemperature, in fact, has a pleiotropic effect on the foldingprocess, destabilising the hydrophobic interactions needed forintermediates aggregation [3]. On the basis of the aboveconsiderations, a rational alternative to mesophilic organisms isthe use of naturally cold-adapted bacteria as hosts for proteinproduction at low temperature (even at around 0˚C).Results: The development of a shuttle genetic system for thetransformation of the cold adapted Gram-negative bacteriumPseudoalteromonas haloplanktis TAC125 (PhTAC125) [4, 5] hasalready been reported. This system has made possible theisolation of constitutive psychrophilic promoters and theconstruction of cold expression systems for the proteinproduction at low temperatures [6]. The described expressionsystem represented the first example of heterologous proteinproduction based on a true cold-adapted replicon [7]. However,the development of an effective cold expression system with


Basic design of a CoMed vector For further information see text.



industrial perspectives needs to be finely tuned possibly using adhoc promoters. Physical separation between bacterial growthphase and expression of the desired proteins, in fact, can notonly improve the productivity of the entire system but can alsoplay an important role in the production of proteins toxic forthe host cells. These goals can only be achieved by usingregulated promoters and efficient induction strategies. Recently,using a proteomic approach and exploiting the informationderiving from the genome sequence of PhTAC125 [8] weisolated and characterized a functionally active two-componentsystem involved in the transcriptional regulation of the genecoding for an outer membrane porin, that is strongly induced bythe presence of L-malate in the medium [9].In this paper we used the regulative region upstream of theporin gene to construct an inducible expression vector, namedpUCRP, that is under the control of L-malate. Performances ofthe inducible system were tested for both psychrophilic andmesophilic protein production using two "difficult" proteins asmodel systems. Moreover, an evaluation of optimal inductionconditions for protein production was also carried out.Conclusion: The inducible expression system was effective inthe production of the psychrophilic �-galactosidase fromPhTAE79 [10] and the mesophilic a-glucosidase from Sacchar-omyces cerevisiae [11] in a fully soluble and active form.References1. Mitra A, Chakrabarti KS, Shahul Hameed MS, Srinivas KV,

Senthil Kumar G and Sarma SP: High level expression ofpeptides and proteins using cytochrome b5 as afusion host. Protein Expr Purif 2005, 41:84–97.

2. Luo ZH and Hua ZC: Increased solubility of glu-tathione S-transferase-P16 (GST-p16) fusion pro-tein by co-expression of chaperones groes andgroel in Escherichia coli. Biochem Mol Biol Int 1998,46:471–477.

3. Jeon YH, Negishi T, Shirakawa M, Yamazaki T, Fujita N,Ishihama A and Kyogoku Y: Solution structure of theactivator contact domain of the RNA polymerasealpha subunit. Science 1995, 270:1495–1497.

4. Birolo L, Tutino ML, Fontanella B, Gerday C, Mainolfi K,Pascarella S, Sannia G, Vinci F and Marino G: Aspartateaminotransferase from the Antarctic bacteriumPseudoalteromonas haloplanktis TAC 125. Cloning,expression, properties, and molecular modelling.Eur J Biochem 2000, 267:2790–2802.

5. Tutino ML, Duilio A, Parrilli E, Remaut E, Sannia G andMarino G: A novel replication element from anAntarctic plasmid as tool for the expression ofproteins at low temperatures. Extremophiles 2001,5:257–264.

6. Duilio A, Madonna S, Tutino ML, Pirozzi M, Sannia G andMarino G: Promoters from a cold-adapted bacter-ium: definition of a consensus motif and molecularcharacterization of UP regulative elements. Extre-mophiles 2004, 8:125–132.

7. Duilio A, Marino G, Mele A, Sannia G and Tutino ML:Sistema di espressione di proteine ricombinanti abasse temperature., Uff It Brev Marchi RM2003/A000155.

8. Medigue C, Krin E, Pascal G, Barbe V, Bernsel A,Bertin PN, Cheung F, Cruveiller S, D’Amico S, Duilio A,Fang G, Feller G, Ho C, Mangenot S, Marino G, Nilsson J,

Parrilli E, Rocha EP, Rouy Z, Sekowska A, Tutino ML,Vallenet D, von Heijne G and Danchin A: Coping withcold: the genome of the versatile marine Antarc-tica bacterium Pseudoalteromonas haloplanktisTAC125. Genome Res 2005, 10:1325–1335.

9. Papa R, Glagla S, Danchin A, Schweder T, Marino G andDuilio A: Proteomic identification of two-componentregulatory system in Pseudoalteromonas haloplanktisTAC125. Extremophiles 2005 in press.

10. Hoyoux A, Jennes I, Dubois P, Genicot S, Dubail F,Francois JM, Baise E, Feller G and Gerday C: Cold-adapted beta-galactosidase from the Antarcticpsychrophile Pseudoalteromonas haloplanktis. ApplEnviron Microbiol 2001, 67:1529–1535.

11. Kopetzki E, Buckel P and Schumacher G: Cloning andcharacterization of baker’s yeast alpha-glucosidase:over-expression in a yeast strain devoid of vacuolarproteinases. Yeast 1989, 5:11–24.

S38Rapid detection of bacteriophage infection andprophage induction using electric biochipsMarcin Los, Joanna Los and Grzegorz WegrzynDepartment of Molecular Biology, University of Gdansk, Kladki24, 80-822 Gdansk, Poland


Background: Bacteria are widely used hosts for production ofmany biotechnologically important substances. Bacteriophagesare viruses that infect bacterial cells. Thus, infection of bacterialcultures by bacteriophages may lead to serious problems,including complete loss of a desired bioproduct and spreading ofbacteriophages throughout the whole laboratory. Althoughphage contamination, and resultant phage infection of bacterialcultures, may cause serious problems in all types of micro-biological laboratories, it is especially dangerous when cultiva-tions are performed on a large scale. Moreover, a number ofcommonly used strains of E. coli contain lambdoid prophagesthat often bear some regulatory genetic elements useful in thecontrol of the expression of cloned genes. However, undercertain conditions a prophage induction occurs that may havesimilar effects on a bacterial culture as phage infection. Evenunder standard cultivation conditions, a spontaneous prophageinduction occurs with low frequency. However, this rareprophage induction results in appearance of infecting phageparticles in amounts ranging from 10�8 to 10�5 pfu (plaqueforming units) per bacterial cell. These numbers seem to be low,but when cultivations are performed on a large scale, e.g.reaching 1010 cells per ml, this means from 102 to 105 phagesper ml. Considering even a very small bioreactor containing onelitre of the culture, this adds up to 108 infecting phage particles.If we consider a 100-litre bioreactor, the number of phages inthe medium may reach 1010. The potential (but, in fact, veryreal) problems described above, indicate a need for rapiddetection of phage infection or prophage induction. However,using traditional methods we can detect the presence of phagesin a bacterial culture unambiguously after several hours afterinfection, at best. Unfortunately, this is usually too late to saveat least a part of the infected culture, and to avoid phagespreading throughout the laboratory. Therefore, it seems that



development of new methods for rapid detection of bacter-iophages in bacterial cultures became crucial.Results: We present novel methods for detection of bacter-iophages, which are based on the use of electric biochips. Theprinciple of this method is to capture the target molecules(either nucleic acids or proteins) on the chip, and the use of thesecondary detection probe which is coupled with an enzymecatalyzing a red-ox reaction. The electric signal appearing as aresult of this reaction is then measured by an micro-electrode.This method gives relatively quick and quantitative results. Twokinds of electric bio-chips were used. One was desired fordetection of bacteriophage genetic material (with DNA probes),and the second was desired for detection of phage virions (withspecific antibodies). In both cases, we were able to detect thepresence of bacteriophages (phages lambda and its derivatives,M13, P1 and T4 were used as models in this study) in amountsbetween 104 to 107 particles/molecules within as short time as25–50 min (the values differed depending on the specific methodused) from sample withdrawal. The results were quantitative ina wide spectrum of concentrations of bacteriophage DNA andvirions.Conclusion: We suggest that electric bio-chips may provide apotentially useful technique for rapid and quantitative detectionof the presence of bacteriophages and for monitoring bacter-iophage infection and prophage induction. It is worth noting thatvery often recurrent infections with the same phage occur inparticular laboratories. This is because many virions can survivein a laboratory even in a dry form. Thus, although for detectionof phage DNA or virions by means of electric bio-chips it isnecessary to prepare specific DNA probes or specific anti-bodies, this method may be especially useful in the case ofrecurrent infections with the same bacteriophage. In such a casesingle isolation of the bacteriophage strain should be enough toprepare specific probes and/or serum.AcknowledgementsThis work was supported by European Commission (projecteBIOSENSE).

S39NIisin Controlled gene Expression (NICE) inLactococcus lactis - versatile applications rangingfrom membrane proteins to large scale processesIgor MierauNIZO food research, Department of Health and Safety, P.O.Box 20, 6710 BA, Ede, The Netherlands


Background: Lactococcus lactis is one of the best studiedbacteria. After its isolation more than 100 years ago, it firstreceived attention as dairy bacterium because of its importancein cheese and butter fermentations. Following the developmentof genetic engineering, it quickly became the paradigm lactic acidbacterium. Today the genomes of three different strains of thegenus L. lactis are elucidated and prototype genome-based completemetabolic models are developed. The development of the NIisinControlled gene Expression (NICE) system about 10 years agogreatly facilitated progress in many areas of research not only inLactococcus itself, but also in all other lactic acid bacteria.Results: The NICE system is a straightforward, easy to usesystem (plug-and-play genetic toolbox) for strictly controlledexpression of homologous and heterologous genes. The

advantages of L. lactis as gene expression system over e.g.E. coli are that it is food grade (including the selection marker),does not produce endotoxins or inclusion bodies, it has verylow protease activity, does not sporulate and it has only onemembrane. At present, the NICE system is quickly growing intoan important tool for expressing and studying prokaryotic andeukaryotic membrane proteins. Furthermore, the NICE systemis growing beyond its initial role as a research tool and is usedfor the production of oral and live vaccines and for the largescale production (3000 L) of pharmaceutical proteins such aslysostaphin.Conclusion: The presentation will describe the principle ofthe NICE system, give an overview of current applications andan outlook on future developments

S40An integral process for the production of virus-likeparticles by insect cellsLaura A Palomares and Jimmy A MenaDepartmento de Medicina Molecular y Bioprocesos. Institutode Biotecnologıa. Universidad Nacional Autonoma de Mexico(UNAM), Cuernavaca, Morelos, 62210, Mexico


Virus-like particles (VLP) are produced when the structuralproteins of a non-enveloped virus are expressed in arecombinant system. As a result, particles identical to the nativevirus, but devoid of the viral genetic material, are obtained. VLPare useful as vaccines, as carriers, and for basic research. Theirproduction represents a challenge, as several proteins need tobe simultaneously expressed and assembled into a complexstructure. Other aspects that complicate process developmentinclude the difficulty of accurately quantifying complete VLP, aswell as possible intermediates, and the difficulty of designingeffective purification procedures.The versatility of the insect cell-baculovirus expression vectorsystem has made it one of the most frequently used systems forthe production of VLP. In this work, our recent advances on theproduction of double-layered rotavirus-like particles (dlRLP) byinsect cells will be presented. The use of a viral vector, such asthe baculovirus, allows the manipulation of the concentration ofthe recombinant proteins by manipulating the multiplicity ofinfection, allowing the production of rotavirus proteins at astoichiometry that maximizes protein assembly [3]. In vivoanalysis of the accumulation of rotavirus proteins in insect cellsshowed that assembly occurs intracellularly. However, differ-ences in the intracellular distribution of rotavirus proteins whenthey were individually expressed suggest that the formation ofRLP involve more than only the contact between two proteins[2]. We will present in vivo studies of the assembly or rotaviralproteins, where we have identified and characterized the limitingsteps and intermediaries in the assembly process. Finally, toolsfor the characterization and quantification of dlRLP andassembly intermediates will be presented [1], along with thedevelopment of a purification scheme for the ’preparativecharacterization of dlRLP. The results obtained and the severalstrategies presented are an example of the characteristics,limitations and specific requirements of animal cells in culture.AcknowledgementsFinancial support by SAGARPA-CONACyT 103, CONACyT-Morelos 2004-C02-058 and DGAPA-UNAM 223805.



References1. Mena JA, Ramırez OT and Palomares LA: Quantification

of rotavirus-like particles by gel permeation chro-matography. J Chromatogr B Analyt Technol Biomed Life Sci2005, 824:267–276.

2. Mena JA, Ramırez OT and Palomares LA: Intracellulardistribution of rotavirus structural proteins andvirus-like particles expressed in the insect cell-baculovirus system. J Biotechnol in press.

3. Palomares LA, Lopez S and Ramırez OT: Strategis formanipulating the relative concentration of recom-binant rotavirus structural proteins during simulta-neous production by insect cells. Biotechnol Bioeng 2002,78:635–644.

S41Fast generation of high producer cho cell lines byan iterative transfection processP-A Girod1,2, M Grandjean1, D Calabrese1,2, D Martinet3,J Beckmann3 and N Mermod11Laboratory of Molecular Biotechnology, University ofLausanne, 1015 Lausanne Switzerland2Selexis SA, Geneva, Switzerland3Division of Genetics, Lausanne University Hospital (CHUV),Swtizerland


Background: Isolation of mammalian cell clones for high-levelprotein production remains usually impeded by the time-consuming processes of selection, gene amplification and theanalysis of many clones that is required to identify one withfavorable properties, while maintaining proper protein proper-ties and consistency. Expression variability results in part fromthe site of transgene integration in the host genome, and fromthe variable number of transgene copies that integrate. We andothers have shown that genetic insulator elements such as MARcan be used to shield transgenes from inhibitory effects of thesurrounding chromosomal sequences, alleviating in part integra-tion site effects [1, 2, 3, 4]. However, productivity remainslimited by the number of transgenes that can be intergrated inthe host genome. Thus, it would be useful to increase thenumber of integrated transgenes, and to render this integrationprocess more frequent and more reproducible.Results: In this presentation, we will describe a multipletransfection procedure that allows simple generation of cell lineswith high and stable levels of recombinant protein production.We will show that this technique improves significantlytransgene expression, up to 10-fold in polyclonal population ofCHO cells, and that this effect results in part from increasedtransgene integration. Using various combinations of vectorelements, we show that improved transgene expressionrequires homologous DNA sequences in the successivelytransfected DNAs. Using FISH studies, we demonstrate thatthe DNAs integrate at a random, but unique, position within thecell genome, and that high productivity is achieved withoutchromosome rearrangement nor transgene amplification. Over-all, our studies imply that homologous recombination mediateshigh efficiency integration of many transgene copies within thecellular genome. When this process is coupled to the use ofchromatin control elements such as MARs, productivities of upto 80 picogram/cell/day can be achieved when expressing IgGs.

Conclusion: Overall, these results indicate that mammaliancell clones displaying very high productivities can be obtained ata high frequency when using an efficient transfection processcombined with effective vector elements.References1. Zahn-Zabal M, Kobr M, Imhof MO, Chatellard P, de Jesus M,

Wurm F and Mermod N:Development of stable cell linesfor production or regulated expression using matrixattachement regions. J Biotechnol 2001, 87:29–42.

2. Girod PA and Mermod N: Use of Scaffold/Matrix-Attachment Regions for protein production. NewComprehensive Biochemistry, Gene transfer and expression inmammalian cells 2003, 38:359–379.

3. Girod PA, Zahn-Zabal M and Mermod N: Use of Matrixattachment regions to generate high producer celllines. Biotechnol Bioeng 2005, 91:1–11.

4. Girod PA, Nguyen D, Calabrese D, Grandjean M,Martinet D, Beckmann J, Bucher P and Mermod N: Insilico identification of potent Matrix attachmentregions from genomes and use to generate highproducer cell lines. Manuscript in preparation.

S42Zeraâ, a novel technology for stable accumulationand easy recovery of recombinant proteins ineukaryotic protein-production hostsI Llop1, M Torrent1, P Marzabal2, M Bastida2, B Llompart2

and D Ludevid11Consorcio CSIC-IRTA. Jordi Girona 18. 08034 Barcelona,Spain2ERA Biotech. Parc Cientıfic Barcelona. Josep Samitier 5.08028 Barcelona, Spain


Background: Subcellular targeting of proteins in host cells isone of the most important issues in protein production.Stability, folding, and post-translational modifications dependon where proteins are sorted. Besides secretion, ER appears asan efficient compartment to accumulate proteins, as demon-strated by the frequent use of K/HDEL fused to the C-terminusof a protein to retain and accumulate recombinant proteins inthe endoplasmic reticulum (ER) of the cell hosts.Results: We present a novel technology (Zeraâ assemblerpeptides) we have developed for recombinant protein produc-tion based on their in vivo accumulation in ER-derived artificialstorage organelles. The Zeraâ peptide is a proline-rich domainof a plant storage protein with a) self-assembling and b) proteinbody formation properties. Zeraâ peptide, as a fusion partner ofproteins of interest, reaches a conformation stage that inducesthe formation of novel dense organelles derived from the ER:Protein Bodies (PBs). The recombinant proteins remain stablyaccumulated within these PBs in host cells. A family of Zeraâ

fusion proteins have been engineered and expressed in differenteukaryotic systems such as yeast, mammalian cells and plants. Inall systems, recombinant proteins accumulate in the newlyformed PBs where they are protected from degradation. Thehigh density of these organelles permits to implement optimizedrecovery and purification processes of the protein product,partially by achieving a ultra-high, pre-purification concentrationor the product.



Conclusion: Our results indicate that the Zeraâ peptide is apowerful tool for both high accumulation of recombinantproteins in eukaryotic cells and their recovery from biomass.

S43Design of improved membrane proteinproduction experiments in yeast: quantitationof the host responseNicklas Bonander1, Kristina Hedfalk2, Christer Larsson2,Petter Mostad3, Celia Chang4, Lena Gustafsson2

and Roslyn M Bill51Department of Cell and Molecular Biology/Microbiology,Goteborg University, Box 462, 405 30 Goteborg, Sweden2Department of Chemistry and Bioscience/MolecularBiotechnology, Chalmers University of Technology, Box 462,405 30 Goteborg, Sweden3Department of Mathematics, Chalmers University ofTechnology, 41296 Goteborg, Sweden4The Wistar Institute, 3601 Spruce Street, Philadelphia,Pennsylvania 19104, USA5School of Life and Health Sciences, Aston University, AstonTriangle, Birmingham B4 7ET, UK


Background: Eukaryotic membrane proteins cannot be pro-duced in a reliable manner for structural analysis. Consequentlyresearchers still rely on trial-and-error approaches, which mostoften yield insufficient amounts. This means that membraneprotein production is recognized by biologists as the primarybottleneck in contemporary structural genomics programs.Here we describe a study to examine the reasons for successesand failures in recombinant membrane protein production inyeast – a eukaryotic production organism – at the level of thehost cell, by systematically quantifying cultures in high-perfor-mance bioreactors under tightly-defined growth regimes.Results: In a first step to taking a systematic, quantitativeapproach to membrane protein production in yeast, we wentback to first principles to collect a data set for our targetprotein, the glycerol channel Fps1p, in high-performancebioreactors under tightly-defined growth regimes. We choseto study expressing cultures at 20, 30 and 35˚C, and pH 5 or 7,with the FPS1 gene under the control of the TPI1 promoter.Changes in temperature clearly affected the production time-course, which had a different profile under different conditions.Protein in both the total extract and the membrane-boundfraction was predominantly produced in the glucose phase. Thedata highlight major differences in production throughout thegrowth curve under a single condition, most pronounced at 35˚C, pH 5 (7-fold in the total extract and 4-fold in the membranefraction). Comparing all tested growth conditions, the overalldifference in production was 10-fold in the membrane fraction(35˚C pH7 vs. 20˚C pH5 3). Importantly, it was clear that thereis no correlation between the total production yield and theyield of membrane-localized protein, most pronounced at 35˚CpH7. This result should be of particular interest to those settingup high-throughput platforms, since it is clear that a ’quick anddirty’ analysis of total extracts can be very misleading since it isnot representative of membrane-inserted protein yields.In order to explain our membrane protein yield data, we firstquantified the corresponding transcript levels. When weperformed real time quantitative PCR (Q-PCR) on TPI1 and

the plasmid-borne FPS1 genes, it was clear that the observedyields could not be explained by variations in the TPI1 or FPS1transcripts. We therefore decided to identify genes (and theircorresponding gene products) that are expressed or repressedin the host cell under specific culture conditions leading to agiven yield of functional membrane protein, as this should allowfor a better understanding of the critical parameters involved.To our knowledge, this approach has not been used to analyzeany membrane protein production experiments: only a micro-array analysis of soluble LuxA production in E. coli has beenreported [1]. We therefore performed an analysis using yeastminiarrays to rationalize the observations that changing theculture conditions from 30˚C pH5 to either 35˚C pH7 or 35˚CpH5 gave good yields of total Fps1 protein, but dramaticallyreduced membrane-bound Fps1 protein yields. In essence, wesought to understand what was failing in the conversion ofimproved total Fps1 protein yields to membrane-insertedprotein under these conditions. 84 genes changed theirexpression level on going from 30˚C pH 5 to 35˚C pH 5 and111 on going from 30˚C pH 5 to 35˚C pH7, with 39 varying ongoing from 30˚C pH 5 to 35˚C pH 5 and 30˚C pH 5 to 35˚C pH7(p < 0.05). Results for the genes, MFa1 and HOR7 provided auseful validation of our data set, as it is has already beenreported that upregulation of MFa1 results in down-regulationof HOR7, a gene of unknown function [2].Both the increase in total Fps1 protein production at 35˚C pH5compared to 30˚C pH5, and the similar total Fps1 protein yieldat 30˚C pH5 compared to 35˚C pH7 were not reflected inmembrane-bound yields. This was accompanied by down-regulation of three genes involved in ribosome biogenesis(RPP1A, CGR1 and BMS1), a membrane component of the ERprotein translocation apparatus, SEC62, and two genes involvedin vacuolar trafficking, APM3 and VTC3. Warner and coworkershave found that transcription of genes encoding both ribosomalproteins and rRNAs is repressed when the secretory pathway isdefective [3], providing the cells with a mechanism for cellularstress adaptation [4]. This hypothesis is also consistent with ourobserved down-regulation of SEC62, since yeast sec62 mutantsare defective in the translocation of several secretory precursorproteins into the lumen of the endoplasmic reticulum, includinga-factor precursors and certain membrane proteins [5].Interestingly, upregulation of MFa1 also indicates that theprotein secretory pathway is compromised [6, 7]. Indeed, theS. cerevisiae a-factor prepro-peptide leader sequence has beenused to confer secretory competence to proteins such as insulin[8], and constructed leaders have been developed for efficientsecretory expression in Pichia pastoris.Other genes related to defects in the secretory pathway couldalso be correlated to poor membrane-bound yields of Fps1p,including SRP102 which encodes the �-subunit of the signalrecognition particle (SRP) receptor. Srp102p has been suggestedto co-ordinate the release of the signal sequence from the SRPwith the presence of the translocon [9], and appears to regulatethis process through a ’switch cycle’ of GTP/GDP binding [10].The remaining genes have roles in key metabolic events, but notin protein quality control; specifically not in ubiquitination.We compared our dataset of 39 genes to those published on theresponse of yeast cells to environmental changes [11, 12], andspecifically on moving cells from 27˚C to 37˚C [11]. Apart fromthe well-documented up-regulation of HSP82 on raising culturetemperature, we found no overall correlation between the



datasets, lending further support to the fact that the genomicprofile we observe is linked to failure in membrane proteinproduction, rather than merely resulting from an increase intemperature.Conclusion: Our data show that the most rapid growthconditions of those chosen are not the optimal productionconditions. Furthermore, the growth phase at which the cellsare harvested is critical: we show that it is crucial to grow cellsunder tightly-controlled conditions and to harvest them prior toglucose exhaustion; just before the diauxic shift. The differencesin membrane protein yields that we observe under differentculture conditions are not reflected in corresponding changes inmRNA levels of FPS1, but rather can be related to thedifferential expression of genes involved in membrane proteinsecretion and yeast cellular physiology.In the search for generic membrane protein production systems,several solutions have been proposed. High-throughputapproaches – which involve trying many conditions chosenessentially at random – do not succeed in generating generichosts since proteins that are not produced using this format arediscarded without understanding why. We suggest that a ’smart-throughput’ approach should enable a more focused, strategicmethod of recombinant eukaryotic membrane protein produc-tion, through the identification and quantitation of theparameters critical for success, thereby allowing productionon a milligram scale. In this study we have specifically identifiedthe importance of a functional secretion pathway in host cellsgrown under tightly-controlled conditions. This should ulti-mately contribute to understanding the critical parameters thatdefine a successful membrane protein production experiment.AcknowledgementsThis work was supported by the European Commission viacontracts LSHG-CT-2004-504601 (E-MeP) to RMB, and QLG2-CT-2002-00988 (SPINE) to LG and RMB. We also acknowledgeChalmers Bioscience Initiative and the Wallenberg Foundationthrough their support of the Membrane Protein Centre,Lundberg Laboratory, Goteborg, Sweden. The miniarray datawas performed in collaboration with Dr L. Showe, W. Horngand L. Gudipati of the Wistar Institute, Philadelphia, USA.References1. Oh MK and Liao JC: DNA microarray detection of

metabolic responses to protein overproduction inEscherichia coli. Metab Eng 2000, 2:201–209.

2. Seidel J and Tanner W: Characterization of two newgenes down-regulated by alpha-factor. Yeast 1997,13:809–817.

3. Mizuta K and Warner JR: Continued functioning of thesecretory pathway is essential for ribosome synth-esis. Mol Cell Biol 1994, 14:2493–2502.

4. Schmidt T, Chen PS and Pellegrini M: The induction ofribosome biosynthesis in a nonmitotic secretorytissue. J Biol Chem 1985, 260:7645–7650.

5. Stirling CJ, Rothblatt J, Hosobuchi M, Deshaies R andSchekman R: Protein translocation mutants defec-tive in the insertion of integral membrane proteinsinto the endoplasmic reticulum. Mol Biol Cell 1992,3:129–142.

6. Caplan S, Green R, Rocco J and Kurjan J:Glycosylation andstructure of the yeast MF alpha 1 alpha-factorprecursor is important for efficient transport throughthe secretory pathway. J Bacteriol 1991, 173:627–635.

7. Avaro S, Belgareh-Touze N, Sibella-Arguelles C, Volland Cand Haguenauer-Tsapis R: Mutants defective in secre-tory/vacuolar pathways in the EUROFAN collec-tion of yeast disruptants. Yeast 2002, 19:351–371.

8. Kjeldsen T: Yeast secretory expression of insulinprecursors. Appl Microbiol Biotechnol 2000, 54:277–286.

9. Pool MR: Getting to the membrane: how is co-translational protein targeting to the endoplasmicreticulum regulated?. Biochem Soc Trans 2003,31:1232–1237.

10. Schwartz T and Blobel G: Structural basis for thefunction of the beta subunit of the eukaryotic signalrecognition particle receptor. Cell 2003, 112:793–803.

11. Gasch AP, Spellman PT, Kao CM, Carmel-Harel O,Eisen MB, Storz G, Botstein D and Brown PO: Genomicexpression programs in the response of yeastcells to environmental changes. Mol Biol Cell 2000,11:4241–4257.

12. Serrano R, Ruiz A, Bernal D, Chambers JR and Arino J:The transcriptional response to alkaline pH inSaccharomyces cerevisiae: evidence for calcium-mediated signalling. Mol Microbiol 2002, 46:1319–1333.

POSTER PRESENTATIONS

P1Comparative transcriptional profiling of thebacterial stress response in temperature andchemically-induced recombinant E. coli processesDaniela Bohm and Ursula RinasGBF – German Research Centre for Biotechnology,Braunschweig, Germany

Microbial Cell Factories 2006, 5(Suppl 1):P1

Background: Production of heterologous proteins results ina number of metabolic and physiological changes in the host cellsduring the course of a production process, namely the induction ofstress responses and corresponding alterations in gene expressionprofiles [1].Results: This study focuses on quantitative monitoring of theadaptation of E. coli to recombinant protein production on thetranscriptome level by a bead-based RNA sandwich hybridisa-tion assay, a rapid novel method based on the detection ofhybridisation events between specific oligonucleotide probesand the target nucleic acids [2, 3].The expression profiles of selected genes including the productgene, anabolic and stress responsive genes were quantitativelyanalyzed in cells producing the human basic fibroblast growthfactor (hFGF-2), a protein that partially aggregates into inclusionbodies. Transcriptome profiles during temperature- and IPTG-induced synthesis of hFGF-2 using the K12 strain TG1 and BL21(DE3) as production hosts, respectively, were compared.References1. Hoffmann F and Rinas U: Stress induced by recombi-

nant protein production in Escherichia coli. AdvBiochem Eng Biotechnol 2004, 89:73–92.

2. Gabig-Ciminska M, Holmgren A, Andresen H, Barken K,Wumpelmann M, Albers J, Hintsche R, Breitenstein A,Neubauer P and Los M, et al: Electric chips for rapid



detection and quantification of nucleic acids. BiosensBioelectron 2004, 19:537–546.

3. Soini J, Falschlehner C, Mayer C, BohmD, Panula J, Vasala A andNeubauer P: Transient increase of ATP as a response totemperature up-shift in Escherichia coli. Microb Cell Fact2005, 4:9.

P2FT-IR spectroscopy for the study of bacterialmembrane stress induced by recombinant proteinproductionDiletta Ami1, Antonino Natalello1, Pietro Gatti-Lafranconi1,Tina Schultz2, Marina Lotti1, Ario de Marco2

and Silvia Maria Doglia11Department of Biotechnology and Biosciences, University ofMilano-Bicocca, Piazza della Scienza 2, 20126 Milano, Italy2EMBL Scientific Core Facilities, Mayerhofstr. 1, D-69117,Heidelberg, Germany


Background: Microorganisms respond to environmental stressesregulating their membrane fluidity by changing the lipid fatty acidcomposition. In particular, in bacterial cells the alteration ofmembrane lipid composition plays an important role in response toheat and toxic stresses [1, 2]. It is known that at increasingtemperatures the regulation of membrane fluidity occurs throughthe incorporationofmoresaturated fattyacids, longeracyl chains and,in some cases, by changing the unsaturated fatty acids from cis to transconformation. Among different stresses, production of heterologousproteins is of particular interest for its applications in biotechnology.Results: In this work we show the potential of FT-IR micro-spectroscopy to monitor changes in membrane composition duringrecombinant protein production. In particular, we studied theinfrared absorption of E. coli strains expressing several proteins withdifferent amount of soluble and aggregated fractions. In addition, as acontrol experiment, we have studied E. coli strains expressing betagalactosidase, as reporter gene, under the chaperone IbpBpromoter [3].In all model systems examined, an increase in the band intensities at2850 cm�1 and at 2925 cm�1 was observed in presence of a highlevel of stress [4]. As these two bands are due to CH2 stretchingvibrations [5], this spectral behaviour may indicate that longer acylchain and /or more saturated fatty acids are incorporated in cellmembranes. The protein expression analysis indicates that theenzymes that lead to the accumulation of Acetyl Co-A (precursor offatty acids) are strongly accumulated whereas the phospholipidedegradation seems being inhibited.Indeed, when the protein is expressed also in a soluble form – asindicated by the FT-IR protein response in the amide I region andconfirmed by the SDS-PAGE analysis – the twoCH2 bands are moreintense than in the case of IB formation. This behaviour seems toindicate that the presence of the soluble recombinant proteininduces a stress in the cell, suggesting that IBs could limit the toxicityof overexpressed foreign proteins.Conclusion: This work highlights the potential of FT-IRspectroscopy not only to study protein aggregation in IBs [6,7, 8], but also to monitor the membrane response induced bythe heterologous production.AcknowledgementsThis work was supported by INFM (Istituto Nazionale Fisicadella Materia) grant to SMD.

References1. Yuk HG and Marshall DL: Heat adaptation alters

Escherichia coli O157:H7 membrane lipid composi-tion and verotoxin production. Appl Environ Microbiol2003, 69:5115–5119.

2. Heipieper HJ, Meinhardt F and Segura A: The cis-transisomerase of unsaturated fatty acids in Pseudomonasand Vibrio: biochemistry, molecular biology andphysiological function of a unique stress adaptivemechanism. FEMS Microbiol Lett 2003, 229:1–7.

3. Lesley SA, Graziano J, Cho CY, Knuth MW and Klock HE:Gene expression response to misfolded protein as ascreen for soluble recombinant protein. Protein Eng2002, 15:153–160.

4. Schuster KC, Mertens F and Gapes JR: FT-IR spectro-scopy applied to bacterial cells as a novel method formonitoring complex biotechnological processes.Vibrational Spectroscopy 1999, 19:467–477.

5. Arrondo JLR and Goni FM: Infrared studies of protein-induced perturbation of lipids in lipoproteins andmembranes. Chem Phys Lipids 1998, 96:53–68.

6. Ami D, Bonecchi L, Calı S, Orsini G, Tonon G andDoglia SM: FT-IR study of heterologous proteinexpression in recombinant Escherichia coli strains.Biochim Biophys Acta 2003, 1624:6–10.


8. Ami D, Natalello A, Taylor G, Tonon G and Doglia SM:Structural analysis of protein inclusion bodies byFourier transform infrared microspectroscopy. Bio-chim Biophys Acta 2006, in press.

P3Regulation of the secretion pathway of CHO cellsfor altered recombinant Mab production ratesduring the course of MTX amplificationYuan Sheng Yang1, Janet Chusainow1, Yan Ying Mao1,Steven CL Ho2 and Miranda GS Yap1,31Bioprocessing Technology Institute, Biomedical SciencesInstitutes, 20 Biopolis Way, #06-01 Centros, Singapore1386682Division of Bioengineering, Nanyang Technological University,Nanyang Avenue, Singapore, 6397983Department of Chemical & Biomolecular Engineering,National University of Singapore, 10 Kent Ridge Crescent,Singapore 119620


Background: Monoclonal antibody (Mab) production bymammalian cells is a complex, multiple-step process which isregulated at transcriptional, translational, and post-translationallevels. A detailed understanding of how cells regulate thispathway is a prerequisite for designing genetic strategies forincreasing antibody production [1]. Methotrexate (MTX), whichis widely used in the creation of high-producing stable cell linesby amplification of gene copy number, provides an effectivemeans to alter Mab production rates for mechanistic studies ofthe regulation of this pathway [2].



Results: In this work, stable CHO DG44 cell lines expressinga human anti-D Mab were created and single-cell clones wereamplified to obtain a series of cultures with varying productionrates. During the course of amplification, changes in the Mabgene copy numbers, transcriptional levels of Mab mRNAs, andaccumulated intracellular Mab peptides were examined for eachclone. In addition, changes in expression levels of representativegenes with function in translation, folding, assembly, anddegradation were determined. Gene copy number and tran-scription level were quantified by quantitative real time PCR,and the intracellular Mab peptides were quantified by westernblotting and ELISA.Conclusion: Results obtained in this work could help identifythe rate-limiting steps and factors that are significant in limitingproduction rate for high-producing clones.AcknowledgementsWe thank Toh Poh Choo and Jessna Yeo who created the celllines used in this work.References1. Gonzalez R, Andrews BA and Asenjo JA: Metabolic

control analysis of monoclonal antibody synthesis.Biotechnol Prog 2001, 17:217–226.

2. Kim SJ, Kim NS, Ryu CJ, Hong HJ and Lee GM: Char-acterization of chimeric antibody producing CHOcells in the course of dihydrofolate reductase-mediated gene amplification and their stability inthe absence of selective pressure. Biotechnol Bioeng1998, 58:73–84.

P4Microarray-based analysis of recombinant proteinproduction in E. coliRonan O’Dwyer1, Xuejun Hu1, Mattia Pelizzola2,Olga Kolaj1, Maria Foti2, Paola Ricciardi-Castagnoli2

and J Gerard Wall1,31Department of Chemical and Environmental Sciences,University of Limerick, Limerick, Ireland2School of Biotechnology and Bioscience, University ofMilan-Bicocca, Milan 20126, Italy

3Materials and Surface Science Institute, University ofLimerick, Limerick, Ireland


Background: The production of heterologous proteins in E.coli is a powerful tool in the generation of many importantbiotechnological and medical products. Despite its widespreaduse as an expression host, however, yields of correctly folded,functional protein are frequently low in E. coli. This is due largelyto the formation of insoluble protein aggregates and topremature lysis of the bacterial cells. We, and others, havepreviously shown that the cell lysis phenomenon associated withrecombinant protein production in E. coli is not a direct result ofsynthesis of heterologous proteins [1, 2]. Instead, proteinproduction triggers a global stress response in the bacterium,but the mechanism by which cell lysis subsequently occursremains unclear [3].We have carried out a microarray-based study of the responseof E. coli to production of two recombinant proteins. In thisanalysis, a murine scFv antibody fragment and a human renalenzyme were produced in the E. coli periplasm, followed by co-production in turn of the cation efflux protein CzrB fromThermus thermophilus and E. coli disulfide bond isomerase DsbC.These latter proteins had previously been demonstrated in ourgroup to delay lysis of the host E. coli cells and increase yields ofthe two proteins [1, 4].Results: Growth and functional yields of the two recombinantproteins were studied using standard techniques. Co-expressionof czrB and dsbC led to delayed lysis of host E. coli cells and toimprovements in functional yields of recombinant proteins (seeFigure 1).Subsequent to mRNA purification and microarray analysis, datamining identified a number of genes whose expression wassignificantly altered upon recombinant protein production.Phage shock proteins and numerous chaperones were signifi-cantly upregulated, while OmpF was the main downregulatedprotein. Genes whose expression reverted towards pre-induction levels upon co-production of CzrB and/or DsbCwere also identified. We report results of manipulation of

Figure 1 (abstract P4)

A Growth of E. coli cells producing the murine2H12 scFv fragment with (diamonds) and without (squares) co-production of DsbC. B Immunoblotdetection of 2H12 scFv produced in the presence and absence of DsbC overproduction. Lane 1. Molecular weight marker; lanes 2–3. no DsbC; lanes4–5. + DsbC; lanes 2,4. insoluble scFv; lanes 3,5. soluble scFv.



expression of a number of these genes in an attempt to increasefunctional yields of the two recombinant proteins in vivo.Conclusion: A microarray-based analysis of recombinantprotein production was utilised to identify changes in geneexpression in E. coli upon induction. Manipulation of expressionof a number of these genes has been used to increase functionalprotein yields in vivo.References1. Spada S, Pembroke JT and Wall JG: Isolation of a novel

Thermus thermophilus metal efflux protein thatimproves E. coli growth under stress conditions.Extremophiles 2002, 6:301–8.

2. Knappik A and Pluckthun A: Engineered turns of arecombinant antibody improve its in vivo folding.Protein Eng 1995, 8:81–9.

3. Hoffmann F and Rinas U: Stress induced by recombi-nant protein production in Escherichia coli. AdvBiochem Eng Biotechnol 2004, 89:73–92.

4. Hu X, O’Dwyer R and Wall JG: Cloning, expressionand characterisation of a single-chain Fv antibodyfragment againstdomoic acid in Escherichia coli.J Biotechnol 2005, 120(1):38–45.

P5A sensor of the Unfolded Protein Response tostudy the stress induced in Yarrowia lipolyticastrains by the production of heterologous proteinsCatherine Madzak and Jean-Marie BeckerichUMR1238 Microbiologie et Genetique Moleculaire, INRA/CNRS/INA-PG, CBAI, 78850 Thiverval-Grignon, France


Background: A critical step in the production of heterologousproteins in yeasts seems to be the efficiency of protein folding

and assembly in the endoplasmic reticulum (ER). Saturation ofthe machinery involved in these steps leads to an accumulationof unfolded and misfolded proteins in the ER, activating theunfolded protein response (UPR). The UPR pathway controlsthe expression of genes for ER-resident chaperones andfoldases, together with genes encoding diverse functions suchas translocation, protein secretion and processing, and proteindegradation. It is generally admitted that the UPR pathway istriggered by the lowering of free Kar2/Bip level in the ER, due tothe binding of this chaperone to misfolded proteins. Thetitration of Kar2/Bip liberates Ire1p kinase, which auto-activates,becoming able to maturate the intron from HAC1 mRNA. Thisprocessing enables the translation of the transcriptionalactivator Hac1p, which enhances the expression of the UPRpathway genes via the recognition of an UPR element in theirpromoter.Results: In order to analyse the UPR in the non-conventionalyeast Yarrowia lipolytica, especially in relation with the produc-tion of heterologous proteins, we designed an UPR-sensorcarried on an integrative vector (pINA1300). This sensorcomprises the promoter from KAR2 gene (containing 3 putativeUPR elements) directing the expression of the lacZ reportergene (see Figure 1). The induction of UPR in Yarrowia strainscarrying the sensor will result in beta-galactosidase production,easily detectable and measurable using coloured reactions.The UPR-sensor pINA1300 vector was used to transformseveral Yarrowia strains sharing the same genetic background:a non-producing control strain (Po1g [1]), and two strainsproducing different heterologous proteins. We first performedvalidation experiments on the UPR-sensor control strain (Po1g+ pINA1300), in order to check if growth conditions known toinduce UPR were able to increase beta-galactosidase productionabove the background level. The results are displayed in theFigure 2.


Map of pINA1300 vector and scheme of the experiments.



Validation of the UPR-sensor: exponential phase cultures ofUPR-sensor control strain (Po1g + pINA1300) were treated ornot with either 20 micrograms per mL tunicamycin, or 20micromolar dithiothreitol, for 3 hours, and beta- galactosidaseproduction in the cells was measured as previously described[1]. The treatment, beta-galactosidase activity, and increaseabove the background level are indicated under each bar.We performed similar validation experiments on the two UPR-sensor strains carrying heterologous gene expression cassettesand obtained similar results (data not shown – note that in theconditions of the validation experiment, the heterologous geneswere not, or very poorly, expressed due to the growth-dependent characteristics of the promoter used [1]). Thus, ourvalidation experiments showed that beta-galactosidase activityreflects the induction of UPR in Yarrowia strains carrying theUPR-sensor.Conclusion: The UPR is extensively studied in Saccharomycescerevisiae, but we found it interesting to analyse it in Yarrowialipolytica, in which the secretion system is co-translational, as inmammalian cells. Moreover, an efficient expression/secretionsystem has been developed in this yeast [2], and the dataobtained could contribute to improve further its performances.The use of the pINA1300 vector will allow us to detect UPR inYarrowia strains, and to measure its level of induction. This UPR-sensor will be used to analyse UPR induction in variousconditions, such as the stress imposed on Yarrowia strains bythe production of different heterologous proteins.

References1. Madzak C, Treton B and Blanchin-Roland S: Strong hybrid

promoters and integrative expression/secretionvectors for quasi-constitutive expression of hetero-logous proteins in the yeast Yarrowia lipolytica. J MolMicrobiol Biotechnol 2000, 2:207–216.

2. Madzak C, Gaillardin C and Beckerich JM: Heterologousprotein expression and secretion in the non-conven-tional yeast Yarrowia lipolytica: a review. J Biotechnol2004, 109:63–81.

P6Native and heterologous protein oxidationand subsequent degradation in a recombinantfilamentous fungus Aspergillus niger B1-DQiang Li, Linda M Harvey and Brian McNeilStrathclyde Fermentation Centre, Department of Bioscience,University of Strathclyde, 204 George Street, Glasgow G11XW, UK


Filamentous fungi have attracted extensive research interest dueto their abilities to secrete large amounts of high-valuedrecombinant proteins. Despite the fact that proteases havebeen recognized as one of the main problems in proteinproduction, little is known about the regulation of them. In the




present study, we hypothesized that reactive oxygen species(ROS), unavoidable by-products in all aerobic cultures, maycause protein oxidation and induce proteolysis in filamentousfungi. To test this hypothesis, we used a variety of oxidativestressors, oxygenation, menadione, hydrogen peroxide, hydro-gen peroxide with copper ion, to study the induction ofintracellular proteolytic activities in A. niger B1-D, a recombinantfilamentous fungus secreting hen egg white lysozyme. Proteincarbonyl content was monitored as a bio-marker for proteinoxidation. Our results show that oxygenation and metal-catalyzed oxidation significantly induce carbonyl groups tobovine serum albumin (BSA). We also found that proteolyticactivities and carbonyl content increase on the addition of thesestressors to the whole broth in A. niger B1-D, and the responsesare dose-dependent. In conclusion, ROS overproductioncorrelates with protein oxidation and proteolysis in A. nigerB1-D. It is advisable to decrease ROS production in thebioprocess of filamentous fungi in order to achieve a higherproductivity of heterologous proteins.

P7Low growth temperatures improve theconformational quality of aggregation pronerecombinant proteins in both soluble andinsoluble E. coli cell fractionsAndrea Vera, Nuria Gonzalez-Montalban,Elena Garcia-Fruitos, Anna Arıs and Antonio VillaverdeInstitut de Biotecnologia i de Biomedicina and Departament deGenetica i de Microbiologia, Universitat Autonoma deBarcelona, Bellaterra, 08193 Barcelona, Spain


Background: Protein aggregation is a major bottleneck in thebacterial production of recombinant proteins. Among others,induction of gene expression at suboptimal growth tempera-tures (for instance, below 37˚C in Escherichia coli), has beenrepeatedly observed as a convenient procedure to enhance thesolubility of aggregation-prone proteins and to minimizeinclusion body formation. However, the effect of low growth

temperatures in protein features, other than mere solubility, hasbeen rarely explored.Results: In this work, we have determined the folding statusand functionality of an engineered GFP variant when producedat 37, 30 and 16˚C. The strain used was BL21 (DE3) and theplasmid pET21-b(+). Gene expression was induced by IPTGusing different production times for each temperature: 2 h at 37˚C, 5 h at 30˚C and 16 h at 16˚C, which are equivalent to thesame biomass increase.After induction, fluorescent microscopy analysis was carriedout, and the cellular extract was fractioned in order to analyseeither the soluble or insoluble fraction using fluorimetry. Theamount of recombinant GFP protein was calculated by WesternBlot to obtain the specific fluorescence emission. PlasmidlessBL21 (DE3) was used as a negative control. Fluorimetry analysisshowed that fluorescence emission of the soluble fraction wasapproximately five times higher at 16˚C than at 37˚C. On theother hand, fluorescence emission of the insoluble fraction wasapproximately 1.5 times higher at 16˚C than at 37˚C. However,when these fluorescence values are related to the quantity ofprotein (Figure 1), the results of the soluble fraction are similar,but surprisingly in the insoluble fraction an increased fluores-cence emission (10 times higher) is observed at 16˚C withrespect to 37˚C.Conclusion: As expected, solubility of a recombinant GFP waslargely improved at low temperatures. On the other hand, andvery interestingly, the quality of GFP, as reflected by its specificfluorescence emission, was largely enhanced at 16˚C not only inthe soluble cell fraction but also in the residual inclusion bodies.This intriguing observation indicates that the physicochemicalconditions governing protein folding act in parallel on bothsoluble and aggregated protein forms. Therefore, proteinmisfolding and aggregation are clearly not coincident events,what strongly supports the hypothesis that incorrect folding isnot a straightforward cause of protein deposition.AcknowledgementsThis work has been funded by BIO2004-00700 from MEC,Spain and 2005GR-00956 (AGAUR). Andrea Vera Barron isrecipient of a doctoral fellowship from UAB, Spain, Nuria


Specific Fluorescence Emission of the engineered GFP variant. Insoluble cell fraction (A) and Soluble cell fraction (B).



Gonzalez-Montalban and Elena Garcia-Fruitos are recipients of adoctoral fellowships from MEC, Spain.

P8Zea mays L. transglutaminase expressionin Escherichia coliPatricia Carvajal1, Enrique Villalobos2,Alexandre Campos2, J Ma Torne2, Eduard Barbera1

and Mireya Santos21Department of Chemical Engineering, Biotechnology lab.GQBB group, Institut Quımic de Sarria (IQS), UniversitatRamon Llull, Via Augusta 390, 08017, Barcelona, Spain2Institut de Biologıa Molecular de Barcelona-Consejo Superiorde Investigaciones Cientıficas (IBMB-CSIC), Jordi Girona18–26, 08034, Barcelona, Spain


Background: Transglutaminase (protein-glutamine:amine�-glutamyl-transferase, E.C. 2.3.2.13) catalyses acyl-transferreactions between �-carboxyamide groups of glutamine resi-dues and the " -amino group of lysines in proteins, leading tointer- or intramolecular cross-linking. Transglutaminases (TGs)have been found in mammals, plants, fish, nematodes andbacteria. Two maize cDNA clones (TGZ15 and TGZ21) thatexpressed active transglutaminase localized in chloroplasts wereisolated [1, 2].

Results: A TGZ sequence was subcloned into pET (Novagen)vector (named as pET28a+TGZ4). The expression assays in E.coli BL21 DE3 cells showed that the main fraction of the protein(>80%) was found in the inclusion bodies (see Figure 1). Thepurification under denaturing conditions in FPLC systemfollowed by a refolding step was a suitable procedure to obtainfunctional TGZ4p (see Figures 2,3). In addition, a specific


Western blot of TGZ4p. S = soluble, IB = inslusion bodies


TGZ4p FPLC purification under denaturing conditions.



antibody against TGZ4p was obtained in the laboratory for theimmunolocalization of this protein in E. coli cells (see Figure 4).Conclusion: TGZ4p was expressed in E. coli mainly asinclusion bodies. The purification under denaturing conditionsand refolding in vitro was a suitable procedure to obtainfunctional TG.AcknowledgementsThanks to the Spanish National Project MCYT BFI2003-003318and CEUB-IQS fellowship.References1. Villalobos E, Santos M, Talavera D, Rodrıguez-Falcon M and

Torne J: Molecular cloning and characterization of a

maize transglutaminase complementary DNA. Gene2004, 336:93–104.

2. Torne JM, Santos MA, Talavera D and Villalobos E: Maizenucleotide sequense coding for a protein withtransglutaminase activity and use thereof. PCT/ES03/00247. Patent number WO03102128 2002.

P9Cellular toxicity triggered by bacterial inclusionbodiesNuria Gonzalez-Montalban1,2, Antonio Villaverde1,2

and Anna Aris1,21Institut de Biotecnologia i de Biomedicina, UniversitatAutonoma de Barcelona, Bellaterra, 08193 Barcelona, Spain2Departament de Genetica i de Microbiologia, UniversitatAutonoma de Barcelona, Bellaterra, 08193 Barcelona, Spain


Background: The cytotoxicity associated with inclusionbodies and its possible impairment to recombinant proteinproduction processes in E. coli, have been poorly studied so far[1]. To explore these possible toxicity mechanisms we haveemployed a well-described system used with other kinds ofprotein aggregates to evaluate deleterious effects in animal cells.Cytotoxicity in animals has been associated not only with arange of proteinaceous aggregates in several degenerativediseases but also with prefibrilar aggregates of protein unrelatedto any clinical diseases since they have been reported to harmcell viability by affecting biochemical parameters such asintracellular redox status and free Ca2+ levels [2]. In thiscontext this work contributes to support the hypothesis thatmisfolded proteins cause negative cellular effects as a result ofexposition of hydrophobic patches or other aggregate char-acteristic structures on its surface.Results: In this study we analysed the cellular toxicityassociated with inclusion bodies formed in MC4100 E. coli,producing the misfolding-prone polypeptide VP1LAC, consistingon a N-terminal �-galactosidase fusion containing the VP1 capsidprotein of foot-and-mouth disease virus [3]. The proteinaggregates produced during either 1 h or 5 h were added at arange of final protein concentrations (from 1 �M to 8.5 �M) toNHI-3T3 cells. We demonstrated by confocal microscopyanalysis that bacterial inclusion bodies bind and enter into thecells. Moreover its cytotoxic effect was evaluated by MTTreduction assay, a standard indicator of cell physiological state.As shown in Figure 1, the experiments reveal that IB significantlyimpairs cell viability mainly when were incubated with 5 h-inclusion bodies. Interestingly the thermal aggregates of �-galactosidase obtained in vitro by a temperature shift to 96˚C andfurther incubation at room temperature, do not significantlymodify MTT reduction in comparison to control wells or to cellsincubated with soluble �-galactosidase.Conclusion: In the present study, we prove that bacterialinclusion bodies are clearly toxic for mammalian cells. Despitethe fact that it has been previously suggested that the mosthighly cytotoxic aggregates are the early prefibrilar assembliesrather than mature fibrils [2], in our case the more structured 5h-aged inclusion bodies show a more pronounced toxic effectcompared to those formed only during 1 h. However, the �-galthermal aggregates, which have been shown to present a fibrilarpattern, behave as expected since they have not been associated


TGase activity of refolded TGZ4p against Tris and phosphate buffer.


TGZ4p TEM Immunolocalization, S=soluble, IB=inclusion body.



with any extent of toxicity. Overall, these results support thehypothesis that different kinds of aggregates are deleterious butencourage us to study the rules that regulate the proteinaceousaggregates’ toxicity.References1. Gonzalez-Montalban N, Carrio MM, Cuatrecasas S, Aris A

and Villaverde A: Bacterial inclusion bodies arecytotoxic in vivo in abscence of functional chaper-ones DnaK or GroEL. J Biotechnol 2005, 118:406–412.

2. Bucciantini M, Calloni G, Chiti F, Formigli L, Nosi D,Dobson CM and Stefani M: Prefibrillar amyloid proteinaggregates share common features of cytotoxicity.J Biol Chem 2004, 279:31374–31382.

3. Corchero JL, Viaplana E, Benito A and Villaverde A: Theposition of the heterologous domain can influencethe solubility and proteolysis of �-galactosidasefusion proteins in E. coli . J Biotechnol 1996, 48:191–200.

P10DnaK-J are limiting for proper recombinantprotein folding only at low production rates andwhen the physiological heat-shock stress responseis not triggeredMonica Martınez-Alonso, Andrea Vera, Elena Garcıa-Fruitos, Nuria Gonzalez-Montalban, Anna Arısand Antonio VillaverdeInstitut de Biotecnologia i de Biomedicina and Departament deGenetica i de Microbiologia, Universitat Autonoma deBarcelona, Bellaterra, 08193 Barcelona, Spain E-mail:[email protected]


Background: It is known that the bacterial production ofrecombinant, misfolding-prone proteins triggers the heat shock


Percentage of cell viability respect to control cells in NIH-3T3 cultures incubated for 24 hours with 4 M of 1 h- or 5 h-aged inclusion bodies, soluble -galand thermal aggregated -gal. Cells incubated with platinum are included in the experiment as a citotoxicity positive control.



response, as observed through the monitoring of several markergenes [1]. On the other hand, the co-production of selectedchaperones along with the recombinant protein has been largelyexplored as a strategy to minimize aggregation of the product,with rather unpredictable and not always consistent results [2, 3,4]. The reasons for the limited success of this approach could liein suspected although not proven different folding requirementsof specific protein species and the occurrence of differentialbottlenecks in their in vivo folding pathways. Moreover, it has notbeen discarded that the protein-induced heat-shock responsecould eventually eclipse the rise of functional plasmid-encodedchaperones resulting from co-production. To explore thispossibility and to offer more light on the functional mechanicsof recombinant protein folding we have quantitatively deter-mined the intracellular levels of DnaK under different conditions,during the production of a misfolding-prone GFP variant.Results: The production in E. coli MC4100 of an engineeredGFP protein (VP1GFP), controlled by the Trc promoter (inpTVP1GFP) and triggered by 1 mM IPTG results in an importantlevel of aggregation, with VP1GFP occurring in both the solubleand insoluble cell fractions at similar extents (42.2% ± 1.6 and57.8% ± 1.6 respectively). When the producer strain carriedpBB535 [5] (containing both IPTG-inducible PA1/lac-O1 controlleddnaK-J chaperone genes) as a second plasmid, the addition ofIPTG promotes the co-expression of both the chaperone geneset and the vp1gfp gene. However, this does not result in anydetectable shift in the fractioning of VP1GFP, which still occurs

in the insoluble cell fraction in an important level (48.2% ± 3.6).In the same line, the specific fluorescence of VP1GFP is notmodified by dnaK-J co-expression in both soluble (143.1 ± 31.5fluorescence units/�g VP1GFP versus 125.4 ± 29.7) andinsoluble (26.2 ± 6.7 versus 30.6 ± 5.4 fluorescence units/�gVP1GFP) cell fractions. Also, the total fluorescence determinedby OD unit is not significantly affected by coexpression (378.0 ±20.1 fluorescence units/ml of culture·OD550 versus 315.0 ± 8.5fluorescence units/ml of culture·OD550).The amounts of DnaK were determined in all cases by means ofWestern blot analysis as indicative of the chaperone setproduction, and the plasmidless MC4100 strain and MC4100/pBB535 were added as controls for this experiment. Asobserved in Figure 1, the levels of DnaK were slightly lower inpresence of pTVP1GFP than in its absence (MC4100/pBB535versus MC4100/pTVP1GFP/pBB535), probably due to a genedosage effect of plasmid coexistence as previously suggested forother plasmid sets [6]. However, DnaK amounts in MC4100/pTVP1GFP/pBB535 upon IPTG addition were still three-foldhigher than in MC4100/pTVP1GFP. In this last case, the heatshock response promoted by the production of VP1GFP itself,although clearly detectable, only doubled the intracellular levelsof the chromosomal-encoded DnaK present in plasmidlessMC4100 (Figure 1). This clearly indicates that the VP1GFP-mediaded induction of the heat shock response is not eclipsingthe phenotypic effect of additional DnaK-J amounts provided byan encoding plasmid.


Estimation of the intracellular amounts of DnaK in the different strains used in this work, under 1 mM IPTG induction.



Interestingly, at low IPTG doses (0.02 mM), the levels of DnaKcannot be distinguished between MC4100/pTVP1GFP/pBB535 andMC4100/pTVP1GFP (not shown), but the specific fluorescence ofVP1GFP is almost doubled in presence of pBB535 (Figure 2).Conclusion: DnaK-J are limiting for the proper folding of lowamounts of VP1GFP, in absence of any endogenous heat-shockresponse. However, under strong production conditions, aphysiological heat-shock response is triggered and an additionalincome of DnaK-J does not promote any detectable effect onVP1GFP protein quality. This indicates that the DnaK-J levelsreached as a response to protein production are high enough tooffer a sufficient supply of such chaperones. Therefore, theproper folding of VP1GFP under these conditions is probablyrestricted by a limiting factor other than DnaK-J, probably aheat-shock product or other cell element whose levels remainmodest during recombinant protein production.AcknowledgementsWe appreciate the generous gift of pBB535 from Prof. B. Bukau.This work has been supported by grants BIO2004-0700 (MEC)and 2005SGR-00956 (AGAUR), Spain. AV, EGF and NGM arerecipient of predoctoral fellowships from UAB, MEC and MECrespectively.References1. Jurgen B, Lin HY, Riemschneider S, Scharf C, Neubauer P,

Schmid R, Hecker M and Schweder T: Monitoring of

genes that respond to overproduction of an inso-luble recombinant protein in Escherichia coli glu-cose-limited fed-batch fermentations. Biotechnol Bioeng2000, 70:217–224.

2. Baneyx F and Palumbo JL: Improving heterologousprotein folding via molecular chaperone and foldaseco-expression. Methods Mol Biol 2003, 205:171–197.

3. Thomas JG and Baneyx F: Protein misfolding andinclusion body formation in recombinant Escherichiacoli cells overexpressing Heat-shock proteins. J BiolChem 1996, 271:11141–11147.

4. Thomas JG and Baneyx F: Divergent effects of chaper-one overexpression and ethanol supplementation oninclusion body formation in recombinant Escherichiacoli . Protein Expr Purif 1997, 11:289–296.

5. Tomoyasu T, Mogk A, Langen H, Goloubinoff P andBukau B: Genetic dissection of the roles of chaper-ones and proteases in protein folding and degrada-tion in the Escherichia coli cytosol. Mol Microbiol 2001,40:397–413.

6. de Marco A and de Marco V: Bacteria co-transformedwith recombinant proteins and chaperones clonedin independent plasmids are suitable for expressiontuning. J Biotechnol 2004, 109:45–52.


Specific fluorescence of the soluble VP1GFP in the different strains used in this work, under 0.02 mM IPTG induction. Insoluble VP1GFP was notfluorescent.



P11Potato virus A genome-linked protein is a nativelyunfolded proteinKimmo Rantalainen and Kristiina MakinenDepartment of Applied Chemistry and Microbiology, Universityof Helsinki, Helsinki, Finland


Background: The target of the study is Potato virus A (PVA,genus Potyviridae) and its genome- linked protein (VPg). Most ofthe PVA proteins are multifunctional, interacting with each otherand with host proteins. Many of the functions are still unclear andsome completely unknown. VPg is a 23 kDa protein interactingfor example with viral polymerase. It has NTP-binding and nuclearlocalization signals overlapping with each other in the N-terminalend of the protein. Growing evidence shows that genome-linkedproteins belong to a class of natively unfolded proteins [1].Descriptive for this class is regions without fixed structure in thecorrectly folded and ready-made protein. Interactions betweenthe unfolded region and its natural substrate usually launchesfolding but structural changes can be regulated also by otherreactions such as phosphorylation.Results: Bioinformatic analysis of PVA VPg was carried out usingseveral different softwares all predicting partly unfolded nature forVPg. Prediction presented in Figure 1A was obtained usingPONDRâ software http://www.pondr.com and VSL1 algorithm.CD spectroscopy was used to get general view of VPg structure.Far-UV spectra shows typical characteristics for unstructuredprotein (see Figure 1B), namely low ellipticity at 222 nm and strongnegative ellipticity near 200. Negative minimum around 208 nmsuggests that considerable amount of a-helixes is also present.Elution profile of size exclusion chromatography gives evidencefor either dimeric or unstructured status of VPg (data notshown). Peak corresponding to the smallest protein hadapproximated size of 49 kDa when calculated size of VPgmonomer is 23 kDa. Bulk of the soluble VPg came out in twopeaks both corresponding to sizes over 200 kDa indicating VPg’stendency to oligomerize.Conclusion: Consistently with the predictions our experi-mental data so far supports the natively unfolded structure of

PVA VPg. In addition, CD spectral data supports the predictionthat VPg probably has some stabile structural elements as well.Since VPg is a multifunctional protein, the partly unfolded natureputatively gives possibilities to regulate the VPg function duringthe different stages of infection. For example, structuralstabilization launched by PVA polymerase NIb or nucleatidyla-tion of VPg [2] might be the key regulatory events leading toinitiation of replication. However, the possible initiators ofstructural stabilization at the unfolded region remains to besolved.References1. Satheshkumar PS, Gayathri P, Prasad K and Savithri HS:

"Natively unfolded" VPg is essential for Sesbaniamosaic virus serine protease activity. J Biol Chem 2005,280:30291–30300.

2. Puustinen P and Makinen K: Uridylylation of thepotyvirus VPg by viral replicase NIb correlateswith nucleotide binding capacity of VPg. J Biol Chem2004, 279:38103–38110.

P12RNA Interference mediated knockdown of genesin order to increase protein production using thebaculovirus expression systemColin Hebert1, Eun Jeong Kim1, Shannon F Kramer2,James J Valdes3 and William E Bentley1,41Center for Biosystems Research, University of MarylandBiotechnology Institute, College Park, MD 20742, USA2Department of Biomedical Sciences, Baylor College ofDentistry, Texas A&M University System Health ScienceCenter, 3302 Gaston Ave., Dallas, TX 75246, USA3U. S. Army Edgewood Chemical Biological Center, AberdeenProving Ground, Maryland 21010, USA4Department of Chemical and Biomolecular Engineering,University of Maryland College Park, College Park, MD 20742,USA


Background: The baculovirus expression system has provento be a robust and versatile system for recombinant protein


Prediction and experimental evidence of natively unstructured nature of Potato virus A VPg A, Prediction of unfolded regions in PVA VPg based on aminoacid sequence. Region from N’ -terminal to Asn56 is here predicted to be unfolded using PONDR software. B, Far-UV spectra of PVA VPg.



production in insect cells. A wide range of promoters is available forthe facile expression of transgenes, and yields of up to 50% of totalprotein have been reported [1]. However, in many cases yield isdecreased as a result of proteases and host cell apoptosis [2]. Pastefforts to overcome this problem include co-expressing chaperoneproteins to assist with folding [3], anti-apoptotic proteins to reducecell death, or adding chemical protease inhibitors to the culturemedia [4]. However, these methods may have non-specific effects,prove too costly to be practical, or impose an undue metabolicburden on an already stressed cell. An alternative approach toincreasing protein production is through the application of RNAinterference (RNAi) to knockdown viral and host genes responsiblefor decreasing the yield of recombinant protein. Potential targetsinclude proteases, cell-death proteins, and cell cycle regulators. Byaltering the metabolic landscape of cells prior to the introduction ofthe baculovirus, protein production can be improved.Results: Initially, double stranded RNA (dsRNA) was producedin vitro against 19 gene targets; including acidic juvenile hormone-sensitive protein (AJHSP1) and Elongation factor 2 in Trichoplusiani (T. ni) larvae, and a virus-encoded cathepsin-like protease,v-cath, in Spodoptera frugiperda (Sf-9) cell culture. The knockdownof these three genes increased the yield of Green FluorescentProtein (GFP) produced via recombinant baculovirus. In the caseof v-cath, the increase in infected cell culture was ~3-fold (Figure1). Coincident with GFP yield, v-cath dsRNA also prolonged cellviability by over a day in infected Sf-9 cells (Figure 1). Ongoingstudies are focused on identifying new host cell gene targets inboth Sf-9 and T. ni cell culture, investigating combinations ofknockdown targets, and integrating the most promising targetsinto expression vectors for the production of in vivo RNAi.Conclusion: Overall, our results support the application ofRNAi as a metabolic engineering tool, specifically for enhancingprotein productivity in the baculovirus expression system.References1. Miller LK: The baculoviruses. New York: Plenum Press;

1997.2. Kost TA, Condreay JP and Jarvis DL: Baculovirus as

versatile vectors for protein expression in insect andmammalian cells. Nat Biotechnol 2005, 23:567–575.

3. Ailor E and Betenbaugh MJ: Modifying secretion andpost-translational processing in insect cells. Curr OpinBiotechnol 1999, 10:142–145.

4. Kato T, Murata T, Usui T and Park EY: Improvement ofGFPuv-beta 3GnT2 fusion protein production bysuppressing protease in baculovirus expressionsystem. Biosci Biotechnol Biochem 2003, 67:2388–2395.

P13Heterologous overexpression of a halophilica-amylaseVanesa Bautista, Julia Esclapez,Rosa Ma Martınez-Espinosa, Francisco Perez-Pomares,Monica Camacho and Ma Jose BoneteDepartment of Biochemistry and Molecular Biology, Universityof Alicante, 03080 Alicante, Spain


Background: Extracellular hydrolytic enzymes such as a-amylases are widely used in diverse applications in differentindustrial areas. a-amylase (EC 3.2.1.1) is an important endo-type carbohydrase that hydrolyzes a-1,4 glycosidic linkages ofD-glucose oligomers and polymers. This enzyme has been foundin organisms of the three Domains, being a key enzyme ofcarbohydrate metabolism. Haloferax mediterranei is an extremelyhalophilic Archaea that requires high salt concentrations togrow. This microorganism is able to grow in a minimal mediumwith ammonium acetate as the only source of carbon andnitrogen.H. mediterranei shows a-amylase extracellular activitywhen grows in this minimal medium in the presence of starch.The main role of this enzyme is the starch metabolism in theextracellular medium, so a lot of microorganisms depend onamylases for survival [1].Results: The extracellular halophilic H. mediterranei a-amylasehas been purified to electrophoretic homogeneity [2]. Theenzyme has been digested in the presence of trypsin to analysethe resulting peptides using the technique of nanoelectrospray LC/MS. From the obtained peptides and by sequence homology withother a-amylases, the H. mediterranei a-amylase gene has been


Effect of silencing of v-cath on the specific growth rate (A), the viability (B), and GFP production (C) in baculovirus infected Sf-9 cells. Cell counts weremeasured using a hemocytometer and viable cells were detected using trypan blue. Cell viability was defined by the ratio of the viable cell number tothe total cell number. () cells treated without dsRNA; () cells treated with dsRNA. GPF was measured quantitatively (C) using a fluorescencespectrometer. (black bar) cells without dsRNA; (white bar) cells treated with dsRNA.



isolated and sequenced, using a library of genomic DNA from H.mediterranei in bacteriophage lambda EMBL3. The molecular massestimated from the deduced amino acid sequence was similar to thecalculated by SDS-PAGE. This enzyme is rich in acidic amino acidswith a 16% Asp and Glu content which is also the case in otherhalophilic enzymes [3]. The PCR product for a-amylase was ligatedin the plasmid pSTBlue1 and cloned into E. coli NovaBlue cells. Theinsert was digested and subcloned in expression vector pET3a in E.coliNovaBlue cells, and introduced in the E. coli BL21(DE3) cells forexpression. The recombinant protein was mainly obtained asinclusion bodies, although a small amount of protein was presentedin the cytoplasmic soluble fraction (Figure 1). The protein obtainedas inclusion bodies was refolded by solubilisation in 8 M ureafollowed by dilution into a high salt concentration buffer in thepresence of calcium (Figure 2), and the protein of the solublefraction was obtained in active state.Furthermore, four genes have been sequenced by primer walking,which match up with four proteins belonging to ABC maltosetransport system.Conclusion: The a-amylase gene from H. mediterranei has beencloned, sequenced and overexpressed. The recombinant proteinhas been obtained in large amounts and refolded with a highefficiency.References1. Jones RA, Jermiin LS, Easteal S, Patel BKC and Beacham IR:

Amylase and 16S rRNA genes from a hyperthermo-philic archaebacterium. J Appl Microbiol 1999, 86:93–107.

2. Perez-Pomares F, Bautista V, Ferrer J, Pire C, Marhuenda-Egea FC and Bonete MJ: Alpha-amylase activity fromthe halophilic archaeon Haloferax mediterranei.Extremophiles 2003, 7(4):299–306.

3. Pire C, Esclapez J, Ferrer J and Bonete MJ: Heterologousoverexpression of glucose dehydrogenase from thehalophilic archaeon Haloferax mediterranei, anenzyme of the medium chain dehydrogenase family.FEMS Microbiol Lett 2001, 200:221–227.

P14Performance of beta-galactosidase inclusionbodies in enzymatic bioprocessesElena Garcıa-Fruitos, Anna Arıs and Antonio VillaverdeInstitut de Biotecnologıa i de Biomedicina and Departament deGenetica i de Microbiologia, Universitat Autonoma deBarcelona, Bellaterra, 08193 Barcelona, Spain


Background: Inclusion body formation is a common eventduring bacterial over-expression of recombinant genes. Thisphenomenon represents a great matter of concern in biotech-nology, because it has restricted the spectrum of proteinsmarketed in this field. In a previous work, we have observed thatrecombinant enzymes produced in bacteria are not completelyinactivated when deposited as inclusion bodies [1] and thataggregation as inclusion bodies does not necessarily split proteinpopulation into active and inactive fractions. Therefore, wedecided to further explore and fully characterize the behaviourof purified beta-galactosidase inclusion bodies in presence ofsubstrate, during a small-scale bioprocess.Results: In this work we have analysed the state of the inclusionbodies formed by an engineered E. coli beta-galactosidase fused tothe aggregation-prone foot-and-mouth disease virus (FMDV) VP1capsid protein (VP1LAC). Inclusion bodies were resuspended in


SDS-PAGE for expression cell fractions.(a) Lane 1: molecular weight standards; Lanes 3 and 4:E. coli BL21(DE3) containing pET3a; Lanes 2 and 5: E. coliBL21(DE3) containing pET3a-Amy; Lanes 2 and 3: total cell proteins; Lanes 4 and 5: soluble cytoplasmic fractions.(b) Lane 1: molecular weightstandards; Lanes 2 and 3: inclusion bodies E. coli BL21(DE3) containing pET3a-Amy with different concentrations.



Z buffer and incubated at 37˚C in agitation in presence of ONPGsubstrate (and in its absence as internal control). Under theseconditions, the beta-galactosidase embedded in inclusion bodiesefficiently hydrolyses ONPG (Figure 1A), while no product appearsin absence of the substrate (Figure 1B). To quantify the activityremaining in inclusion bodies and that eventually present in the

soluble fraction, during the incubation of this aggregates withONPG, samples were taken at two points (t2 min and t30 min) andafter centrifugation, the supernatant and the pellet were used for asecond enzymatic analysis with CPRG as substrate (Figure 2) [1, 2].The amount of protein was also quantified in both soluble andinsoluble fractions at the times chosen (Table 1).


(a) Effect of NaCl concentration on the folding efficiency of -amylase inclusion bodies dissolved in 8 M urea. Refolding buffer: 20 mM Tris-HCl pH 7.4with salt. (b) Effect of calcium concentration on the folding efficiency of -amylase inclusion bodies dissolved in 8 M urea. Refolding buffer: 20 mM Tris-HCl pH 7.4, 3 M NaCl and calcium.


A. Product formed by inclusion bodies (quadruplicate) through ONPG hydrolysis as determined at 414 nm. B. Control (inclusion bodies withoutONPG).



The results obtained, comparing the samples incubated with andwithout ONPG (named control in Figure 2), suggest that thepresence of substrate in the suspension might positivelyinfluence the solubilisation of the aggregated protein.Conclusion: We could conclude that, interestingly, when anenzyme aggregated as inclusion bodies is incubated with itssubstrate, part of this protein might be spontaneously solubilisedin a process that seems to be eventually favoured by thepresence of substrate. Moreover, this soluble protein showsconsiderable enzymatic activity that is a major contributor tothe enzymatic process initiated by inclusion bodies.AcknowledgementsThis work has been funded by BIO2004-00700 from MEC, Spainand 2005SGR-00956 (AGAUR). Elena Garcıa-Fruitos is recipientof a doctoral fellowship from MEC, Spain.References1. Garcıa-Fruitos E, Gonzalez-Montalban N, Morell M, Vera A,

Ferraz RM, Arıs A, Ventura S and Villaverde A: Aggrega-tion as bacterial inclusion bodies does not implyinactivation of enzymes and fluorescent proteins.Microb Cell Fact 2005, 4:27.

2. Ferraz RM, Aris A and Villaverde A: Profiling theallosteric response of an engineered beta-galactosi-dase to its effector, anti-HIV antibody. Biochem BiophysRes Commun 2004, 314:854–860.

P15Production of recombinant mink growth hormonein E. coliJolanta Sereikaite1, Alina Statkute1, Mindaugas Morkunas1,Vitaliano Borromeo2, Camillo Secchi2 and Vladas-Algirdas Bumelis11Department of Chemistry and Bioengineering, VilniusGediminas Technical University, Vilnius, Lithuania2Department of Veterinary Pathology, Hygiene and Health,Biochemistry and Physiology Unit, University of Milan, Italy


Background: Growth hormones are produced by the ante-rior pituitary gland of vertebrates. Apart from stimulating linear

body growth, it plays an important role in variety of metabolicand physiological processes. There is almost no information onmink growth hormone (mGH), which probably could be a usefulfactor in mink fur production, synthesis in E.coli and futurerecovery of bioactive protein. It is known that mGH gene wascloned and sequenced [1, 2]. We report the possibility toproduce large amounts of mGH using recombinant DNAtechnology.Results: The host strain E. coli BL21(DE3) harbouring theplasmid pET21a+/mGH was grown in batch fermentationprocess at 37˚C using nutrient rich medium to produce mGH.The target protein expression was induced with 0.2 mM or 1mM of isopropyl-�-D-thiogalactoside at OD600 of 2.0. In bothcases after 3 hours of induction the expression level was similarand equal to 23% and 27% of the total cellular protein,respectively. mGH when overexpressed in E. coli accumulatedas inclusion bodies. After cell disruption by sonication inclusionbodies were purified by washing with water, then with cleaningsolution (2 M urea, 1 M NaCl, 5 mM EDTA, 1 mM PMSF in 0.1 MTris-HCl buffer pH 9.0) and once more with water. The washedinclusion bodies were found to contain approximately 80% ofmGH. 8 M urea solution was used for its solubilization. mGHwas refolded by dilution protocol in the presence of glutathionepair. The mGH conformational state was analyzed by RP-HPLC.Two-step purification process comprising of ion-exchangechromatography on Q-Sepharose and hydrophobic chromato-graphy on Phenyl-Sepharose was developed. The biologicalactivity of the purified mGH was assessed in vitro using a mousemyeloid cell line transfected with the full length ovine GHreceptor [3]. Stimulation of cells was assessed using the MTT-formazan dye assay, that monitors both metabolic and mitogenicactivity. 25–30 mg of highly purified and biologically active mGHwas obtained from 4 g of biomass.


A. Product formed by soluble fraction (A) and inclusion bodies (B) through CPRG hydrolysis as determined at 540 nm.

Table 1 (abstract P14)

Protein (%) Inclusion bodies Soluble

t2 min t30 min t2 min t30 minsamples 100 28 <1 72



Conclusion: The method presented here allows producinglarge quantities of mGH and considering initiation of scientificinvestigations on mGH availability in fur industry.References1. Harada Y, Tatsumi H, Nakano E and Umezu M: Cloning

and sequence analysis of mink growth hormonecDNA. Biochem Biophys Res Commun 1990, 173:1200–1204.

2. Shoji K, Ohara E, Watahiki M and Yoneda Y: Cloning andnucleotide sequence of a cDNA encoding the minkgrowth hormone. Nucleic Acids Res 1990, 18:6424.

3. Beattie J, Phillips K and Borromeo V: Differentialinhibition of recombinant bovine GH (rbGH) activ-ity in vitro by in vivo enhancing monoclonal anti-bodies. Mol Cell Biochem 2001, 220:103–108.

P16Comparative analysis of E. coli inclusion bodiesand thermal protein aggregatesNuria Gonzalez-Montalban1,2, Elena Garcıa-Fruitos1,2,Salvador Ventura1,3, Anna Arıs1,2

and Antonio Villaverde1,21Institut de Biotecnologıa i de Biomedicina, UniversitatAutonoma de Barcelona, Bellaterra, 08193 Barcelona, Spain2Departament de Genetica i de Microbiologia, UniversitatAutonoma de Barcelona, Bellaterra, 08193 Barcelona, Spain3Departament de Bioquımica i de Biologıa Molecular,Universitat Autonoma de Barcelona, Bellaterra, 08193Barcelona, Spain


Background: In bacteria, inclusion bodies are commonlyobserved during overexpression of plasmid-encoded recombi-nant genes, and represent a great matter of concern inbiotechnology [1]. Bacterial inclusion bodies are also connectedto the protein quality control [2] and to the prevention ofcytotoxicity associated to aberrantly folded proteins [3, 4]. Onthe other hand, these protein aggregates are dynamic struc-tures, since they grow as the result of an unbalanced equilibriumbetween protein deposition and removal [5, 2]. Therefore,there is not any physiological evidence of bacterial inclusionbodies being structures well organized to facilitate embedded

protein removal by chaperones or proteases. We havecomparatively analyzed the molecular organization and dyna-mism of a recombinant E.coli �-galactosidase and its derivativeVP1LAC [6] when either deposited as inclusion bodies or asaggregates resulting from in vivo thermal denaturation in alaboratory wild type strain E.coli MC4100 and its derivativesDnaK�and GroEL44 (namely JGT20 and BB4565, respectively).The expression of both lacZ and VP1LAC genes is triggered bytemperature up shift from 28˚C to 42˚C.Results: A small part of the recombinant �-galactosidasepresent in the cell (~5%) was found in the insoluble cell fractionas a result of a heat shock at 42˚C and remained nearly constantduring the 3-hours heat shock. However, a progressively higherfraction of VP1LAC (up to 45% at 3 h) occurred as inclusionbodies (data not shown). Nevertheless, this compositionalevolution was parallel to a structural evolution (see Figure 1)since polypeptides embedded in inclusion bodies undergo acontinuous formation of extended, intermolecular �-sheetstructure. This was deduced from the evolution of the bandsapproximately 1627 cm�1 and 1692 cm�1 relative to that at1652 cm�1. On the other hand, recombinant �-galactosidaseonly represents around 3% of the protein species found ininsoluble fraction, while VP1LAC accounted for 90% of theinclusion body material. In fact, inclusion bodies were enrichedwith VP1LAC species, especially in those native-like forms (seeFigure 1) peaking approximately at 1638–1640 cm�1.The formation of �-galactosidase thermal aggregates andVP1LAC inclusion bodies was explored in absence of eitherthe main cytoplasmatic chaperones DnaK (JGT20) and GroEL(BB4565). As expected (see Table 1), the soluble �-galactosidasewas more active than the soluble engineered version VP1LAC.Despite this fact, protein aggregated as inclusion bodies wasmuch more active (from 2 to 8 fold) than that occurring inthermal aggregates (up to 10 fold in wild type cells), indicating ahigher occurrence of properly folded protein. While, GroELseems to be fairly relevant, this event it is clearly depending onDnaK, as in JGT20, insoluble VP1LAC is less active thaninsoluble �-galactosidase.Conclusion: Thermal denaturation of �-galactosidase resultsin the formation of heterogeneous aggregates that are ratherstable in composition during the heat shock stress. On the


FTIR of -galactosidase aggregates (left) and VP1LAC inclusion bodies (right) formed during 1 hour (continuous), 3 hours (dotted) or 5 hours (dashed).



contrary, protein deposition as inclusion bodies rendershomogeneous but strongly evolving structures. In this context,the specific activity of enzyme-based inclusion bodies is muchhigher than in the equivalent thermal aggregates, by a mechanismthat might be controlled by the chaperone DnaK. Proteindeposition as inclusion bodies is then a cell driven complexprocess through which misfolded protein forms but alsofunctionally competent polypeptides are efficiently packaged.AcknowledgementsThis work has been funded by BIO2004-00700 from MEC, Spainand 2005SGR-00956 (AGAUR). Nuria Gonzalez-Montalban andElena Garcıa-Fruitos are recipients of doctoral fellowships fromMEC, Spain, and Salvador Ventura is supported by a "Ramon yCajal" project awarded by the MCYT an co-financed by theUniversitat Autonoma de Barcelona.References1. Baneyx F and Mujacic M: Recombinant protein folding

and misfolding in Escherichia coli. Nat Biotechnol 2004,22:1399–1408.

2. Carrio MM and Villaverde A: Construction and decon-struction of bacterial inclusion bodies. J Biotechnol2002, 96:3–12.

3. Hunke S and Betton JM: Temperature effect oninclusion body formation and stress response inthe periplasm of Escherichia coli. Mol Microbiol 2003,50:1579–1589.

4. Gonzalez-Montalban N, Carrio MM, Cuatrecasas S, Aris Aand Villaverde A: Bacterial inclusion bodies arecytotoxic in vivo in absence of functional chaperonesDnaK or GroEL. J Biotechnol 2005, 118(4):406–12.

5. Carrio MM and Villaverde A: Protein aggregation asbacterial inclusion bodies is reversible. FEBS Lett 2001,489:29–33.

6. Corchero JL, Viaplana E, Benito A and Villaverde A: Theposition of the heterologous domain can influencethe solubility and proteolysis of beta-galactosidasefusion proteins in E. coli. J Biotechnol 1996, 48:191–200.

P17Point mutation of serine 179 in the humanProlactin (PRL) affects recombinant proteinexpression, folding and secretion, abolishes PRLnickel (II)-binding and increases heparin bindingcapacitiesEric Ueda1, Carlos Soares1, Ameae Walker2

and Paolo Bartolini11Biotechnology Department, IPEN-CNEN, CidadeUniversitaria, Sao Paulo, Brazil

2Division of Biomedical Sciences, The University of California,Riverside, CA, 92521, USA


Background: S179D prolactin (S179D PRL) is a pseudopho-sphorylated form of human prolactin (PRL) which has inhibitoryeffects on tumor growth [1] and angiogenesis [2]. The S179DPRL preparations used for these experiments consisted ofproperly refolded inclusion bodies (IB) from Escherichia coli [3].Trying to attain a better folded mutant, we used secretionexpression based systems. However, single point mutations canaffect protein periplasmic expression [4], and secretion frommammalian cells [5]. We observed that upon a mutation ofSerine 179 to an Aspartate, expression was nearly abolishedwhen compared with PRL in E. coli periplasm, while thecytoplasmic product was more prone to proteolysis. Usingeukaryotic cells we were able to produce preparationscomparable to IBs in terms of bioactivity. We also demonstratedthat this mutant had a higher affinity for heparin and lowerbinding capacity towards divalent metals (M (II)).Results: S179D PRL periplasmic expression was very lowwhen compared to PRL. Use of different promoters, differentsignal peptides or different activation temperatures had noeffect (Figure 1).MALDI-TOF spectrometry was carried out for identity ofS179D PRL in the extracts (Figure 2).BL21 strain was used (Figure 1B) without improvements forS179D PRL expression (Table 2).We used BL 21 codon plusâ in order to investigate the GC-,AT-rich sequence of the PRLs influence on expression. Thisstrain did not rescue expression of S179D PRL or PRL (Figure1C). pTac induction at lower temperatures should encourageprotein solubility and folding in the cytoplasm [6]. We carriedout cytoplasmic expression with an Origami B strain, in whichcytoplasm folding is facilitated [7]. Surprisingly, when S179D PRLwas produced in soluble form, unlike PRL, low molecular formswere observed (Figure 3A and 3B), and also in BL21, cleavedforms and soluble high molecular aggregates were present(Figure 3A). pL constructs had very low yields for both PRLs(Figure 3).An eukaryotic expression system was chosen to successfullyproduce soluble, monomeric, recombinant S179D PRL.B-casein bioassays were carried out to check S179D PRL folding.(Figure 5).Moreover S179D PRL had a decreased affinity towards Ni (II)ans Zn (II). On the other hand it had an increased affinitytowards heparin.

Table 1 (abstract P16) Specific activity (in U/ng) of -galactosi-dase and its derivative VP1LAC produced in different strains, inthe soluble and insoluble fractions.

Strain Solublefraction Insolublefraction

MC4100/pJCO46 628.2 ± 40.5 6.3 ± 0,3MC4100/pJVP1LAC 234.1 ± 52.9 65.2 ± 19,4BB4565/pJCO46 689.7 ± 164.9 63.6 ± 2,2BB4565/pJVP1LAC 230.2 ± 25.7 129.6 ± 45,9JGT20/pJCO46 888.9 ± 179.3 175.2 ± 34,9JGT20/pJVP1LAC 12.5 ± 3.8 10.3 ± 6.3

Table 1 (abstract P17) Protein expression yield (g/mL/OD) andfinal optical densities (OD600) of different strains with pLpromoter.

E. colistrain Protein yield(g/mL/OD)

Final OD600

PRL W3110 1.3 ± 0.2 4.0 ± 0.3BL21 1.9 ± 0.4 1.3 ± 0.2

BL21 codon plus 1.4 ± 0.3 1.0 ± 0.2S179D PRL W3110 0.34 ± 0.03 3.8 ± 0.6

BL21 0.35 ± 0.5 1.3 ± 0.5BL21 codon plus 0.40 ± 0.3 1.2 ± 0.1



Conclusion: We tried to produce a correctly folded form ofS179D PRL, already obtained as refolded IBs [3]. Unexpectedly,this point mutation of PRL impaired protein expression, and wasnot related to the strain, protease degradation of our protein,or preferential codon usage (Figure 1). To avoid proteolysis andmisfolding we used lower temperatures during protein produc-tion [8], but it failed to produce S179D PRL. Low levels ofS179D PRL were only detected by immunoblots (Figure 1) andby immunoassay (table 1). Expression of soluble S179D PRL inthe cytoplasm of E.coli was not efficient either, as denoted bysoluble aggregates and cleaved S179D PRLs. Eukaryotic expres-sion systems have a better folding machinery, being difficult-to-fold proteins more easily expressed [9]. Thus, we successfullyproduced S179D PRL at RP-HPLC detectable levels (Figure 4).MALDI-TOF analysis showed that all samples had the expectedmolecular weight (Figure 2). RP-HPLC demonstrated thatS179D PRL had a different folding than PRL. The bioactivityassay showed that all preparations of S179D PRL were correctlyfolded. S179D PRL also showed physical-chemical differences,having a lower M (II)-affinity and a higher heparin-affinity. Thisconfirms reports of PRL mutants with low Zn (II) affinity that arepoorly secreted [4] and also could account for its anti-angiogenic effect [2, 10].

References1. Xu X, Wu W, Williams V, Khong A, Chen YH, Deng C and

Walker AM: Opposite effects of unmodified prolactinand amolecularmimic of phosphorylated prolactin onmorphology and the expression of prostate specificgenes in the normal rat prostate. Prostate 2003, 54:25–33.

2. Ueda E, Ozerdem U, Chen YH, Yao M, Huang KT, Sun H,Martins-Green M, Bartolini P and Walker AM: Amolecular mimic demonstrates that phosphory-lated human prolactin is a potent anti-angiogenichormone. Endocr Relat Cancer 2006, 13:95–111.

3. Chen TJ, Kuo CB, Tsai KF, Liu JW, Chen DY andWalker AM: Development of recombinant humanprolactin receptor antagonists by molecular mimi-cry of the phosphorylated hormone. Endocrinology1998, 139:609–16.

4. Duenas M, Ayala M, Vazquez J, Ohlin M, Soderlind E,Borrebaeck CA and Gavilondo JV: A point mutation in amurine immunoglobulin V-region strongly influ-ences the antibody yield in Escherichia coli . Gene1995, 158:61–6.

5. Sun Z, Lee MS, Rhee HK, Arrandale JM and Dannies PS:Inefficient secretion of human H27A-prolactin, a


A, B and C, Immunoblots of periplasmic extracts.



mutant that does not bind Zn2+. Mol Endocrinol 1997,11:1544–51.

6. Reyes LF, Sommer CA, Beltramini LM and Henrique-Silva F: Expression, purification, and structuralanalysis of (HIS)UBE2G2 (human ubiquitin-conju-gating enzyme). Protein Expr Purif 2006, 45:324–8.

7. Bessette PH, Aslund F, Beckwith J and Georgiou G:Efficient folding of proteins with multiple disulfidebonds in the Escherichia coli cytoplasm. Proc Natl AcadSci USA 1999, 96:13703–8.

8. Makrides SC: Strategies for achieving high-levelexpression of genes in Escherichia coli . Microbiol Rev1996, 60:512–38.

9. Ellgaard L and Helenius A: Quality control in theendoplasmic reticulum. Nat Rev Mol Cell Biol 2003,4:181–91.

10. Ricard-Blum S, Feraud O, Lortat-Jacob H, Rencurosi A,Fukai N, Dkhissi F, Vittet D, Imberty A, Olsen BR and vander Rest M: Characterization of endostatin bindingto heparin and heparan sulfate by surface plasmonresonance and molecular modeling: role of divalentcations. J Biol Chem 2004, 279:2927–2936.

P18Models for the study of inclusion bodies formationas a function of fermentation conditions andprotein sequencePietro Gatti-Lafranconi1, Diletta Ami1,Antonino Natalello1, Gaetano Invernizzi1, Ario deMarco2, Silvia Maria Doglia1 and Marina Lotti11Department of Biotechnology and Biosciences, University ofMilano-Bicocca, Piazza della Scienza 2, 20126 Milano, Italy2EMBL Scientific Core Facilities, Mayerhofstrasse 1, D-69117,Heidelberg, Germany


Background: The building of aggregates of variable complexity isoften observed in bacterial host cells upon over-expression ofrecombinant proteins. This event is thoroughly studied as it impactson the production of recombinant proteins and also as a model toinvestigate the molecular and physiological factors producing proteinaggregation in living cells [1]. In E. coli, a network of molecularchaperones assists protein folding and re-folding [2]. Recentexperiments showed that aggregation reversion can be improvedas protein synthesis is interrupted and that the ratio protein/


Molecular masses determined by MALDI-TOF-MS.



chaperons as well as the kind of chaperons enclosed in inclusionbodies varies according to the physiology of overproduction [1, 3, 4].Stability, solubility and propensity of a protein to aggregate bothin inclusion bodies and in amyloid structures have been relatedto its polypeptide sequence [5]. This study aims at gaining adeeper insight in the composition and kinetics of aggregateformation and to relate this information to the molecularfeatures of the recombinant expressed proteins.Results: We have studied the behaviour of three differentproteins over-expressed in E. coli by commercial expressionvectors regulated by IPTG. The three model polypeptidesdisplay different features: (i) the cold active lipase fromPseudomonas fragi is very unstable even at moderate tempera-ture and, therefore, is a very sensitive tool to investigate thetemperature-dependent aggregation development [6]; (ii) thegreen fluorescent protein-glutatione S- transferase fusionprotein enables to monitor residual fluorescence in aggregatedproteins and, as a consequence, to evaluate the extent ofresidual native structure; (iii) wild type and mutated lactoglo-bulins have been used as a probe to test the effect of changes inthe amino acid sequence on the protein stability duringrecombinant expression. Ratio of soluble to insoluble proteinshas been evaluated by SDS PAGE and activity has been measuredin both fractions, whenever applicable. Additional information

has been provided by structural analysis performed by Fouriertransform infrared spectroscopy. In all the models thefermentation temperature has been identified as a majordeterminant of the total amount and rate of aggregation aswell as of the complexity, compactness and residual native- likestructure of inclusion bodies [4, 5, 6, 7]. Moreover, concentra-tion of DnaK inside inclusion bodies has been followed byWestern-blot analysis and a correlation with the amount ofinsoluble protein has been detected.Conclusion: Kinetics of aggregation, content and residualprotein structure and activity in E. coli inclusion bodies arefeatures highly protein specific but also dependent on theconditions under which aggregation occurred. This last observa-tion suggests that the precise monitoring of recombinant proteinaggregation during the fermentation can lead to a system in whichthe optimisation of the growth conditions is automatically setwith the biotechnologically relevant increase of soluble proteinyields. Furthermore, the monitoring of the aggregation dynamicsspecific for the different mutants might provide valuableindications to engineer species with higher solubility.References1. Baneyx F and Mujacic M: Recombinant protein folding

and misfolding in Escherichia coli. Nat Biotechnol 2004,22(11):1399–1408.


Immunoblots of soluble fractions of E. coli lysates.



2. Deuerling E and Bukau B: Chaperone-assisted folding ofnewly synthesized proteins in the cytosol. Crit RevBiochem Mol Biol 2004, 39:261–277.

3. Carrio MM and Villaverde A: Construction and decon-struction of bacterial inclusion bodies. J Biotechnol2002, 96(1):3–12.

4. Schrodel A and de Marco A: Characterization of theaggregates formed during recombinant proteinexpression in bacteria. BMC Biochem 2005, 6:10.

5. Ventura S: Sequence determinants of protein aggre-gation: tools to increase protein solubility. Microb CellFact 2005, 4:11.

6. Ami D, Natalello A, Gatti-Lafranconi P, Lotti M andDoglia SM: Kinetics of inclusion body formationstudied in intact cells by FT-IR spectroscopy. FEBSLett 2005, 579(16):3433–3436.

7. Ami D, Natalello A, Taylor G, Tonon G and Doglia SM:Structural analysis of protein inclusion bodies byFourier transform infrared microspectroscopy. Bio-chim Biophys Acta 2006 in press.

P19Addition of Repressor in inducible promotersystem improves soluble expression ofrecombinant protein in E. coliKyung-Hwan JungDepartment of Food and Biotechnology, Chungju NationalUniversity, Chungju 380-702, Chungbuk, Korea


Immunoblot of conditioned medium. B, C, RP-HPLC analysis.


-casein bioassay. * p < 0.05 versus control; ** p < 0.01 versus control.AU, arbitrary unit.




Background: Insoluble protein aggregate (inclusion body) isfrequently accumulated during the heterologous protein expressionby the bacterial inducible promoter system. In this study, althoughmany reports have proposed the methodology to circumvent theaggregate formation [1, 2, 3], we tried to control the transcriptionrate by an addition of the repressor for inducible promoter. Theaddition of repressor was tried just after the inducer was added, inorder to increase the soluble expression level.Results: To improve the soluble expression level of recombi-nant interferon-alpha (IFN-alpha) in E. coli, repressor (glucose)was added after induction. In this system, arabinose-induciblepromoter (pBAD) controlled the transcription of IFN-alphagene. The fractionation of soluble and insoluble part of theinduced E. coli by B-PERII solution (Pierce) showed that glucoseaddition after induction resulted in improvement of the solubleexpression, otherwise IFN-alpha was expressed mostly ininsoluble portion (see Figure 1). Finally, over 60% of the totalprotein expression was found in the soluble fraction of total celllysate. Probably this principle might be able to apply to otherheterologous protein expression which is prone to a proteinaggregate formation in the cytoplasm.Conclusion: The glucose (repressor) addition improved thesoluble expression level in arabinose-inducible promoter systemin E. coli.This principle might be able to apply to a heterologous proteinexpression which is prone to a protein aggregate formation inthe cytoplasm.References1. Baneyx F: vivo folding of recombinant proteins in

Escherichia coli. Manual of Industrial Microbiology andBiotechnology Demain AL, et al 1999, ASM.

2. Swartz JR: Advances in Escherichia coli production oftherapeutic proteins. Curr Opin Biotechnol 2001, 12:195–201.

3. Sorensen HP and Mortensen KK: Soluble expression ofrecombinant protein in the cytoplasm of Escherichiacoli. Microb Cell Fact 2005, 4:1.

P20Production of proteins in Bacillus subtilis can beimproved by engineering components affectingposttranslocational protein folding anddegradationMarika Vitikainen, Hanne-Leena Hyyrylainen,Anna Kivimaki, Vesa P Kontinen and Matti SarvasLaboratory of Infection Pathogenesis, National Public HealthInstitute, Mannerheimintie 166, FIN-00300 Helsinki, Finland


Background: Bacillus species have a high capacity to secretetheir own proteins into the extracellular medium and aretherefore considered as attractive hosts for producing hetero-logous proteins. However, the secretion is often inefficientsuggesting that there are bottlenecks in the secretion pathway.This research focuses on the posttranslocational events inprotein secretion. Three putative bottleneck components (seeResults) affecting the extracytoplasmic protein folding anddegradation were genetically engineered and the effects on theprotein secretion were studied [1].Currently we are participating in EuroSCOPE program onScience of Protein Production for Functional and StructuralAnalysis. The project will continue to further develop andexploit B. subtilis as a host especially for the production of


Western blot analysis; (A) Soluble and insoluble expression of recombinant interferon-alpha by arabinose induction (0.05%), (B) Soluble and insolubleexpression of recombinant interferon-alpha by arabinose induction (0.05%) and 0.5% glucose addition after induction.




The change of soluble and insoluble fraction after arabinose induction. Glucose (repressor) was added after induction (,). In other two cases (,), glucosewas not added. Fraction was obtained from image analysis of Figure 1.



protein complexes and membrane proteins. The aim is to findout balanced sets of quality control and protein folding factorsto be able to optimise protein production. In the EuroSCOPEprogram membrane proteins of B. subtilis as well as gram-positive pathogen representing potential vaccines and targets fornovel antimicrobial drugs are used as model proteins. Theproject has been initiated by producing a membrane protein B.subtilis, histidine kinase YxdK.Results: The first component modulated is the amount ofPrsA lipoprotein on the outer surface of the cytoplasmicmembrane. PrsA is an essential membrane-bound folding factorwith peptidyl prolyl isomerase activity. Previous studies haveidentified that secretion of some exoproteins is dependent onthe PrsA lipoprotein. Dependency of model proteins on thePrsA was studied by lowering the PrsA level below the wild typelevel. The effect of PrsA overproduction was also investigated.The second component is the increase of the net negativecharge of the cell wall generated by mutating the dlt operon andconsequently preventing the D-alanine substitution of anioniccell wall polymers. The third component modulated is the levelof HtrA-type quality control proteases at the membrane-cellwall interface that serve as cleaning proteases to degradeaccumulated and misfolded proteins. The effect on the secretionof 11 industrically interesting heterologous proteins was studiedby western blotting and enzymatic assays [1]. The results areshown in table 1. The secretion of four of proteins wasdependent on the PrsA lipoprotein and the overproduction ofPrsA enhanced the secretion of two of them, a-amylase of B.stearothermophilus (4-fold) and pneumolysin (1.5-fold). Themutation in the dlt operon enhances the secretion of oneprotein, pneumolysin, about 1.5-fold. Decreasing the level ofHtrA proteases caused harmful effects on growth and did notenhance secretion. Pertussis toxin subunit S1 was found to be asubstrate for HtrA-type proteases and its secretion wasdependent on these proteases.Conclusion: In this research a fairly large number of modelproteins were used allowing us to make more generalconclusions on how secretion of heterologous protein in B.subtilis can be improved. Results indicate that some hetero-logous proteins are secreted at enhanced levels when compo-nents involved in the late stages of protein secretion aremodulated. PrsA lipoprotein and its overproduction are goodcandidates to be tested when optimising the secretion of anexoprotein in B. subtilis. Modulation of the net charge of the cell

wall may also have role in biotechnological applications.However lowering the level of HtrA proteases does not seemto be a means to enhance protein secretion.Reference1. Vitikainen M, Hyyrylainen HL, Kivimaki A, Kontinen VP and

Sarvas M: Secretion of heterologous proteins inBacillus subtilis can be improved by engineering cellcomponents affecting posttranslocational proteinfolding and degradation. J Appl Microbiol 2005, 99:363–375.

P21Production of cysteine-rich proteinsin E. coli – the challenge of WntsAnu Mursula, Ulf Liebal and Peter NeubauerBioprocess Engineering Laboratory, Department of Processand Environmental Engineering and Biocenter Oulu, Universityof Oulu, P.O. Box 4300, FIN-90014 Oulu, Finland


Background: The Wnt family consists of secreted extracel-lular glycoproteins that induce an intracellular signaling pathwayinvolved in many events during embryogenesis and adult tissuemaintenance. As they are involved in a large variety of cellularprocesses, errors in the Wnt-signaling pathways can lead forexample to degenerative diseases and cancer and in embryos todevelopmental defects. So far there are 19 genes encoding forWnt-proteins found in both mouse and human. Wnt-proteinsare highly conserved, both with each other as well as betweenspecies. They all consist of about 350–400 amino acids, havemultiple cysteines, are hydrophobic (possibly due to a conservedpalmitoylation) and have a high pI.Recombinant Wnt proteins are needed as important tools forexample in basic research concerning developmental biology andWnt function as well as for medical applications (e.g.therapeutics, stem cells). However, production of recombinantWnt proteins in E. coli is very challenging, because theirhydrophobicity and several disulfide bonds make their properfolding extremely difficult. In this study, strategies for theexpression of Wnt proteins in E. coli are investigated.Results: A process for production of active murine Wnt1 inE. coli has recently been developed in our laboratory [1]. Thiswas the first time a Wnt protein has been produced in an activeform using a prokaryotic expression system. Here Wnt1 was

Table 1 (abstract P20) Effect of PrsA, dlt mutation and depletion of HtrA proteases on secretion of heterologous proteins studied.

Protein and origin PrsA dependency PrsA Over- expression Dlt mutation Depletion of HtrA protease

-amylase (B. stearothetmophilus) yes(+) no effect-glucanase (B. licheniformis) no no effectPenicillinase (B. licheniformis) no no effect no effectPneumolysin (S. pneumoniae) yes(+)Diphteria toksoid (C. diphteriae) no no effectStaphylokinase (S. aureus) yes(-)Pectinase (E. carotovora) yes(+) no effectPectin methylesterase (E. chrysanthemi) no no effect no effect-lactamase (E. coli) yes(+) no effectPertussis toxin S1 (B. pertussis) noPertussis toxin S4 (B. pertussis) no

yes(+), positive dependency, yes(-) negative dependency, , increase in secretion and , decrease in secretion.



targeted to the periplasmic space which has more oxidativeconditions for proper folding than the bacterial cytoplasm.Currently, processes for the expression of other Wnt proteinsare developed.Two different expression strategies are being followed. First,since the folding in the bacterial cytosol for disulfide bond-containing proteins is very difficult, one option is to let therecombinant proteins aggregate as inclusion bodies and refoldthem later in vitro. Advantages of using inclusion bodies forexpression include for example larger yield of recombinantprotein and fewer purification steps.The second strategy utilizes cytoplasmic expression andthioredoxin reductase deficient host strains. In these hoststrains the cytoplasm is more oxidative than in wild type E. colifacilitating disulfide bond formation and folding.For both strategies some encouraging preliminary results havebeen obtained. The expression conditions are currentlyoptimized followed by protein purification (and refolding) aswell as activity tests.AcknowledgementsThis work is financed by the TEKES NEOBIO program andAcademy of Finland.Reference1. Fahnert B, Veijola J, Roel G, Karkkainen MK, Railo A,

Destree O, Neubauer P and Vainio S: Murine Wnt-1 withan internal c-myc tag recombinantly produced inEscherichia coli can induce intracellular signaling ofthe canonical Wnt pathway in eukaryotic cells. J BiolChem 2004, 279:47520–47527.

P22Modulation of inclusion body (IB) formationkinetics by different induction regimes in E. colifed batch cultivationsMonika Cserjan-Puschmann, Franz Clementschitsch,Gerald Striedner, Florentiner Potschacher, Jurgen Kernand Karl BayerDepartment of Biotechnology, Institute of AppliedMicrobiology, University of Natural Resources and Applied LifeSciences, Vienna, Austria


Background: Heterologous protein expression often resultsin the accumulation of non-native insoluble, quite homogenousprotein deposits inside bacterial cells, called inclusion bodies(IBs) [1, 2]. Currently there is a strong trend to favour IBformation in industrial recombinant protein production pro-cesses, because IBs are a source of relatively pure and stabilisedproteins. Therefore, the major objectives of process optimisa-tion are to achieve a controllable, stable and nearly complete IBformation and IBs of high quality.IB formation is a complex multifactorial synthetic pathway(transcription, translation, folding, aggregation) that depends onthe specific host/vector system, the intrinsic characteristics ofthe protein, the physiology of the host cell and the processoperation (cultivation temperature, growth rate, media compo-sition, etc). However, the precise mechanisms of the in-vivoprotein aggregation process remain poorly understood. Thebalance between heterologous and host protein production rateplays a fundamental role in IB formation. Therefore, varying

metabolic loads gained by different induction regimes are appliedto control kinetics of IB formation.Host/vector system: E. coli HMS174(DE3) pET30a producing acodon-optimised GFP fusion protein (47 kDa) showing strongtendency to form IBs. To investigate IB formation kinetics aseries of exponential carbon-limited fed-batch cultivations wereperformed at different induction levels of recombinant proteinexpression. Transcription rate control was achieved bycontinuous supply of limiting amounts of the inducer (IPTG) ina constant ratio to biomass [3]. During cultivation yieldof recombinant protein, composition of IBs, distribution ratioof soluble and aggregated proteins, IB quality in consideration ofthe subsequent downstream procedures, cellular growth(BDM), total cell number (TCN) were monitored. In additionthe metabolic load on the host cells exerted by different inducerconcentrations was quantified by the signal molecule ppGpp [4].Results: In all cultivations of this model protein the impact ofinducer level on the ratio of soluble and aggregated proteins wasnot significant, more than 96% of the total recombinant proteinwas deposited in IBs showing low concentrations of host cellproteins.The key results of this series of fed batch cultivationsdemonstrated that the specific content of recombinant proteinincreases according to the inducer concentrations, whereby ahigher yield of total biomass is obtained under low inducingconditions. The outcome of these experiments is thatapproximately the same total amount of recombinant proteincould be produced whereby the IB formation rate showedstrong differences.In addition to the target protein (47 kDa) fragments of it with alength of about 20–35 kDa as confirmed by Western blots wereobserved. This inhomogeneity caused by incorrect proteintranslation account up to 40% of the expressed recombinantprotein at the end of cultivation in particular at high inductionlevels. These results clearly demonstrate the benefial impact oflower metabolic burden on increased IB quality.The varying induction levels differently affected the host cellsystem. During a non-induced cultivation the cells are exponen-tially growing and the ppGpp level is constant. Due to thesynthesis of recombinant protein stringent response wastriggered and in succession the cells lost their capability todivide in relation to the metabolic burden. IB formation kineticscan be classified into three phases: a growth associated productformation (phase I), a partial growth decoupled pase (II) withoutfurther cell division and a complete growth decoupled IBformation (phase III), where the recombinant protein isexpressed at the expense of the cellular protein. However,cell viability could be maintained at low induction levels thatcause a tolerable metabolic load.Conclusion: The performed experiments showed close inter-relationships between the level of induction, cellular growth, IBformation with respect to metabolic load. In summary it can besaid that the inducer feed strategy that permits tuning ofrecombinant gene expression in relation to the metabolicpotential of the host cell synthesis machinery is a valuable toolto attain maximal yield and quality of IBs. In future the alterationof the host cell physiology due to different induction regimes willbe assayed using genomic and proteomic analysis.AcknowledgementsThis work was supported by the Austrian Center of Biophar-maceutical Technology. http://www.acbt.at



References1. Baneyx F and Mujacic M: Recombinant protein folding

and misfolding in Escherichia coli. Nat Biotechnol 2004,22:1399–1408.

2. Villaverde A and Cario MM: Protein aggregation inrecombinant bacteria: biological role of inclusionbodies. Biotechnol Lett 2003, 25(17):1385–1395.

3. Striedner G, Cserjan-Puschmann M, Potschacher F andBayer K: Tuning the transcription rate of recombi-nant protein in strong Escherichia coli expressionsystems through repressortitration. Biotechnol Prog2003, 19:1427–1432.

4. Cserjan-Puschmann M, Kramer W, Durrschmid E,Striedner G and Bayer K: Metabolic approaches forthe optimisation of recombinant fermentation.Microbiol Biotechnol 1999, 53:43–50.

P23Nonclassical inclusion bodies in Escherichia coliSpela Peternel1, Marjan Bele2, Vladka Gaberc-Porekar1

and Viktor Menart1,31Laboratory for Biosynthesis and Biotransformation, NationalInstitute of Chemistry, Ljubljana, Slovenia2Laboratory for Materials Electrochemistry, National Instituteof Chemistry, Ljubljana, Slovenia3Biopharmaceuticals, Lek Pharmaceuticals d.d., Ljubljana, Slovenia


Background: Formation of inclusion bodies (IBs) during over-expression of recombinant proteins in Escherichia coli is ofcommon occurrence. The target protein inside inclusion bodiesis usually misfolded and a series of denaturation/renaturationsteps is necessary for isolation of biologically active protein.Purification protocol must be optimized case by case. Due tolow efficiency of most denaturation/renaturation procedures alot of trials have been performed to obtain soluble and properlyfolded target proteins inside E. coli cytoplasm with the aim ofincreasing the yield of the target protein.In our laboratory we are exploring another approach based on ourrecent observation, that Granulocyte Colony-Stimulating Factor (G-CSF), when produced in E. coli at lower temperature (around 25˚C),forms inclusion bodies containing large amount of correctly foldedprotein precursor [1]. Such inclusion bodies have remarkablydifferent properties than the classical inclusion bodies therefore weproposed the term ’’nonclassical inclusion bodies’’ (ncIBs). Here wewould like to expose some typical characteristics of the ncIBs.Most of our studies were performed on G-CSF but similar resultswere also found for His7dN6TNF-a(His-tagged, N-terminallytruncated form of tumor necrosis factor) and GFP (greenfluorescent protein) indicating that the phenomenon of ncIBs is ofbroader occurrence.Results: Nonclassical inclusion bodies have some interestingproperties. They are more fragile and more soluble in milddetergents than classical inclusion bodies. Methods often used fordisruption of bacterial cells during the isolation of IBs, such asenzymatic lysis, sonication and high pressure homogenization werecompared to check whether bacterial cell disruption method hasany influence on mechanical stability and solubility of IBs.Enzymatic lysis of bacterial cells appears to be mild enoughdisruption method not influencing the integrity of IBs and thesupernatant of the lysed sample does not contain any target protein.

As judged from SDS-PAGE analysis, enzymatic treatment does notassure complete release of the cytoplasmic material, including themajority of soluble host proteins, although under the opticalmicroscope only IBs are observed in a highly clustered form.Another drawback of this method is that the enzyme (lysozyme)diffuses into the pores of ncIBs and stays there entrapped, which canrepresent a problem during protein isolation.On the other hand, homogenisation and sonication, methods usuallyused for bacterial cell disruption, enable total release of IBs, but theyseem to be quite harmful for ncIBs. Surprisingly, a noticeabledisassembling and partial or sometimes complete solubilization ofncIBs was observed. Therefore, optimization of sonication andhomogenisation for isolationof ncIBS frombacterial cells is necessary.Nonclassical IBs are very soluble in classical washing solutionscontaining detergents (e.g. 1% deoxycholate, octylglucoside orTriton X-100). Even washing of studied ncIBs (G-CSF, GFP,His7dN6TNF-a with lower detergent concentrations, e.g., 0.1%deoxycholate results in significant protein loss.Another interesting property of ncIBs, initially observed at macrolevel during centrifugation, is irreversible contraction when thebuffer pH is reduced from neutral (pH 7) to acidic (pH 4). This wasfurther confirmed by electron microscopy and the size of ncIBs atvarious pH was determined. Preliminary results show that inclusionbodies have rather porous structure, which seems to depend on thebuffer pH. The smaller size of the pores at acidic pH are probably thecause for less efficient extraction of the target protein from IBs atlow pH. As a consequence of contraction, one would also expectthe increase of density, which was actually qualitatively confirmed incentrifugation experiments.Probably the most important and useful property of ncIBs is largeamount of properly folded target protein or its precursor. Thetarget protein can be extracted in non denaturing conditions usinglow concentration of mild detergents or even simple buffers.Conclusion: Nonclassical IBs compared to classical are char-acterized by higher fragility, higher solubility, irreversible contrac-tion at acidic pH and most importantly, a high amount of correctlyfolded target protein or its precursor. When employing classicalisolation procedure on preparative scale the described propertiescan lead to substantial loss of the target protein. However, on theother hand, using these properties as an advantage for thedevelopment of simplified cost-effective downstream processes,without denaturing solvents and with no need for renaturation,poses a challenge for the future.Reference1. Jevsevar S, Gaberc-Porekar V, Fonda I, Podobnik B,

Grdadolnik J and Menart V: Production of nonclassicalinclusion bodies from which correctly folded proteincan be extracted. Biotechnol Prog 2005, 21(2):632–639.

P24Interfacing Pichia pastoris cultivation withexpanded bed adsorptionMehmedalija Jahic, Josef Knoblechner,Theppanya Charoenrat, Sven-Olof Enforsand Andres VeideDepartment of Biotechnology, Royal Institute of Technology,KTH, Stockholm, Sweden


Background: For improved interfacing of the Pichia pastoris fed-batch cultivation process with expanded bed adsorption (EBA)



technique, a modified cultivation technique was developed. Themodification included thereductionof themediumsalt concentration,which was then kept constant by regulating the medium conductivityat low value (about 8mS cm-1) by salt feeding. Before loading, the lowconductivity culture broth was diluted only to reduce viscosity,caused by high cell density. The concept was applied to a one-steprecovery andpurification procedure for a fusion protein composedofa cellulose binding module (CBM) from Neocallimastix patriciarumcellulase 6A fused to lipase B from Candida antarctica (CALB).Results: The modified cultivation technique resulted in lower celldeath and consequently lower concentration of proteases and othercontaminating proteins in the culture broth (see Figure 1). Flowcytometry analysis showed 1% dead (propidium stained) cellscompared to 3.5% in the reference process. During the wholeprocess of cultivation and recovery, no proteolysis was detectedand in the end of the cultivation the product constituted 87% of thetotal supernatant protein. The lipase activity in the culturesupernatant increased at an almost constant rate up to a valuecorresponding to 2.2 g L-1 of CBM-CALB. In the EBA process nocell-adsorbent interaction was detected but the cell density had tobe reduced by a two-times dilution to keep a proper bed expansion.At flow velocity of 400 cm h-1, the breakthrough capacity was 12.4 gL-1, the product yield 98%, the concentration factor 3.6 times, thepurity about 90%, and the productivity 2.1 g L-1 h-1.Conclusion: Our achievements in the modified cultivation stagethat increased the quality of the feedstock for the separation stageswere: higher target protein to total protein concentration ratio in theculture broth, low protease activities, and lower salt concentrationand conductivity. The low salt and conductivity should also make thefeedstock more suitable for loading on ion exchanger EBA media.These achievements made the expanded bed adsorption techniquemore efficient for initial recovery of CBM-CALB in particular butshould also make the EBA technique more attractive for therecovery of other recombinant proteins from P. pastoris system.AcknowledgementsThis work is part of the BiMac Enzyme Factory programme andfinanced by the Sodra Skogsagarnas Stiftelse for Forskning,Utveckling och Utbildning.

P25Human Prolactin (hPRL) and Growth Hormone(hGH) distinct behavior under bacteriophagelambda PL promoter controlCarlos RJ Soares, Eric KM Ueda, Tais L Oliveira,Susana R Heller and Paolo BartoliniInstituto de Pesquisas Energeticas e Nucleares, IPEN-CNEN/SP, Centro de Biotecnologia, Sao Paulo, Brazil


Background: When producing recombinant protein in E. colifor therapeutic use, it is desirable not only to obtain substantialamounts of it, but also make sure that potential contaminantssuch as antibiotics or inducing agents (isopropyl-beta-D-thioga-lactopyranoside, IPTG or nalidixic acid) will not taint the finalproduct. To prevent this shortcoming we can use expressionsystems where the promoter is activated by temperature shift,which denatures the controlling repressor protein cIts, allowingpromoter activity. While in our hands hGH was successfullyexpressed and secreted in E. coli periplasm with yields in generalwell above 1 �g/mL/A600, after a temperature shift from 30˚C to42˚C [1], attempts to express a related protein hormone (hPRL)with basis on the same protocol were not successful, providing0.03 �g/mL/A600 at the most. Knowing that hPRL compared withhGH is a much more labile protein, we tried to obtain it from thesame strain, but without the presence of the repressor proteinand under optimized temperature conditions.Results: Human growth hormone periplasmic secretion in abacterial host that has also been transformed with the plasmidpRK-248cIts, which contains the thermosensitive transcriptionrepressor (cIts) gene [2, 3] has been studied at different activationtemperatures (Table 1).We can observe that the �PL promoter isalmost totally repressed up to 37˚C, while at 42˚C itsderepression permits a useful hGH periplasmic secretion that isacceptable according to previously established parameters [1]. InTable 1 we also can see the results obtained in a set of differentexperiments observing that without repressor still a quite highhGH secretion (1 �g/mL/A600) is obtained at 30˚C while anapparently higher secretion is obtained at 42˚C. Under the sameconditions, considering the described expression vector intowhich the hGH gene has been substituted by the hPRL gene, stillwith the presence of cIts, an approximately 130–180-fold lowersecretion level for this hormone is observed at 42˚C. This led usto better study the behaviour of our hPRL-producing E. colistrains, either transformed or not with the plasmid pRK-248cIts.Considering not only that hPRL has been found a particularlylabile protein but also that the bacterial periplasmic enviromentcan even be more detrimental to protein stability, especially athigh temperatures [4], it was decided to carry out a study onhPRL periplasmic secretion by "activating" at 30˚, 35˚, 37˚ and 42˚C, with and without the presence of the repressor. The"activating" temperature of 37˚C, and the bacterial strain lackingthe cIts repressor, thus provided the highest hPRL secretionlevel, i.e. approximately 30-fold higher that those obtained withthe equivalent strain containing the repressor gene. In Table 2 wecan appreciate the statistical difference for prolactin yieldsobtained under different temperatures. As already observed forhGH (Table 1) the strain lacking the repressor gene is producing asignificantly higher (P < 0.001) amount of hPRL, even at 42˚C.Since it has been reported that the lack of repressor could easily leadto plasmid loss [3], a study was carried out determining hPRL


SDS-PAGE analysis of samples withdrawn from the bioreactor in two P.pastoris mTLFB cultivation processes. 20 L of the culture supernatant wasloaded in each lane. The two strong upper bands, in the lanes 2 to 6, representprotein of interest, which appears as two diffuse bands due to glycosylation.Lanes 7 to 10 correspond to samples from reference cultivation of the same P.pastoris strain, transformed with linear vector lacking DNA coding for theprotein of interest (thus, the contaminating proteins).



periplasmic yield in the strain lacking cIts after two growth periodscorresponding to 10 and 50 E. coli generations, obtaining 0.64 ± 0.05and 0.78 ± 0.03 �g hPRL/mL/A600 respectively. Also the presence ornot of antibiotic (amp) did not influence the specific expression yieldfor at least 40 generations.This same strain is being utilized for setting up a rapid and flexiblefeed batch fermentation in a laboratory bioreactor, obtaining upto now ~7 �g hPRL/mL with an optical density of 42.4 A600.Conclusion: A relatively high hPRL periplasmic secretion (upto 0.9 �g/mL/A600), never reported before, has been obtained byconstitutive expression of the unrepressed �PLpromoter, at 37˚C. The expression level is approximately 10-fold higher thanthat obtained in previous work [5] by using an IPTG-activatedtac promoter. We can conclude that these data open the way tothe utilization of E. coli instead of insect or mammalianexpression systems for the production of an authentic andhighly homogeneous hPRL.AcknowledgementsSupported by FAPESP and CNPq.References1. Soares CRJ, Gomide FIC, Ueda EKU and Bartolini P:

Periplasmic expression of human growth hormonevia plasmid vectors containing the �P promoter: useof HPLC for product quantifications. Protein Eng 2003,16:1131–1138.

2. Bernard HU and Helinski DR:Use of the phage promoterPL to promote gene expression in hybrid plasmidcloning vehicles. Methods Enzymol 1979, 68:482–492.

3. Crowl R: Expression of human interferon genes in E.coli with the lambda PL promoter. Methods Enzymol1986, 119:376–383.

4. Makrides SC: Strategies for achieving high-level expres-sion of genes in E. coli. Microbiol Rev 1996, 60:512–538.

5. Morganti L, Soares CRS, Affonso R, Gout PW andBartolini P: Synthesis and characterization of recom-binant authentic human prolactin secreted into theperiplasmic space of Escherichia coli. Biotechnol ApplBiochem 1998, 27:63–70.

P26Two-compartment bioreactor as a scale-downmodel to study the effect of glucose overflow andanaerobiosis on large-scale recombinant proteinproduction processesJaakko Soini1, Ulla Pajulampi1, Janne Sandqvist1,Arne Matzen2 and Peter Neubauer11Bioprocess Engineering Laboratory, Department of Process andEnvironmental Engineering, University of Oulu, Oulu, Finland2Sanofi-Aventis, Germany


Background: In high-cell density fermentations the host cells areoften subjects of transient changes in microenvironment aroundthem. This is true especially in large-scale bioreactors. The changescan be for example substrate gradient, differences in oxygenavailability and pH variations. Our aim is to obtain more informationabout physiological changes of E.coli W3110 and its recombinantvariants in such conditions for better understanding of thebottlenecks in recombinant protein production processes.To mimic the conditions in large-scale fermentations, we have set-up a two-compartment bioreactor [1], in which cells are circulatedbetween a regular stirred tank reactor (STR) and a plug-flow reactor(PFR) using a peristaltic pump. The glucose is fed into the bottom ofthe plug-flow reactor with the aim of maintaining the glucoselimitation in the STR part.The advantage of the model is that the conditions of the zoneswhere the changes occur can bemeasured.We take samples from 4positions A, B, C andD (See Fig 1) of the PFR and additionally from 1sample position of the STR. The normal process parameters such aspH, DOT and temperature are measured from the STR andadditionally we have placed DOT and pH sensors in two positions ofthe PFR (S1 and S2).Results: From the initial fermentation experiments in the STR-PFRreactorwehave seen that themodel is a goodsimulator for conditionsin large-scale fermentors.When the STRpart onlywasmonitored, nosigns from anaerobic conditions or pH variations were observed.However, in the PFRpart glucosewasmeasured and the highest value

Table 1 (abstract P25) hGH periplasmic secretion level activating at different temperature and utilizing hGH-secreting W3110strains, with or without the repressor gene (cIts).

temperature phGH-DsbA-PL+ pRK-248 cIts (g/mL/A600 ± SD) phGH-DsbA-PL(g/mL/A600 ± SD)

30˚C 0.01 1.0 ± 0.14 (n = 2)35˚C 0.02 -37˚C 0,06 -42˚C 1,31 ± 0.38 (n = 4) 1.61 ± 0.11 (n = 3)

Table 2 (abstract P25) hPRL periplasmic secretion level in E. coli W3110, at different temperatures utilizing a vector containing(phPRL-DsbA-cIts -PL) and one not containing (phPRL-DsbA-PL) the repressor gene.

temperature phPRL-DsbA-PL+ pRK-248 cIts (g/mL/A600 ± SD) phPRL-DsbA-PL(g/mL/A600± SD) Statistical significance a

30˚C 0.001 (n = 1) 0.14 ± 0.02 (n = 6) -35˚C - 0.73 ± 0.07 (n = 5) P < 0.00137˚C 0.03 (n = 1) 0.92 ± 0.10 (n = 6) P < 0.0139˚C - 0.60 ± 0.12 (n = 8) P < 0.00142˚C 0.02 (n = 1) 0.19 ± 0.05 (n = 4) P < 0.001

a Student’s T-test comparing each value to the previous one



was obtained close to the feeding point decreasing then towards theend of the plug-flow reactor. Rapid formate accumulation wasobserved and 150mg/Lof formatewas produced in fewminutes. Lowamountsof oxygenwasmonitored fromPFRbefore the feed start butafter it variable dropped down to zero at bothmeasuring points. AlsopH decreased more than 0.5 units in the plug-flow compartment.Reference1. George S, Larsson G and Enfors SO: A scale-down two-

compartment reactorwith controlled substrate oscilla-tions Saccharomyces cerevisiae. Bioprocess Engineering 1993.

P27Automated fed-batch cultivations using baseconsumption for real time biomass determinationduring production of heterologous proteinsMarkus Ganzlin, Benjamin Gerwat and Philipp GarbersGlobal Protein Science & Supply, AstraZeneca, Macclesfield, UK


Background: Automation can improve efficiency for fast devel-opment of reproducible bioreactor processes required for drugdiscovery. Target protein production in bioreactors can also benefitfrom online biomass determination, since the problem of undefinedmaximum specific growth rates for a high through-put of newly-designed recombinant E.coli strains can be addressed. In order torealise online biomass monitoring a variety of different technicalapproaches are commercially available. Some of them are quitecomplex and expensive. In contrast to this an effective method ofreal time biomass monitoring for E.coli fed-batch cultivations wasdescribed previously [1].Results: A software tool was programmed in MATLABâ andlinked to the SCADA system MFCS/winâ via an OPC Client.Using this tool with user-friendly graphical interfaces, automatedinduction and exponential increasing mass feeding strategies

were carried out successfully. A reproducible automated batchend detection was implemented and successfully validated fortwenty cultivations. The batch end detection triggered theautomatic start of an exponential increasing feed strategy. Duringthe development of the detection method a suitable set of logicalconditions based on online cultivation data was determined.These conditions were used for a loop algorithm, which ensuredthat all conditions were true for a period of three minutes, whicheliminated interference of outlier online signals. For all fed-batchcultivations a good linear correlation between base (NH3)consumption and biomass production was determined. Thebiomass yield coefficient with respect to ammonia was constantduring the course of cultivation and remained the same afterprotein production was induced. The same yield coefficient (YX/

NH3 = 5.9 g g-1) was determined for five different feedingstrategies. These bioprocesses used a feed forward strategyaiming at a different constant growth rate (�SET1 = 0.04 h-1, �SET2

= 0.06 h-1, �SET3 = 0.09 h-1, �SET4 = 0.12 h-1 �SET5 = 0.14 h-1).Conclusion: The implementation of a reproducible automatedbatch end detection significantly reduced the requirements forout of hours working time. Using the online base consumptionsignal as an indicator for biomass production improved thequality of decision making during running time.Reference1. Schmidt M, Viaplana E, Hoffmann F, Marten S, Villaverde A and

Rinas U: Secretion-dependent proteolysis of heterolo-gous protein by recombinant Escherichia coli is con-nected to an increased activity of the energy-generatingdissimilatory pathway. Biotechnol Bioeng 1999, 66:61–67.

P28Caspase activation, sialidase release and changesin sialylation pattern of recombinant humanerythropoietin produced by CHO cells in batchand fed-batch culturesKok Hwee Chuan1, Sing Fee Lim1, Laurent Martin2,Chee Yong Yun1, Sophia OH Loh1, Francoise Lasne2

and Zhiwei Song11Bioprocessing Technology Institute, Biomedical Sciences Institutes,20 Biopolis Way, #06-01 Centros, Singapore 1386682Laboratoire National de Depistage du Dopage, 143 AvenueRoger Salengro, 92290 Chatenay-Malabry, France


Product yield and product sialylation are the two most principalyardsticks that determine the commercial viability of mostrecombinant cell culture processes. In this study, we describe astrategy whereby an optimized harvesting time point was establishedbased on product titer and sialylation pattern of recombinant humanerythropoietin (EPO) produced in a CHO cell line. The activation ofvarious caspases, the release of intracellular sialidase and the changesin sialylation pattern of the recombinant product in medium weretracked in both batch and fed-batch cultures. In both setups, allcaspase activities were found to peak at the culture time point atwhich declivity of cell viability was most pronounced. Also, release ofintracellular sialidase and lactate dehydrogenase (LDH) coincidedwith the observed decline in cell viability and the concomitantincrease in caspase activities. By incorporating isoelectric focusing(IEF), coupled with double blotting as the de novo technique in theanalysis of product sialylation, prompt resolution of secreted EPOisoforms in a time course formatwere obtained. The IEF profile of the


Schematic figure of the STR-PFR reactor and its sampling points.



batch culture showed relatively good consistency in productsialylation compared to fed-batch culture, which showed gradualband shifts towards more basic isoforms as the culture progressed.Based on key parameters such as product yield and extent of productsialylation, the optimized harvesting time point was found to be at the91% culture viability mark in both batch and fed-batch cultures. Inaddition, various metabolite analyses were conducted in a bid toidentify potential death inducing factorspresent inbatch and fed-batchcultures. Accumulation of metabolites, coupled with elevatedosmolality in fed-batch culture was found to be sufficient in itspropensity to impede cell growth and tomoderately inducecell death.

P29Set up and optimization of a fermentationprotocol for the production of a human antibodyfragment (Fab’) express in E. coli. Pre-pilot andcGMP pilot scale studiesE Riscaldati, A Ciabini, A Baccante, D Moscatelli,MF Errichetti, A Colagrande, S Cencioni, F Marcocci, V DiCioccio, L Di Ciccio, M Allegretti, F Martin and G MauriziBiotechnology Department and Biotech Pilot Plant – Dompepha.r.ma s.p.a., Via Campo Di Pile, 67100 L’Aquila, Italy


Background: The efficacy of mAb and their fragments in cancertherapy depends on their capacity to recognize tumor associatedantigens. From a clinical point of view, intactmAb are employed in thetreatment of lymphoid tumors whereas antibody fragments such asFabsorF(ab’)2 canbe the therapeuticof choice for solid tumorsdue toshorter circulating half life and a higher tumor penetration, bothcharacteristics well suited in a radio immunotherapy (RIT) approach.Ovarian cancer is the leading cause of death among gynecologicaltumors. Standard first line therapy consists of surgery followed bychemotherapy cycles. About 90% of patients relapse with a 5-yearsurvival rate of 5–20% [1]. So far, second line chemotherapy afterclinical relapse has shown limited efficacy. With the advancement ofthe recombinant biotechnology, a novel approach based on humanmonoclonal antibody immunotherapy has began to be investigated.The alfa folate receptor (aFR), which is a 38000 Dalton anchoredmembrane protein, is over expressed in more than 90% of ovariancarcinoma cells, and in 60% of other gynecological carcinomas [2]. Incontrast to normal epithelial cells, ovarian carcinoma cells expressthis receptor on their external surface making it accessible forbinding to monoclonal antibodies.The basic concept of our project is to produce a full human F(ab’)2fragment for radio immunotherapy to specifically target the aFR. Thecandidate Fab fragment was isolated and cloned into an Escherichia coliexpression vector.Afterclassical shaking flask cultures expression therecombinant strain was taken to lab scale fermentation plant wheredifferent feeding strategies were designed to increase the productionyield [3].Results: Fermentation scale up is aimed at themanufacture of largequantities of product while maintaining the specific yields and quality.We report the development of a Fed-batch fermentation processevaluated at 10 and 300 liters scale, for the production of functionalFab’ monomer in E. coli, at high cell density fermentation, under DO-stat control strategy using a synthetic medium. The construction of astable bicistronic expression vector and a proprietary host-plasmidpair allows us to obtain both high level expression (up to 100 mg/L)and correctly folded protein expressed in the periplasm butstraightforwardly in the broth supernatant were the Fab’ accumulate.

To identify process-specific stress factors and to understand thephysiological responses to the vessel specific physical conditions, theprocess was evaluated at 10 liters scale for animal grade productionand with comparable results, at 300 L scale in a clinical grade cGMPplant. Despite substrate oscillation due to the DO-stat mechanism,whitch final results is a linear feeding, at both scale the plasmidstability and cell vitality, measured by means of a biomass monitor(Aber instrument) were maintained. Neither lysis nor proteaseproduction were detected during the fermentation course.Typical process parameter and analytical data coming from 10 and300 L are reported in Figure 1 (a-d) and in Figure 2 (a-b). In Table 1mean yield coefficient at both scales are calculated.All activities carried out for 300 L production scale are developed in aGMP compliance pilot plant. At the end of fermentation both pH andtemperature are adjusted to trigger contaminant proteins precipita-tion. The remaining soluble proteins were recovered from thebiomass by tangenzial flow filtrations (TFF) and purified to homo-geneity. Fabbiological activitywasmonitoredbyBIACORE, FACSandELISA analysis.Conclusion: Altogether the reporteddata confirmed the technicalfeasibility of 10 to 300 L scale-up along with the process robustnessand recombinant Fab’ production consistency. Several fed-batchmethods for carbon source feeding maintaining low by-productslevels were developed. Most of these are based on mathematicalmodels that foresee growth patterns and expected demand fornutrients. Our results suggest that a DO-stat feeding strategy is anefficient, straightforward scalable, physiological and elegant way ofcarrying out fermentation process to obtain both high expressionlevel and properly folded protein. The Fab’ quantity and qualityproduced by the described process were adequate to justify theproduction of clinical trials material. Moreover some criticalparameters for scale up were identified.References1. Berek JS, Bertelsen K, du BA, Brady MF, Carmichael J,

Eisenhauer EA, Gore M, Grenman S, Hamilton TC,Hansen SW, Harper PG, Horvath G, Kaye SB, Luck HJ,Lund B, McGuire WP, Neijt JP, Ozols RF, Parmar MK, Piccart-Gebhart MJ, van Rijswijk R, Rosenberg P, Rustin GJ, Sessa C andWillemse PH: Advanced epithelial ovarian cancer: 1998consensus statements. Ann Onco 1999, 10:87–92.

2. Lu Y and Low PS: Immunotherapy of folate receptor-expressing tumors: review of recent advances andfuture prospects. J Control Release 2003, 91:17–29.

3. Garcıa-Arrazola R, Siu SC, Chan G, Buchanan I, Doyle B,Titchener-Hooker N and Baganz F: Evaluation of a pH-statfeeding strategy on the production and recovery of Fab’fragments from E. coli. Biochem Eng J 2005, 23:221–230.

P30Evaluation of antifoams in the expression of arecombinant FC fusion protein in shake flask culturesof Saccharomyces cerevisiae & Pichia pastorisWilliam Holmes1, Rodney Smith2 and Roslyn Bill11Aston Academy of Life Sciences, Aston University, AstonTriangle, Birmingham, UK2CTM Biotech Ltd, Babraham Research Campus, Babraham,Cambridge, UK


Background: Optimisation of culture conditions for theexpression and production of important therapeutic biologics



such as recombinant proteins, antibody-fragments and fusionproteins is a key element in the rapid and cost effectivemanufacture of these important molecules [1]. The factors to beconsidered when producing proteins from microorganisms suchas Saccharomyces cerevisiae or Pichia pastoris include: pH,temperature, carbon and nitrogen sources and the essentialoxygen requirement. The demand for oxygen by a microorgan-ism can be met by aerating the medium that it is growing in,which is most often done by sparging sterile air through themedium.

An unfortunate effect of both sparging gas through culturemedia at high rates and intense agitation is the formation offoam. This is a particular problem when surface active speciessuch as proteins are present at high concentrations. Foams aregas/liquid dispersions with >95% gas content [2]. Foamformation can reduce the efficiency of gas exchange at thesurface of the culture, as a barrier is formed between the cultureand the gases in the headspace of the vessel. Foaming can also bedetrimental to the cells: when bubbles burst they exert sheerforces, which can damage cells and/or any secreted proteins.


a)Effect of DO-Stat strategy on: () biomass level (OD600), () residual substrate concentration and () Fab’ accumulation at 10 liters scale fermentation(HPLC analysis); b typical curve of (–) DO, (–) pH, (—) OD600 and (—) Inductor (FEED) during DO-Stat fermentation; c representative curves of (–)conductivity and (–) capacitance in fermentation broth and Oxygen and CO2 in exhausted air; d Western blot of samples taken throughout theinduction phase



Additionally cells and culture medium are lost to the foam phasewhich can lead to a decrease in process productivity. In extremecases a ’foam out’ situation can lead to loss of process sterility[2].In order to minimise the deleterious effects of foaming, antifoamagents are used which prevent foam forming by reducing thesurface tension of the culture [1]. There is a wide range ofantifoam agents available from various suppliers. Examples ofcommonly used antifoams include compounds from the follow-ing chemical types: polyalkylenglycols, alkoxylated fatty acidesters on a vegetable bases, polypropylene glycol (PPG),siloxane polymers, mineral oils and silicates.For this investigation a secreted recombinant protein expressedby both P. pastoris and S. cerevisiae was used as a marker ofprotein production yield. The product protein was producedusing the following expression systems; in P. pastoris the genehad been inserted into a methanol-inducible expressioncassette. In S. cerevisiae (Uracil autotrophic strain) proteinexpression was under the control of the TPI1 promoter. Theprotein itself has a molecular weight of approximately 48 kDa.

We examined the effectiveness of four antifoam agents; Schill &Schelinger’s Struktol SB2121 (Polyalkylenglycol), Schill & Schelinger’sStruktol J673A (an alkoxylated fatty acid ester on a vegetable base),Sigma Antifoam C (Siloxane polymer) and Fluka P2000 (Polypropy-lene glycol), for usewith both P. pastoris and S. cerevisiae.The effect onthe growth rate and the protein production yield for all antifoamtypes at varying concentrations was determined by monitoring thegrowth and target protein production in S. cerevisiae and P. pastoris.Results: The different types of antifoam affect S. cerevisiae and P.pastoris growth in different ways depending on the concentrationandmedium type being used.When Struktol J673A is usedwith YPDmedium for P. pastoris growth, increasing antifoam concentrationincreases optical density (OD) at 595 nm (see Figure 1). Converselywhen Struktol SB2121 is used with SD-URA medium for S. cerevisiae(strain: ALCOFREEä Yeast 01) [3] protein production, increasingantifoam concentration reduces OD measurements of the cultures(see Figure 2). When Antifoam C is used with YPD medium, S.cerevisiae growth is not affected by Antifoam C concentrations upto 8% (see Figure 3). The effect on protein production is lessvariable, with the trend being that concentrations over 1% total


a Effect of DO-Stat strategy on: () biomass level (OD600), () residual substrate concentration and () Fab’ accumulation at 300 liters scale fermentation(HPLC analysis); bWestern blot of different samples taken throughout the induction phase.

Table 1 (abstract P29) Parameters for mean yield coefficient calculation at 10 and 300 L scale fermentation.

Fermentation Time OD600 DCW Substrate Fab Fab DCW Substrate YXS YPX YPSscale (l) (h) g.l-1 g.l-1 g.l-1 g.l-1.h g.l-1.h g.l-1.h g.g-1 g.g-1 g.g-1

(X) (S) (P) (rP) (rX) (-rS)

10 36 137 36.4 186 0.0642 0.00178 1.01 5.16 0.196 0.0018 0.00035

3003 8 10 2.9 15 0.36

30 6 18 5.8 30 0.96300 37 136 36.2 190 0.0685 0.00185 0.98 5.13 0.190 0.0019 0.00036



volume decrease the yield of recombinant protein in the cultures(See Figure 4).Conclusion: The data indicate that antifoam agents can beused at concentrations up to 1% total volume. Higherconcentrations can lead to higher optical densities beingobtained but with a decrease in protein yield. Additionallysome of the antifoam agents become difficult to work with athigher concentrations, producing precipitates which interferewith sampling and analysis. Table 1 highlights the mainconclusions for each individual antifoam and application.AcknowledgementsThe work presented is part of a PhD by William Holmes inconjunction with Aston University. The author would like tothank the Engineering and Physical Sciences Research Council(EPSRC) for supporting this work.

References1. Dow Corning: Dow Corning Antifoam product infor-

mation.http://20057426-FoamContGuideEur.indd, created15/03/2005.

2. Varley J, Brown AK, Boyd JWR, Dodd PW and Gallagher S:Dynamic multi-point measurement of foam beha-viour for a continuous fermentation over a range ofkey process variables. Biochem Eng J 2004, 20:61–72.

3. ALCOFREEä Yeast 01 derived from the CEN. PK strainfamilyGothia Yeast Solutions AB, Terrassgatan 7, 411 33Gothenburg, Sweden; http://www.gothiayeast.com.


Growth curves for P. pastoris in YPD medium at 30˚C with J673Aantifoam.


Growth curves for S. cerevisiae TM6* in YPD medium at 30˚C withAntifoam C


Growth curves for S. cerevisiae TM6* in SD-URA medium at 30˚C withSSB2121 antifoam.


Silver stain of P. pastoris production phase samples 120 hr post methanolinduction with Struktol J673A antifoam: Lane 1 0% J673A, 2 0.5% J673A,3 1% J673A,4 2% J673A,5 4% J673A,6 8% J673A. The arrow indicates therecombinant protein produced in these experiments.



P31Cloning and overexpression of a yeast phytasegene in Pichia pastorisMelanie Ragon, Virginie Neugnot-Roux, Guy Moulinand Helene BozeUMR IR2B, Equipe Genie Microbiologique et Enzymatique,AGRO. M-INRA, 2 Place Viala, 34060 Montpellier Cedex 01,France


Background: Phytate (myo-inositol hexakisphosphate) repre-sents the major storage form of phosphorus in cereal grains,legumes, pollens and oilseeds. However, the bioavailability ofphosphorus-phytate in plant derived feedstuffs is limited becausemonogastric animals lack intestinal phytase at the level neededto hydrolyse phytate and release the necessary inorganicphosphorus [1]. One solution currently employed is amendmentof animal rations with phytase [2]. Phytases (myo-inositolhexakisphosphate 3- and 6-phosphohydrolase) catalyse thehydrolytic degradation of phytate yielding lower inositolphosphates (InsP5 to InsP1), myo-inositol and inorganic phos-phate. Phytases are produced by a wide range of organisms :plant, animal and especially microorganisms.Results: Our aim was to overexpress a new yeast phytasegene in the methylotrophic yeast Pichia pastoris. The ORF codingthis new yeast phytase was isolated from Debaryomyces castellii.The deduced 461-amino-acid protein sequence, correspondingto a 51.2 kDa molecular mass, contained the consensus motif(RHGXRXP) which is conserved among phytases. This proteinshared 21 to 69% sequence similarities with various phytases ofyeast or fungal origin. Heterologous expression of this phytasein the methylotrophic yeast P. pastoris was tested both under theP. pastoris inducible alcohol oxidase (AOX1) promoter and theconstitutive glyceraldehyde-3-phosphate dehydrogenase (GAP)promoter in fermentor. In both cases, the a-factor signalsequence was utilised, resulting in secretion of phytase into theculture medium. Maximum production level obtained was107 U/mL, i.e. 1340 U/g DCW with the AOX1 expressionsystem and 16.5 U/mL i.e. 300 U/g DCW with the GAP one.Conclusion: These productions corresponded to an over-expression by 100 and 10 times respectively compared to thewild production. The biochemical characteristics of therecombinant phytase are identical to those of the native one.Thus, the new yeast phytase gene of Debaryomyces castellii hasbeen successfully expressed in P. pastoris.

AcknowledgementsThe financial support by ADISSEO and ANRT (AgenceNationale de la Recherche Technologique) to M. R., in theform of doctoral grant, is gratefully acknowledged.References1. Wodzinski RJ and Ullah AHJ: Phytases. Adv Appl Microbiol

1996, 42:263–303.2. Stahl CH, Roneker KR, Thornton JR and Lei XG: A new

phytase expressed in yeast effectively improves thebioavailability of phytate phosphorus to weanlingpigs. J Anim Sci 2000, 78:668–674.

P32Optimisation of substrate feeding in shake flaskcultures of Pichia pastoris for recombinant proteinproductionMonika Bollok1, Maria Ruottinen1, Mirja Krause1,Antti Vasala1, Eija-Riitta Hamalainen1, Antje Neubauer2,Johanna Myllyharju2 and Peter Neubauer11Bioprocess Engineering Laboratory, Dept. Process &Environm. Engin2Biocenter Oulu, P.O. Box 4300, University of Oulu,FIN-90014 Oulu, Finland E-mail: [email protected]


Background: Pichia pastoris is used as a common host forproduction of recombinant proteins. Gene expression is mostlycontrolled by the AOX promoter through methanol addition.The aim of this study was to investigate whether the generallyapplied methanol addition protocol is optimum and whether theexpression in shake flasks can be improved by applying adifferent feeding scheme.Therefore we applied the recently described wireless on-linemonitoring wireless system (SENBITâ which allows the applica-tion of standard sensors such as pH and pO2 in shake flasks [1].Moreover, a sensitive sandwich hybridization technology wasused for the quantitative analysis of the expression level forprocess relevant marker genes which to provide data about thephysiological state of the cultures and hereby a better under-standing of the microbial responses.Results: The impact of the feeding protocol was studied in shakeflask cultures of Pichia pastoris for recombinant collagen productionwith methanol as inducer and carbon source. On-line measurementof pO2 revealed that the standard methanol feeding protocol is notfavourable. The culture is starved for long times for methanol and

Table 1 (abstract P30) Summary of main conclusions

Antifoam name Antifoam type Pichia pastoris Saccharomyces cerevisiae

Growth Production Growth Production

SB2121 Polyalkylenglycol No effect < = 8%[SB2121]

No effect < = 8%[SB2121]

Optimal when[SB2121] >0% <4%

Decreased by[SB2121] >1%

J673A Alkoxylated fatty acidester on a vegetablebase

Increases with[J673A]

Decreased by [J673A]>1%

Increases with[J673A]

Decreased by [J673A]>1%

Antifoam C Siloxane polymer No effect < = 8%[Antifoam C]

TBC No effect < = 8%[Antifoam C]

TBC

P2000 Polypropylene glycol Decreased by[P2000] >1%

TBC Increases with[P2000]

TBC



also oxygen may be depleted shortly after a methanol pulse. A fed-batch like feeding procedure was developed by applying a computercontrolled micropump system for semi-continuous addition ofmethanol. As a result the amount of collagen was improved.Furthermore, also the expression of collagen prolyl 4-hydroxylase, acollagen modifying enzyme was strongly increased which resulted incollagen of higher stability. The improvement of the cultureconditions with the new feeding protocol were verified by semi-quantitative analysis of different cellular mRNAs.Conclusion: Regular feeding of small amounts of methanol in asemi-continuous way improves the behaviour of recombinantcultures of Pichia pastoris and increased the amount and qualityof collagen in our study. We propose that this method isgenerally favourable for the optimisation of gene expression inPichia pastoris shake flask cultures.AcknowledgementsThis study was supported by the TEKES "Neobio" programmeand a grant to MB by the Academy of Finland.Reference1. Vasala A, Panula J, Bollok M, Illman L, Halsig C and

Neubauer P: A new wireless system for decentralisedmeasurement of physiological parameters fromshake flasks. Microb Cell Fact 2006, 5:8.

P33Increasing the ease and speed of eukaryoticprotein expression: a new cell-free in vitrotranslation system based on Sf insect cell extractsFrank Schaefer, Annette Zacharias, NicoleBrinker-Krieger and Uritza von GrollQIAGEN GmbH, QIAGEN Strabe 1, 40724 Hilden, Germany


Background: For researchers looking for fast access to theirprotein of interest, cell-free expression systems are anattractive option, as they do not require specialized equipment,are open systems, and offer fast screening of expressionconstructs for expression efficiency, yield, solubility and othercriteria (e.g. requirement of cofactors).

A broad range of eukaryotic proteins require posttranslationalmodifications such as phosphorylation, glycosylation, or signalpeptide cleavage to display full functional activity.Results: We will describe the most important features of anew system for recombinant eukaryotic protein expression thatretains the speed and economy inherent to cell-free methods.Data showing the ability of the system to generate complexposttranslational modifications (PTMs), including highly efficientglycosylation of EPO and ORM1 proteins (Figure 1) andphosphorylation of AKt1 kinase (Figure 2) will be presented.The robustness of the system is demonstrated by the expressionof membrane proteins (e.g., OGCP) and the synthesis of severalproteins, such as human clotting factors, that could not beexpressed in E. coli systems.Conclusion: Expressed proteins with the EasyXpress ProteinSynthesis Insect Kit are post-translationally modified with highefficiency.

P34Impact of apoptosis gene targeting onrecombinant protein glycosylationDanny Chee Furng Wong1, Niki Soo Ching Wong1,John Soo Yang Goh1 and Miranda Gek Sim Yap1,21Bioprocessing Technology Institute, Biomedical SciencesInstitutes, 20 Biopolis Way, #06-01 Centros, Singapore1386682Department of Chemical & Biomolecular Engineering,National University of Singapore,10 Kent Ridge Crescent,Singapore 119260


Background: Chinese Hamster Ovary (CHO) cell is one ofthe major cell lines used for complex recombinant proteinproduction. During CHO cell culture, loss in viability attributedto apoptosis often results in lower recombinant protein yieldand affects protein quality. Through the individual targeting offour apoptosis genes identified via expression profiling studies,four apoptosis resistant CHO GT (Gene Targeted) cell lineswere constructed. These cell lines enabled prolonged cultureviability, higher maximum viable cell densities and significantincrease in recombinant human interferon gamma (IFN-�) yields.Furthermore, it was observed that the IFN-� from CHO GTcells has a higher level of sialic acid content.Results: To further characterize the impact of apoptosis genetargeting on recombinant protein glycosylation, a detailed


6xHis-tagged EPO (erythropoietin) and ORM1 (Alpha-1-acid glycopro-tein 1) were expressed as 14C-Leu-labeled proteins using the EasyXpressProtein Synthesis Insect Kit. After expression an aliquot of each wastreated with the endoglycosidase EndoH, which removes glycan moietiesform glycosylated proteins. Aliquots of treated and untreated proteinseparated by SDS_PAGE and proteins visualized by autoradiography.


Akt1 was expressed in triplicate reactions using the EasyXpress ProteinSynthesis Insect Kit. 2.5 l aliquots were separated by SDS-PAGE andprotein detected by Immunoblotting using an antibody specific for akt1phosphorylated at Ser 473. NTC: no template control; M: marker.



analysis of the micro-heterogeneity of IFN-� was performed. Inaddition, a quantitative real-time PCR (qRT-PCR) involving 24N-glycosylation-related genes from CHO cells was establishedto profile the changes in expression of these genes across theexponential, stationary and death phase of the four CHO GTcell lines and compared to the parental cell line.IFN-� glycan analysis revealed that the CHO GT cell lines havehigher proportions of tri- and tetra-antennary glycan branchingas well as higher proportions of fully sialylated glycoforms.Interestingly, these changes in IFN-� correlated with changes inN-glycosylation gene expression. Compared to the parentalcells, higher expression of N-acetylglucosaminyltransferases IVand V, which are responsible for glycan branching, was observedin CHO GT cell lines. The enzymes involved in sialylation,�-galactoside a2,3-sialyltransferase and CMP-sialic acid trans-porter, were also upregulated in the CHO GT cells.Conclusion: This study showed that apoptosis targeting couldaffect protein glycosylation but further experiments would beneeded to establish the link between apoptosis and glycosyla-tion. Nevertheless, it was demonstrated that the quantitativereal time PCR method could potentially be used to identifypossible ’bottlenecks’ or ’compromised’ pathways in N-glyco-sylation and subsequently allow for the development ofstrategies to improve glycosylation quality.

P35N-glycosylation differences between wild-type andrecombinant strains affect catalytic properties oftwo model enzymes: �-glucosidase andphosphataseIsabelle Mobeche, Melanie Ragon, Guy Moulinand Helene BozeUnite de Genie Enzymatique et Microbiologique, Agro.M – INRA, 2, place Viala, Montpellier, France, 34060


Background: Glycosylation is involved in many proteins’properties and functions such as conformation, thermostability,solubility, protection against proteolysis, intra-cellular migration,secretion. Surexpression and production of proteins by yeastsalready constitute a fabulous technological tool but some in vivodysfunctions are frequently reported (half-life time, allergy...) [1].Study of recombinant glycoproteins glycosylation is then ofcrucial importance: not only for better understanding of therelationship between the glycoprotein properties and itsoligosaccharides but also for being able to orientate theglycosylation process of a given microorganism.This study compares N-glycosylation of two model enzymes,�-glucosidase and phosphatase, between wild-type yeast strainsand heterologous hosts, Pichia pastoris and Schizosaccharomycespombe. It also tries to correlate glycosylation differences toenzyme catalytic properties and to distinguish which part ofglycosylation could be attributed to the nature of microorgan-ism or to the glycoprotein itself (sequence, number ofglycosylation sites...).Results: Gene sequences analysis assumes 11 potentialN-glycosylation sites for the �-glucosidase and 9 for thephosphatase.Glycans part is around 30% of total molecular weight for thenative and recombinant phosphatase while �-glucosidase shows

quantitative glycosylation differences since it represents 45% oftotal molecular weight for the native form and 30% for therecombinant �-glucosidase.Monosaccahrides composition and oligosaccharides type andbranching were studied by capillary electrophoresis for bothproteins [2].Mannose appears as the major component of N-oligosacchar-ides from wild-type and Pichia pastoris strains while Schizosac-charomyces pombe N-oligosaccharides composition dispays alarger range of monosaccharides such as ribose and galactose[3]. Trace of N-acetylglucosamine are also encountered in allproteins.N-oligosaccharides profiles (polymerisation degree) are differ-ent between strains and proteins. Nevertheless, for a givenprotein, oligosaccharide structure (type of linkages betweenmonosaccharides units) and composition are markedly the samebetween wild-type strain and Pichia pastoris whereas Schizosac-charomyces pombe exhibits higher chains and different linkages.Those observed N-glycosylation differences affect enzymecatalytic properties, mainly thermostability.Conclusion: Schizosaccharomyces pombe appears as a singularheterologous host since for both proteins expressed itsglycosylation is widely different from the one of wild-type andPichia pastoris strains. The nature of the microorganism itselfseems to be of major importance for determining monosacchar-ides composition while the gene sequence and culture condi-tions affect mainly oligosaccharides profiles.References1. Bretthauer RK and Castellino F: Glycosylation of Pichia

pastoris derived proteins. Biotechnol Appl Biochem 1999,30:193–200.

2. Evangelista RA, Liu MS and Chen F-TA: Characterizationof 8-aminopyrene-1,3,6-trisulfonic acid derivatizedsugars by capillary electrophoresis with laser –induced fluorescence detection. Anal Chem 1995,67:2239–2245.

3. Ziegler FD, Gemmil TR and Trimble RB: Glycoproteinsynthesis in yeast: Early-events in N-linked oligosac-charides processing in Schizosaccharomyces pombe.J Biol Chem 1994, 269:12527–12535.

P36Cell engineering of Pseudoalteromonas haloplanktisTAC125: Construction of a mutant strain withreduced exo-proteolytic activityErmenegilda Parrilli, Angela Maria Cusano, Maria Giulianiand Maria Luisa TutinoDipartimento di Chimica Organica e Biochimica, Universita diNapoli "Federico II", Napoli, Italia


Background: We have already shown that using cold-adaptedbacteria as host vectors, some "intractable" proteins can beefficiently produced at temperature as low as 4˚C [1, 2].Furthermore, we set up a "cold" gene-expression systemimplemented for the secretion of recombinant proteins in theAntarctic Gram-negative bacterium Pseudoalteromonas haloplank-tis TAC125 (PhTAC125). Such a system could effectivelyconjugate the positive effect of low temperature on therecombinant product solubility with the obvious advantages



linked to extra-cellular protein targeting. This novel systemmakes use of the psychrophilic a-amylase from PhTAB23 [3] assecretion carrier. Several chimerical proteins were producedand used to test the versatility and efficiency of the novelsecretion system. All the chimerical proteins were efficientlyproduced and secreted (Cusano AM, Ph. D thesis 2005Universita di Napoli "Federico II"). However, bacteria belongingto Pseudoalteromonas genus are reported to secrete a wide rangeof exo-proteins, especially proteases. This feature could hamperboth applicability and efficiency of the cold-adapted secretionsystem, due to the possible recombinant product degradation.The PhTAC125 genome sequence [4] was recently determined.The in silico genome analysis highlighted the presence of aputative Type II secretion system (T2SS), while the extra-cellulartargeting of the cold a-amylase depends on a still uncharacter-ized secretion pathway [4].Results: Construction of the PhTAC125 suicide vector:Figure 1 shows the Vs suicide vector constructed to generate

PhTAC125 genomic mutants. It is characterised by the presence of:i) the pJB3-derived oriT (1), a DNA fragment responsible for theinitiation of the conjugative transfer between an Escherichia coli �pirstrain (donor) and the psychrophilic cells (acceptor); ii) the E.coliblaM gene, encoding a mesophilic �-lactamase which is used asselection gene to isolate the first site-specific integration event; iii)pheSGly294, which encodes a mutated version of the E. coli a subunitof Phe-tRNA synthase [5], which renders bacteria sensitive top-chlorophenylalanine. This phenylalanine analog is used ascounterselective agent for the isolation of those strains in whicha second recombination event occurred. To assure a proper levelof pheSGly294 expression, its transcription was subjected to thecontrol of a psychrophilic synthetic promoter (P13).Construction of a PhTAC125 gsp- [�gspCN] strain: Toinactivate the T2SS pathway in PhTAC125 (Figure 2), a deletionstrategy was applied. Two genomic fragments were PCRamplified by using specific oligonucleotides as primers. Theycorrespond to the 5’ 360 bp portion of gspC and 3’ 300 bpportion of gspN respectively. The fragments were suitablydigested and cloned into the Vs vector. The resulting vector(VsCN) was mobilized by intergeneric conjugation [1] intoPhTAC125, and the cells were plated at 4˚C on TYP solidmedium containing 30 �g/ml carbenicellin to select those inwhich a single recombination event occurred. Second recombi-nation event was induced by repeated plating of mutantpsychrophilic cells at 4˚C on minimum solid medium containing20 mM p-ClPhe. The occurrence of the correct deletion waschecked by sequencing the specific PCR fragments.Phenotypic characterization of PhTAC125 gsp- strain:The global exo-proteolytic activity of the PhTAC125 gsp-strainwas analyzed by in gel zymography and compared to that of thewild type strain. As shown in Figure 3, culture supernatant ofgsp- strain contains a reduced number of exo-proteases.Conclusion: We report here a cell engineering approach tothe construction of a Ph TAC125 strain with reduced exo-protease activity. By applying a gene-placements strategy, weobtained a mutant strain in which the gene cluster encoding theT2SS was almost totally deleted. While the growth behaviorand some physiological features of the gsp- mutant areindistinguishable from the wild type ones, the deleted straindisplays a remarkable reduction in the protease content in theculture supernatant. This aspect makes the PhTAC125gsp-

mutant a promising host for the recombinant secretion into thehost extra-cellular medium of proteins with biotechnologicalpotential.


Schematic representation of the PhTAC125 Vs suicide vector. mcs,Multiple cloning site; oriT, origin of conjugative transfer [1]; oriC andAmpR, origin of replication and beta-lactamase encoding gene from thepUC18 vector; P13, synthetic psychrophilic promoter; pheSGly294, E. coligene encoding a mutated version of the subunit of Phe-tRNA synthase.


Genetic organization of the PhTAC125 gsp cluster and gspCN deletion. The PhTAC125 gsp- mutant was generated by deleting a genomic regioncorresponding to that displayed into the dotted rectangle.



AcknowledgementsThis work was supported by grants from Ministero dell’Uni-versita e della Ricerca Scientifica (Progetti di Rilevante InteresseNazionale 2003; FIRB 2001), of Programma Nazionale diRicerche in Antartide 2004 and of Regione Campania L.R. 05/03. Support from the National Center of Excellence inMolecular Medicine (MIUR – Rome) and from the RegionalCenter of Competence (CRdC ATIBB, Regione Campania –Naples) is gratefully acknowledged.References1. Duilio A, Tutino ML and Marino G: Recombinant protein

production in Antarctic Gram negativebacteria.Methods Mol Biol 2004, 267:225–237.

2. Duilio A, Marino G, Mele A, Sannia G and Tutino ML:Ufficio Italiano Brevetti e Marchi n. RM2003/A000155.

3. Tutino ML, Duilio A, Parrilli E, Remaut E, Sannia G andMarino G: A novel replication element from anAntarctic plasmid as a tool for the expression ofproteins at low temperature. Extremophiles 2001,5:257–264.

4. Medigue C, Krin E, Pascal G and Barbe V, et al: Copingwith cold: the genome of the versatile marineAntarctica bacterium Pseudoalteromonas halo-planktis TAC125. Genome Research 2005, 15:1325–35.

5. Kast P: pKSS – a second-generation general purposecloning vector for efficient positive selection ofrecombinant clones. Gene 1994, 138:109–14.

P37Improvement of the energy metabolism ofrecombinant CHO cells by cell sorting for reducedmitochondrial membrane potentialGeorg Hinterkorner, Gudrun Brugger, Dethardt Muller,Friedemann Hesse, Renate Kunert, Hermann Katingerand Nicole BorthInstitute for Applied Microbiology, Department ofBiotechnology, Universitat fur Bodenkultur, Muthgasse 18,1190 Vienna, Austria


Background: Mammalian cells, when cultivated in-vitro, arecharacterised by an inefficient glucose metabolism, whichleads to the production of lactic acid. Specific glucose uptakerates and corresponding lactate production rates aredependent on the cell line as well as on glucose concentrationand growth rate. To ensure sufficient supply of energy, cellsmetabolise glutamine, which improves viability and growthrates, but results in the release of ammonium into themedium. High ammonium concentrations, however, havebeen shown to impair product glycosylation. As themitochondrial membrane potential was shown to correlateto cell specific glucose uptake rates, Rhodamine 123, alipophilic cationic dye was used for cell sorting. Tworecombinant CHO cell lines with known differences in lacticacid production rate were used as model cell lines andsubclones obtained by sorting for low and high mitochondrialmembrane potential, respectively.Results: The two cell lines C2F5 and C2G12, which bothproduce a human monoclonal antibody and which wereobtained by a similar transfection and amplification protocol,differ in their specific glucose uptake and lactate productionrate, as well as in their growth rates and maximum cell counts.To isolate cells with an altered energy metabolism, C2G12 cellswere stained with Rh123 and sorted both for a lower and ahigher mitochondrial membrane potential into 96 well plates.Batch cultures in Spinner bottles were run of the best subcloneof each sort.Conclusion: Using sorting protocol based on a simple stainingmethod for mitochondrial membrane potential we were able toisolate subclones from an established monoclonal antibodyproduction cell line with significantly altered physiologicalproperties. The subclone sorted for lower mitochondrialmembrane potential had a faster growth rate, attained higherfinal cell concentrations in batch cultures, had lower glucose andglutamine uptake and lactate production rates as well as a higherspecific production rate. The subclone sorted for highmitochondrial membrane potential on the other hand had alower growth rate and final cell count, increased glucose andglutamine consumption and lactate production rates. Thesesubclones can now be used for genomic or proteomic analysis ofproperties that characterise a cell line with efficient or inefficientmetabolism. In addition, the method described is a valuable toolfor cell line development and optimisation, offering thepossibility to isolate subclones with both superior and inferiorproperties. Future cell line development programs can now beextended from merely looking for a cell line with a high specificproduction rate, to one, which in addition has optimisedmetabolic properties.


Gelatin zymography of PhTAC125 wt and gsp- supernatants. Psychro-philic cells were grown in TYP medium at 4˚C till late exponential phase.Culture supernatants were recovered by culture centrifugation, 10 timesconcentrated and loaded onto a 10% SDS-PAGE containing bovinegelatin. After the electrophoresis run, the gel was washed to remove theNa-SDS and incubated in the development solution overnight at 15˚C.Finally the gel is stained with Comassie blue and destained. Molecularweight markers were marked in kDa.



P38Production of membrane proteins in yeastRichard AJ Darby, Mohammed Jamshad, Ljuban Grgicand Roslyn M BillSchool of Life and Health Sciences, Aston University, AstonTriangle, Birmingham, UK


Background: Yeast is an important and versatile organism forstudying membrane proteins. It is easy to cultivate and canperform higher eukaryote-like post-translational modifications.S. cerevisiae has a fully-sequenced genome and there are severalcollections of deletion strains available, whilst P. pastoris canproduce very high cell densities (230 g/l).Results: We have used both S. cerevisiae and P. pastoris toover-produce the following His6 and His10 carboxyl terminalfused membrane proteins. CD81 – 26 kDa tetraspanin protein(TAPA-1) that may play an important role in the regulation oflymphoma cell growth and may also act as the viral receptor forHepatitis C-Virus. CD82 – 30 kDa tetraspanin protein thatassociates with CD4 or CD8 cells and delivers co-stimulatorysignals for the TCR/CD3 pathway. MC4R – 37 kDa seventransmembrane G-protein coupled receptor, present onneurons in the hypothalamus region of the brain and predictedto have a role in the feast or fast signalling pathway. Adt2p –34 kDa six transmembrane protein that catalyses the exchange ofADP and ATP across the yeast mitochondrial inner membrane.Conclusion: We show that yeasts are flexible productionorganisms for a range of different membrane proteins. Theyields are such that future structure-activity relationship studiescan be initiated via reconstitution, crystallization for X-raydiffraction or NMR experiments.AcknowledgementsThis work is supported by the European Commission viacontract LSHG-CT-2004-504601 (E-MeP) to RMB. We alsoacknowledge Advantage West Midlands for their support of ourlaboratory.

P39Fast and efficient generation of influenza A viruslike particles from synthetic genesTheresa Schinko1, Haruthai Thaisuchat1,Hendrik Viljoen2, Nisha Padhye2,3

and Reingard Grabherr11Institute of Applied Microbiology, University of NaturalResources and Applied Life Sciences, Vienna, Austria

2Department of Chemical Engineering, University of Nebraska,Lincoln, NE 68588 USA3Megabase Research Products, 2726N.48th Street, Lincoln,NE 68504 USA


Background: With the recent emergence of the bird flu inmany European countries, molecular biologists are challengedmore then ever to advance the present methods of vaccinedevelopment. We have developed a method that is based on thefollowing key elements: safety, efficacy and rapidity. Influenza Avirus like particles (VLP) were generated in insect cells by co-transfection of especially designed plasmids. In order to produceVLPs, four recombinant baculoviruses were generated eachcontaining two influenza genes under control of the Autographacalifornica multiple nuclear polyhedrosis virus (AcMNPV) pH andp10 promotors for high level expression in Sf9 insect cells. VLPscontained 8 of 10 influenza A virus proteins of strain PR8,missing NS1 and NS2. Alternatively, VLPs were generated, byassembly of just three proteins, HA, NA and M1, which areresponsible for induction of the immune system in vivo. Allinfluenza genes have been produced from synthetical oligonu-cleotides, using a rapid thermocycler, the PCRJetâ. Syntheticgenes of new emerging influenza A variants can be producedaccurately and rapidly by using this technology. Recombinantbaculoviruses were generated using the Bac-to-Bac system byhomologous recombination between a transfervector and thebaculoviral shuttlevector (bacmid) in DH10Bac cells. Byoptimizing DNA synthesis and gene transfer into Sf9 cells, weanticipate major improvements in flexibility, speed and yield ofinfluenza vaccine production as compared to available technol-ogies.Results: Co-transfection of bacmids resulted in the generationof influenza A virus like particles in the supernatant of Sf9 cells.VLPs were purified by means of Sucrose gradient centrifugationand the expected results were confirmed by Electron micro-scopy, Western Blot analysis and hemagglutination assays.

P40Protein expression for structural studiesYoav Peleg1,6, Shira Albeck1,6, Yigal Burstein2,6,Orly Dym1,6, Yossi Jacobovitch1,6, Nurit Levy1,6,Ran Meged1,6, Yigal Michael1,6, Jaime Prilusky3,6,Gideon Schreiber4,6, Israel Silman5,6, Tamar Unger1,6

and Joel L Sussman1,61Department of Structural Biology, The Weizmann Institute ofScience, Rehovot 76100, Israel

Table 1 (abstract P36) Numbers in brackets are percent of the corresponding value of the parental cell line

C2F5 C2G12 C2G12 low C2G12 high

Inoculation cell concentration [cells/ml] 0,2 106 0,2 106 0,2 106 0,2 106

Cell count after 4 days [cells/ml] 2,0 106 0,8 106 1,4 106 (170) 0,5 106 (61)Specific growth rate [/day] 0,61 0,39 0,51 (131) 0,26 (67)Specific Glucose uptake [pg/cell/day] 525 900 660 (73) 1170 (130)Specific Glutamine uptake [pg/cell/day] 130 185 165 (88) 245 (132)Specific Lactate production [pg/cell/day] 505 700 420 (60) 1075 (154)Specific mAb production [pg/cell/day] 6,3 3,7 7,3 (195) 4,4 (118)Rh123 fluorescence at 2 g/l glucose [rel.U] 300 455 360 (80) 510 (112)



2Department of Organic Chemistry, The Weizmann Instituteof Science, Rehovot 76100, Israel3Department of Biological Services, The Weizmann Institute ofScience, Rehovot 76100, Israel4Department of Biological Chemistry, The Weizmann Instituteof Science, Rehovot 76100, Israel5Department of Neurobiology, The Weizmann Institute ofScience, Rehovot 76100, Israel6The Israel Structural Proteomics Center (ISPC),The Weizmann Institute of Science, Rehovot 76100, Israel


Background: Protein structure determination is an essentialtool for studying protein function, and assists the design of noveldrugs. The goal of the Israel Structural Proteomics Center (ISPC) isto determine the structures of proteins related to human health intheir functional context http://www.weizmann.ac.il/ISPC[1]. One ofthe bottlenecks encountered, primarily for eukaryotic proteins, isthe production of soluble and correctly folded proteins suitable forcrystallization trials. In order to overcome this obstacle, we haveapplied various expression strategies. E. coli is the expressionsystem of choice, in which different parameters are tested inparallel. In cases in which post-translational modification is essentialfor obtaining a functional protein, eukaryotic expression systemssuch as Pichia pastoris or baculovirus are being employed.Results: Each target is expressed in parallel in severalexpression vectors in E. coli. Design of vectors, which harborthe same restriction sites and code for a cleavable N-terminalHis-tag facilitate DNA cloning and purification, respectively.When necessary, a target protein is co-expressed with itsnatural binding partners or with molecular chaperones. Resultsof a representative protein expression experiment are shownbelow. Figure 1 demonstrates the effect of co-expression ofmolecular chaperones in E. coli on protein solubility. Levels ofsoluble protein were significantly increased when the proteinwas co-expressed with certain combinations of molecularchaperones (Figure 1, C and 1D).

Conclusion: Rapid evaluation of protein expression under small-scale culture conditions is essential for implementing an efficienthigh-throughput protein production process. Parallel utilization of arepertoire of protein expression strategies resulted in a significantincrease in the number of soluble proteins obtained.Reference1. Albeck S, Burstein Y, Dym O, Jacobovitch Y, Levi N, Meged R,

Michael Y, Peleg Y, Prilusky J, Schreiber G, Silman I, Unger T andSussman JL: Three-dimensional structure determinationof proteins related to human health in their functionalcontext at the Israel Structural Proteomics Center(ISPC). Acta Crystallogr D Biol Crystallogr 2005, 61:1364–72.

P41Abstract withdrawn


P42Expression and purification of the D2 dopaminereceptor and the Neurokinin A receptorCedric Fiez-Vandal1, Renaud Wagner1, Franc Pattus1

and So Iwata21UMR 7175, Departement Recepteurs et ProteinesMembranaires, Ecole Superieure de Biotechnologie deStrasbourg, Bd Sebastien Brandt, BP 10413, 67412 IllkirchCedex, France2Membrane Protein Crystallography Group, Wolfson Lab,Biochemistry Building, Department of Biological Sciences,London Imperial College, South Kensington Campus, ExhibitionRoad, London SW72AZ, UK


Background: The D2 dopamine receptor (D2DR) and theNeurokinin A receptor (NK2R) are G-protein-coupled receptors(GPCRs). The first one is a major target of antipsychotics andParkinson’s disease drugs while the second is involved in smoothmuscle contractions. GPCRs, also known as seven transmembranereceptors (7TMRs), represent by far the largest family of plasmamembrane receptors, comprising approximately 1000members in thehuman genome. They regulate virtually all known physiologicalprocesses in mammals including the senses of smell, taste and vision.Indicative of their central importance in current clinical therapeutics isthe very large number of drugs that target these receptors, eitherdirectly or indirectly. Although much progress has been made in thepharmacological characterization of a large number of GPCRs, theonly three-dimensional structure available is that of bovine rhodopsin.A 3D-structure ofD2DRorNK2Rwould increase our understandingof itsmolecularmechanismandof the signal transductionof allGPCRs.Results: In order to produce the large and homogenousreceptor preparations required for structural studies, heterolo-gous production procedures have been established using themethylotrophic yeast Pichia pastoris (D2DR) and SFV- infectedmammalian cell lines (NK2R). The receptors, produced as a fusionprotein with both a Flag-tag and a His10 tag at their Nterminus anda biotinylation domain at their C-terminus for immuno-detectionand purification, shows specific binding activity with their selectiveantagonists (Spiperone for D2DR and SR48,968 for NK2R).Preliminary purification procedures in a scalable fashion weredesigned using a mixture of sugarbased detergents, Cholesteryl-HemiSuccinate and other lipids (POPC, POPE, POPG).


Protein co-expression with molecular chaperones in E. coli. Arrowindicates the position of the protein. A- Expression without chaperones;B–D: Expression with various combinations of molecular chaperones; P,Pellet; S, Soluble fraction; N, Protein following capture on Ni-beads.



P43Efficient, antibody-mediated allosteric activationof an immobilized, E. coli beta-galactosidaserecombinant biosensorRosa M Ferraz1,3, Anna Arıs1, Gregorio Alvaro2

and Antonio Villaverde11Institut de Biotecnologia i de Biomedicina and Departamentde Genetica i de Microbiologia, Universitat Autonoma deBarcelona, Bellaterra, 08193 Barcelona, Spain2Departament d’ Enginyeria Quımica, Universitat Autonomade Barcelona, Bellaterra, 08193 Barcelona, Spain3Departament de Matematica Aplicada IV, UniversitatPolitecnica de Catalunya. Campus Nord, Jordi Girona 1-3,08034, Barcelona, Spain


Background: Allosteric biosensors are based on engineeredreporter enzymes responsive to analyte binding throughdetectable changes in the specific activity [1, 2]. Since antibodiesare efficient allosteric effectors, such devices are especiallyuseful for the diagnosis of infectious diseases. In previousstudies, we have introduced an antigenic peptide from the HIVstructural protein gp41, spanning amino acids 579 to 613 of theEnv precursor [3], into a permissive site of E. coli beta-galactosidase, resulting in the chimeric protein NF795gpC. Inthe presence of immune sera or anti-peptide antibodies, thesoluble enzyme is efficiently activated in a fast and homogeneousimmunoassay [3, 4]. To further develop biosensor devices insolid phases, with wider applicability in field conditions, we havehere explored the allosteric properties of NF795gpC whenimmobilized in an agarose substrate.Results: The immobilization process was optimised by using acommercial beta-galactosidase and different crosslinked types ofagarose, from which we finally considered 4BCL as the optimumfor the assay because it permitted the total accommodation ofthe protein in the agarose porus. We monitored the binding

process through activity assays (at pH 9.5) along time insupernatant and suspension, always controlling the stability ofthe protein [5]. The decrease of activity in the supernatant andnot in the suspension indicates the efficient joining of the solubleprotein to the support.After that, we checked the activation capacity of immobilizedNF795gpC upon the exposure to an anti-peptide antibody. InFigure 1 it is compared the allosteric activation of theimmobilized NF795gpC beta-galactosidase and its solubleversion. Clearly, the protein maintains its allosteric activationproperties even in its immobilized form, although with adifferential, and slightly delayed profile regarding the solubleform.Conclusion: NF795gpC is responsive to the allosteric mod-ification mediated by anti-peptide antibodies even whenimmobilized in an agarose support, proving that the conforma-tional modifications induced by the adaptive binding andsupporting activation do not require the protein in solution.The different activation kinetics observed in soluble andimmobilized enzyme versions could be due to either structuralconstraints to the active site conformational modulation or to adifferential accessibility of the antigenic peptide (the allostericreceptor) to activating antibodies. The obtained results arepromising regarding the possible use of allosteric biosensors insolid-phase platforms.AcknowledgementsThis work has been funded by BIO2004-00700 from MEC, Spainand 2005SGR-00956 (AGAUR). Rosa Maria Ferraz is recipient ofa doctoral fellowship from Departament d’Universitats, Recercai Societat de la Informacio de la Generalitat de Catalunya i delFons Social Europeu.References1. Villaverde A: Allosteric enzymes as biosensors for

molecular diagnosis. FEBS Lett 2003, 554:169–172.2. Ferraz RM, Vera A, Arıs A and Villaverde A: Insertional

protein engineering for analytical molecular sensing.Microb cell Fact 2006, 5:15.


Coloured product of the ONPG hydrolysis formed by immobilized (A) and soluble (B) beta-galactosidase NF795gpC in presence () and in absence () ofan anti-peptide antibody.



3. Ferrer-Miralles N, Feliu JX, Vandevuer S, Muller A, Cabrera-Crespo J, Ortmans I, Hoffmann F, Cazorla D, Rinas U, Prevost Mand Villaverde A: Engineering regulable E. coli beta-galactosidases as biosensors for anti-HIV antibodydetection inhumansera. J Biol Chem2001,276:40087–40095.

4. Ferraz RM, Arıs A and Villaverde A: Profiling theallosteric response of an engineered beta-galactosi-dase to its effector, anti-HIV antibody. Biochem BiophysRes Commun 2004, 314:854–860.

5. Alvaro G, Fernandez-La Fuente R, Blanco RM andGuisan JM: Immobilization-Stabilization of PenicillinG Acylase from Escherichia coli . Appl Biochem Biotechnol1990, 26:181–195.

P44Cost-effective production of labeled recombinantproteins in E. coli using minimal mediumAleksei Rozkov, Bert Larsson, Robert Bjornestedt,Patrik Stromberg, Fredrik Lindqvist and Fritz SchweikartGlobal Protein Science & Supply, DECS, AstraZeneca R&D,Sodertalje, S-15185 Sweden


Background: Application of NMR spectroscopy in drug dis-covery often requires large quantities (hundredsmg) of recombinantproteins labeled with stable isotopes (nitrogen-15, deuterium andcarbon-13 in various combinations). Expression of recombinantproteins in E. coli is traditionally carried out using rich media such asLB due to simplicity, low cost and familiarity. Use of commerciallabeled rich medium with the same characteristics as a conventionalLB is a very attractive option for small-scale production of labeledproteins. However, larger scale cultivation with such media couldbecome cost-prohibitive. In this study we describe how minimalmediumwas used for large-scale cost-effective production of labeledrecombinant proteins in E. coli.Results: Uniform high-level labeling was achieved by using alabeled substance as a sole source of the corresponding element, i.e.ammonium salts as a nitrogen-15 source and glucose as a carbon-13source. In the experiments requiring deuteration, deuterium oxide(99.9%) and ortho-phosphoric acidwere used as deuterium sources.The degree of deuterium incorporation in recombinant proteinachieved by this method ranges from 84 to 89 per cent (remaininghydrogen atoms are derived from glucose). Use of these labeledsubstances allowed large savings of medium costs compared tocommercially available rich medium. The medium cost savings were6-fold for triple labeled (2H, 15N, 13C) and 20-fold for 15N-labeledmedium. Achieved savings for production of 100 gram of triple-labeled biomass, for instance, can reach tens of thousands EUR.While rich medium is an ill-defined complex mixture, a minimalmedium is composed of defined components, which areconsumed at constant specific rates by known metabolicpathways until one of the essential nutrients is exhausted. In

labeling experiments the concentration of the most expensivemedium component is set to be limiting, thus minimizing thecost. Nitrogen requirement can be easily calculated due to itsrelatively constant content in biomass (14%) [1]. This value canincrease only slightly during protein overexpression due tohigher nitrogen content in protein (18%) [2]. Carbon demand(as glucose) cannot be predicted as easily. Although carboncontent in biomass is fairly constant at 50%, glucose also servesas an energy source and is degraded into carbon dioxide andother by-products. These energy requirements for biomassbiosynthesis, as well as for maintenance, vary depending on theplasmid, host strain and induction conditions (Table 1).The culture growing on minimal medium with glucose in shake flaskis much less prone to oxygen limitation as opposed to rich mediumdue to a greatly reduced specific growth rate (1.5 vs. 0.5 1/h) [3].This makes a shake flask culture more suitable for small-scaleexperiments with a good potential for scale-up if needed.Cells growing on minimal medium in batch culture metabolizeglucose with a constant stoichiometry. Therefore, biomass growthis directly proportional to carbon dioxide evolution and oxygen andalkali consumption. These parameters can be used for indirectestimation of biomass in bioreactor and for triggering pre-programmed events such as induction and temperature changes.Unattended operation reduces a need for overtime and to increasereproducibility of the process.Conclusion: Use of minimal medium significantly reduced thecosts of labeled protein production. Defined physiologicalconditions, better process control and monitoring of nutrientsenabled to achieve higher expression yields in fermentercompared to flasks with LB medium (Figure 1).References1. Nielsen J and Villadsen J: Bioreaction Engineering

Principles. Plenum Press, New York; 1994.2. Oura E: Biomass from carbohydrates. Biotechnology

VCH Verlag. Weinheim, Germany: Dellweg H 1983, 3:3–42.3. Gupta A and Rao G: A study of oxygen transfer in

shake flasks using a non-invasive oxygen sensor.Biotechnol Bioeng 2003, 84:351–358.

P45Structural biology of Helicobacter pyloritype IV secretion systemAlessandro Angelini1,2, Laura Cendron1,2, Anke Seydel1,2,Nicola Barison2, Tommaso Tosi2, Roberto Battistutta1,2

and Giuseppe Zanotti1,21Venetian Institute of Molecular Medicine (VIMM), Padua,Italy2Department of Chemistry, University of Padua, Padua, Italy


Background: Helicobacter pylori chronically infects the gastricmucosa of millions of people annually worldwide: it has been

Table 1 (abstract P44) Biomass yield coefficient (OD per g/L glucose) observed during production of 3 recombinant proteins inbatch culture with minimal medium.

BL21(DE3) Protein A BL21(DE3)* Protein B BL21(DE3) Protein C BL21(DE3) Protein C

before induction 1.5 1.2 1.3 1.3after induction 1.5 (0.1mM IPTG) 1.1 (0.5mM IPTG) 1.3 (0.05mM IPTG) 0.9 (0.5mM IPTG)



estimated that over 50% of the world population carries thisinfection. H. pylori has been associated with the development ofseveral diseases, like chronic gastritis, gastric and duodenalulcer, gastric adenocarcinoma and mucosa-associated lymphoma[1, 2, 3].The complete genome sequence of two different isolates of H.pylori (J99 and 26995) is known. The strains that contain a 37 kbforeign DNA region, called cag pathogenicity island (cag-PAI),cause the most severe form of virulence [4].The cag-PAI encodes for a functional type IV secretion apparatushomologous to the VirB/D4 Type IV Secretion System (T4SS) ofthe plant pathogen Agrobacterium tumefaciens and other Gram-negative bacteria [5]. T4SSs are involved in conjugal DNAtransfer, in the DNA delivery to (or uptake from) theenvironment, for instance the release of oncogenic DNA intoinfected plant cells by A. tumefaciens, or in the translocation ofeffector proteins [6, 7].The T4SS encoded by the cag-PAI of H. pylori is responsible for thetranslocation into the host cell of the protein CagA, a majorantigenic virulence factor encoded within the cag-PAI. Once

secreted into the gastric epithelial cells, CagA induces cellularmodifications, such as elongation and spreading of host cells [8].The aim of this structural genomic project is to determine the three-dimensional structure of most of the proteins encoded by the cag-PAI, a task that will allow to elucidate the function and theorganization of the entire T4SS of such a relevant pathogenicbacterium [9].Results: Protein production for structural studies presents oneof the most difficult and challenging tasks for heterologousexpression in E. coli. Generally, the protein must be native, active,soluble, highly pure, and concentrated. We have identifiedprotein insolubility/aggregation as the major bottlenecks in theprocess towards the determination of protein structures by X-ray diffraction. Each protein often needs separate handling andanalysis to determine tag choice, growth and buffer conditions foroptimal solubility. To speed up recombinant protein production,we have adopted a strategy of parallel expression of a proteinfrom a variety of vectors containing different tags and/or fusionpartners, and a variety of E. coli host strains.To this point in time, we have cloned, expressed, and purifiedseveral proteins of the cag pathogenicity island of H. pylori. Theyall have been expressed in E. coli. We have already determinedthe structure of CagZ [10] and CagS, using the Se-Met method.We have also obtained crystals of an other protein, along withcrystallization tests on other cag proteins.Perspectives: We believe these studies will also furnishvaluable information for vaccine production and provide insightsinto the mechanism of H. pylori pathogenesis.References1. Covacci A, Telford JL, Del Giudice G, Parsonnet J and

Rappuoli R: Helicobacter pylori virulence and geneticgeography. Science 1999, 284:1328–1333.

2. Moss SF and Sood S: Helicobacter pylori . Curr Opin InfectDis 2003, 16:445–451.

3. Moss SF and Blaser MJ: Mechanisms of disease:Inflammation and the origins of cancer. Nat ClinPract Oncol 2005, 2:90–97.

4. Akopyants NS, Clifton SW, Kersulyte D, Crabtree JE,Youree BE, Reece CA, Bukanov NO, Drazek ES, Roe BAand Berg DE: Analyses of the cag pathogenicity islandof Helicobacter pylori. Mol Microbiol 1998, 28:37–53.

5. Bourzac KM and Guillemin K: Helicobacter pylori -hostcell interactions mediated by type IV secretion. CellMicrobiol 2005, 7:911–919.

6. Cascales E and Christie PJ: The versatile bacterial typeIV secretion systems. Nat Rev Microbiol 2003, 1:137–149.

7. Christie PJ, Atmakuri K, Krishnamoorthy V, Jakubowski Sand Cascales E: Biogenesis, architecture, and func-tion of bacterial type IV secretion systems. Annu RevMicrobiol 2005, 59:451–485.

8. Hatakeyama M and Higashi H: Helicobacter pylori CagA:a new paradigm for bacterial carcinogenesis. CancerSci 2005, 96:835–843.

9. Remaut H and Waksman G: Structural biology ofbacterial pathogenesis. Curr Opin Struct Biol 2004,14:161–170.

10. Cendron L, Seydel A, Angelini A, Battistutta R andZanotti G: Crystal structure of CagZ, a proteinfrom the Helicobacter pylori pathogenicity islandthat encodes for a type IV secretion system. J MolBiol 2004, 340:881–889.


Expression of 27.5 kDa soluble recombinant protein (marked by arrow).Expression is similar for LB and minimal medium for shake flask culturesand the same induction time. Fermenter cultivation allows a higherexpression level due to longer induction time. Soluble cell fractions wereprepared by chemical/enzymatic lysis. Induction 40 M IPTG at 20˚. Lane1. SeePlus 2+ MW ladder. Lane 2. Shake flask LB, time after induction 17h. Lane 3. Shake flask, minimal medium, time after induction 17 h. Lane 4.Fermenter 2H, 15N 5-litre scale fermentation, minimal medium, timeafter induction 40 h.



P46Expression of genes encoding membrane proteinsfrom the hyperthermophilic Archeon Pyroccocusabyssi in Pichia pastorisCecile Labarre, Karine Blondeau and Herman van Til-beurghIBBMC bat 430, South PARIS University, Orsay cedex, France


Experimental strategies, first results and identifed bottlenecks ofa structural genomics initiative on proteins of hyperthermophilicArchaea Pyroccocus abyssi are discussed here. Five ORFs havebeen cloned by a standard protocol in the methylotrophic Pichiapastoris expression system, using two different constructs, withor without the signal sequence a mating factor of S. cerevisiae.The C-myc epitope and 6 His codons were added at the 3’-endof the targeted genes to allow immunodetection of recombinantproteins and to facilitate their further purification. We haveselected at least one producer clone for each protein of interestand for almost each construction. All the membrane proteins ofinterest were produced in Erlenmeyer flasks culture and in fed-batch cultivation using for large scale cells preparation.Production efficiencies were relatively low in both cases butthe quantities of biomass produced have allowed us to collectsufficient amount of membrane fractions. The five proteins wereextracted, solubilized and partially purified. Further work shouldinvolve structural biology investigations.

P47Recombinant expression of disulfide-rich proteins:carboxypeptidase inhibitors as model proteinsLaura Sanglas, Sılvia Bronsoms, Joan L Arolas,Julia Lorenzo and Francesc X AvilesInstitut de Biotecnologia i Biomedicina and Departament deBioquımica i Biologia Molecular, Universitat Autonoma deBarcelona, 08193 Bellaterra, Barcelona, Spain


Background: A large number of physiologically relevantproteins comprise disulfide bonds: protease inhibitors, pro-teases, nucleases, growth factors, venom neurotoxins, andothers. The recombinant expression of these proteins usuallyrepresents an important challenge due to the presence ofdisulfide bonds, which may affect protein solubility inside the celland lead to misfolding and subsequent aggregation.In the last years, specific inhibitors of carboxypeptidases of theM14A and M14B subfamilies have emerged as potential drugsable to protect plants against insect attack and to improve bloodclot fibrinolysis [1]. However, the small size and high number ofdisulfide bonds present in these molecules strongly influencetheir recombinant production and further biochemical char-acterization. Here, we have tested different systems for thesuccessful expression of four carboxypeptidase inhibitors fromthe potato plant (PCI; 39 residues and 3 S-S), the intestinalparasite Ascaris suum (ACI; 65 residues and 4 S-S), the medicalleech Hirudo medicinalis (LCI; 66 residues and 4 S-S), and the softtick Rhipicephalus bursa (TCI; 75 residues and 6 S-S)[2].Results: The recombinant expression of PCI in the methylo-trophic yeast Pichia pastoris, using the pPCI9 vector (Invitrogen),resulted in a soluble and properly folded protein that was

secreted into the medium to a final yield of 5 mg/liter. Theutilization of the E. coli protease-deficient strain BL21(DE3) andthe vector pBAT4-OmpA [3] routinely yielded 15 mg/liter ofrecombinant protein, 10-fold higher than a previously describedE. coli system (pIN3OmpAIII vector in E. coli strain MC1061) [4].However, the scale-up from flask culture to fed-batch fermentordid not improve the previous system (70 versus 200 mg/liter) [5].Importantly, the PCI expressed in E. coli was completely foldedby addition of redox agents to the culture supernatant (2 mMCys-Cys/4 mM Cys, pH 8.5). Intracellular expression of PCI as athioredoxin fusion protein in E. coli ADA494 using the pET-32bvector led to extremely low yields of soluble protein.Recombinant LCI was cloned and expressed in periplasm usingE. coli BL21(DE3) cells and the pBAT4-OmpA vector. Approxi-mately 5 mg/liter of recombinant LCI were produced in shakerflasks, a moderate improvement compared with the previouslyreported expression systems in E.coli using MC1061 andADA494 cells with the pIN3OmpAIII and pET-32b vectors,respectively, both yielding 3 mg/liter [6]. The addition of redoxagents also rendered native folded protein. Recombinant TCIwas expressed in E. coli BL21(DE3) cells using the pBAT4-OmpAvector, with a yield of 1 mg/liter. Intracellular expressionattempts did not render recombinant TCI. Similar to PCI andLCI, the presence of redox agents was necessary to achieve afolded inhibitor. Scale-up studies for LCI and TCI wereperformed in a fed-batch fermentor using the conditionsoptimized for PCI. However, low yields of recombinant proteinwere obtained for both molecules (<5 mg/liter).Currently, we are working on the recombinant expression ofACI. This protein has been cloned in the pPICZa vector(Invitrogen) for expression in Pichia pastoris and in the pBAT4-OmpA and pET-32b vectors for expression in E. coli. Initialresults point out yeast as the most useful system for theexpression of recombinant ACI. Intracellular expression sys-tems are also being tested in E. coli.Conclusion: The use of BL21(DE3) cells and pBAT4-OmpAvector in combination with redox agents results in soluble andproperly folded protein for all tested inhibitors, indicating thatthe periplasmic expression is a good solution to cope with theproblems related to disulfide bond formation. However, thescale-up of this system does not render high protein yields. Onthe other hand, intracellular expression of these proteins usuallyleads to misfolding, aggregation and inclusion bodies formation.For all proteins, the extracellular expression in the yeastP. pastoris seems to be an adequate alternative to overcome thefolding problems. Finally, the overall results indicate that thehigher the number of disulfide bonds, the lower the proteinexpression yield.AcknowledgementsThis work has been supported by Grant BIO2004-05879(Ministerio de Educacion y Ciencia, Spain) and by the Centrede Referencia en Biotecnologia (Generalitat de Catalunya,Spain).References1. Arolas JL, Vendrell J, Aviles FX and Fricker LD: Metallo-

carboxypeptidases: emerging drug targets in bio-medicine. Curr Pharm Des 2006 in press.

2. Arolas JL, Lorenzo J, Rovira A, Castella J, Aviles FX andSommerhoff CP: A carboxypeptidase inhibitor fromthe tick Rhipicephalus bursa: isolation, cDNA cloning,



recombinant expression, and characterization. J BiolChem 2005, 280:3441–3448.

3. Peranen J, Rikkonen M, Hyvonen M and Kaariainen L: T7vectors with modified T7lac promoter for expres-sion of proteins in Escherichia coli. Anal Biochem 1996,236:371–373.

4. Molina MA, Aviles FX and Querol E: Expression of asynthetic gene encoding potato carboxypeptidaseinhibitor using a bacterial secretion vector. Gene1992, 116:129–138.

5. Marino-Buslje C, Molina MA, Canals F, Aviles FX andQuerol E: Overproduction of a recombinant carbox-ypeptidase inhibitor by optimization of fermenta-tion conditions. Appl Microbiol Biotechnol 1994,41:632–637.

6. Reverter D, Vendrell J, Canals F, J Horstmann, Aviles FX,Fritz H and Sommerhoff CP: A carboxypeptidaseinhibitor from the medical leech Hirudo medicinalis.Isolation, sequence analysis, cDNA cloning, recom-binant expression, and characterization. J Biol Chem1998, 273:32927–32933.

P48Automated purification of soluble histidine taggedintegrase of Tn21 expressed in E. coli cells in lowamountsIoana Grigorescu1, Anna Andersson1, Markus Galin1,Anita Jonsson1, Anders Molin1, Susanne Nyholm-Westin1,Carolina Johansson2, Lars Sundstrom2

and John Flensburg11GE Healthcare, Bio-Sciences AB, Bjorkgatan 30, SE-751 84Uppsala, Sweden2Department of Medical Biochemistry and Microbiology(IMBIM), BMC, Box 582, SE-751 23 Uppsala, Sweden


Background: Deca-Histidine tagged integrase Tn21, a basicDNA-binding protein with a molecular mass of 38 kDa, whenexpressed in E. coli bacteria resulted in inclusion bodies. To obtainnative, biologically active protein, it was decided to purify onlycytoplasmic soluble integrase, present in low amounts. The optimalgrowth conditions, low temperature and 20 hours of growth periodwere used to cultivate the bacterial cells to increase the amount ofthe soluble integrase. LC-MS/MS techniques were used as analyticaltechniques to measure concentration of the expressed proteinduring growth optimization.Results: AKTAxpressä, an automated system, which allowsmulti-step purification of protein, was used to purify theenzyme. Additional ready-to-run protocols increased theflexibility of combining different chromatographic techniques(affinity and ion exchange chromatography, gel filtration anddesalting) in the desired order.All purifications were performed at + 6˚C as the protein is labile.The method consisted of an immobilized metal chelatingchromatography as capture step. A column prepacked with NiSepharoseä High Performance was used to capture the deca-histidine tagged integrase. The system allowed the automatedcollection of peaks in the storage loops and the transfer of themajor peak to a gel filtration column (Hi Loadä 16/60Superdexä 75 prep grade). The purified protein was analyzedusing SDS-PAGE and MS techniques.

Conclusion: Using the optimized experimental conditions,5 mg of pure labile integron integrase of Tn21 was purified,sufficient enough for functional studies as well as crystallizationscreening.

P49Transcriptional analysis of protein production andinduction of unfolded protein response in Pichiapastoris expressing a Rhizopus oryzae lipase underthe FLD1 promoterDavid Resina1, Monika Bollok2, Francisco Valero1,Peter Neubauer2 and Pau Ferrer11Department of Chemical Engineering, Escola TecnicaSuperior d’Enginyeria, Universitat Autonoma de Barcelona,08193-Bellaterra (Cerdanyola del Valles), Spain2Bioprocess Engineering Laboratory, Department of Processand Environmental Engineering and Biocenter Oulu, Universityof Oulu, Finland


Background: Methanol-free high cell density fed-batch culti-vation strategies for the Pichia pastoris expression system havebeen recently developed by expressing a Rhizopus oryzae lipase(ROL) under the transcriptional control of FLD1 promoter (PFLD1)[1]. These cultivation strategies were based on the use ofsorbitol and methylamine as carbon and nitrogen source,respectively, during the induction phase of the cultivationprocess. The specific growth rate proved to be an importantparameter in the productivity of secreted ROL. Moreover,intracellular active product accumulation and a decrease in thespecific product secretion rate were observed along theinduction phase of the fermentation process. These resultssuggested the presence of a bottleneck(s) throughout thesynthesis and secretion process of the heterologous lipase.Recently [2], flow cytometry analyses of intracellular ROL levelsconfirmed that a fraction of the product was retained within thecell. Further, this intracellular product accumulation wasconcomitant with an increase on the BiP protein, a chaperoneof the HSP70 class that plays an important role in the unfoldedprotein response (UPR). Notably, the increase of BiP and ROLcontent in the cell was detected soon after the beginning of theinduction phase. Interestingly, the intracellular BiP and ROLprofiles were different depending on the specific growth rate ofthe cells.In this study, we report the application of a sandwichhybridization assay-based technique [3] for quantification ofspecific mRNAs levels during the extracellular production ofROL in P. pastoris under the transcriptional control of PFLD1.These studies have been carried out in fed-batch cultures at twodifferent specific growth rates.Results: Fed-batch fermentations were performed at twodifferent controlled specific growth rates, namely at a limitedspecific growth rate of about 0.005 h-1 (20% of �max) and undercarbon excess (i.e. achieving a near- �max specific growth rate ofabout 0.02 h-1). These cultivations were performed with twodifferent strains: i) a P. pastoris X33-derived strain expressingROL under the transcriptional control of the P FLD1 and, 2) aP. pastoris GS115H-derived strain co-expressing the inducedform of the S. cerevisiae’s UPR transcription factor Hac1p geneunder the control of the constitutive GAP promoter and, theROL gene under the PFLD1 control [2].



The transcriptional levels of key genes involved in P. pastoris’ C1carbon compounds and amines metabolism (formaldehydedehydrogenase, FLD1, and alcohol oxidase, AOX1), proteinfolding (the BiP-encoding gene, Kar2, and protein disulfideisomerase gene, PDI), as well as the ROL gene were monitoredthroughout the fed-batch phase of cultivations.As expected, the quantitative RNA analyses demonstrated thattranscriptional levels of AOX and FLD are growth-ratedependant, remaining relatively low and constant at growth-limiting rates, but clearly induced by 2~3-fold during theinduction phase (growth on sorbitol and methylamine as solecarbon and nitrogen sources, respectively). Notably, inductionlevels were higher in the X33-derived strain than in GS115H/HAC1.PDI mRNA levels and profiles were also �-dependant. PDImRNA levels remained low and constant at low growth rate,whereas they were clearly induced (about 2-fold) under carbonexcess growth conditions, reaching a maximum after 70 h ofinduction in the X33-derived strain; after that point, PDI mRNAlevels decreased to the initial basal level towards the end of theinduction phase. Such induction was more moderate in theGS115H/HAC1 strain. Interestingly, PDI mRNA profiles fol-lowed a similar pattern as ROL mRNA levels, which clearlyincreased during the induction phase under carbon excessgrowth conditions, reaching a maximum after 60–70 h ofinduction. In contrast, Kar2 profiles were strikingly different,particularly in the X33-derived strains: at growth-limitingconditions Kar2 mRNA levels were sharply increased by 2~3fold, reaching a maximum soon after the onset of the inductionphase. After reaching this maximum, Kar2 mRNA levelsdecreased exponentially along the induction phase. This profilewas similar but less pronounced under carbon excess condi-tions. In contrast, Kar2 mRNA levels in the GS115H/HAC1strain remained rather constant along the induction phase of thecultivation.Conclusion: Overall, the bead-based mRNA sandwich hybri-dization assay is a useful instrument to follow transcriptionalevolution of key genes in protein production processes. In thisstudy, a clear cellular (stress) response to ROL production isshown at the transcriptional level, consistent with previous dataon intracellular stress protein markers levels [2]. Also, this studyprovides new insights on the interactions between physiologicalstate (growth rate) and protein expression in P. pastoris. Thesepreliminary results strongly suggest that the decrease in ROLsecretion rates observed along the induction phase of fed-batchcultivations [2] may be partially related with a downregulation ofROL transcription levels at later stages of the induction phase.AcknowledgementsThe present work has been supported by the SpanishProgramme on Chemical Processes Technologies (ProjectCTQ2004-00300) and the grant No. 2005SGR 00698 from theDURSI (Generalitat de Catalunya) as well as by projects fromAcademy of Finland (project no. 203839) and the FinnishFunding Agency for Technology and Innovation (TEKES, CORFproject of the Neobio program). The Department of ChemicalEngineering of the Universitat Autonoma de Barcelona is theUnit of Biochemical Engineering of the Centre de Referencia enBiotecnologia de la Generalitat de Catalunya. D.R. acknowledgesthe Spanish MEC for a predoctoral grant. We thank Dr. Bernard

de la Cruz (KGI, U.S.A.) for providing the putative nucleotidesequence from the Kar2 gene from P. pastoris.References1. Resina D, Cos O, Ferrer P and Valero F: Developing high

cell density cultivation strategies. Biotechnol Bioeng2005, 80:65–69.

2. Resina D, Cos O, Gasser B, Mauer M, Valero F,Mattanovich D and Ferrer P: Analysis and engineeringof bottlenecks in Rhizopus oryzae lipase productionin Pichia pastoris using the nitrogen source-regu-lated FLD1 promoter. in press.

3. Rautio J, Barken KB, Lahdenpera J, Breitenstein A, Molin Sand Neubauer P: Sandwich hybridisation assay forquantitative detection of yeast RNAs in crude celllysates. Microb Cell Fact 2003, 2:4.

P50Determination of plasmid content in eukaryoticand prokaryotic cells using Real-Time PCRAdriano Azzoni1, Elisabete Carapuca1,D Miguel F Prazeres1, Gabriel A Monteiro1

and Filipe Mergulhao1,21Centro de Engenharia Biologica e Quımica, Instituto SuperiorTecnico, 1049-001 Lisbon, Portugal2LEPAE, Faculty of Engineering of the University of Porto,Chemical Engineering Department, 4200-465 Porto, Portugal


Background: Determination of the plasmid content in pro-karyotic cells during plasmid DNA (pDNA) production and ineukaryotic cells after transfection is crucial for DNA vaccinedevelopment. In Escherichia coli, pDNA is usually determinedafter plasmid extraction, either by UV absorbance or bydensitometry of ethidium bromide-stained agarose gels. Fluor-escence microscopy techniques are mainly used in eukaryoticcells. These techniques are time-consuming and labour-intensiveand can not be used for process control. Thus, a Real-Time PCRmethod was developed to monitor the plasmid content of E. coliand Chinese Hamster Ovary (CHO) cells.Results: Real-Time PCR with a 108 bp amplicon enabled thedetection of quantities as low as 4 copies of pDNA per cell inE. coli and 100 copies/cell in CHO cells transfected with highcopy-number plasmids (see Figure 1). The procedure can beperformed in less than 30 minutes and requires no sample pre-treatment. Analysis of pDNA number in E. coli harbouring aColE1 plasmid revealed that copy number reached a maximumduring exponential phase of growth and that this numberdecreased up to 80% upon entry into stationary phase.Additionally, the half-life of pDNA in transfected CHO cellswas 20 hours and around 100 copies of plasmid were stilldetected 6 days after transfection.Conclusion: Monitoring the pDNA content on producing andrecipient cells is crucial for DNA vaccine development. TheReal-Time PCR method developed on this work provides quasi-online results and is suitable for process control and optimisa-tion. The procedure was first developed for E. coli and wasquickly adapted to CHO cells. It is therefore likely that it can bemodified for application with other prokaryotic and eukaryoticsystems.



P51HYBMFA: a bioinformatics’ tool for batch-to-batch bioprocess optimisation supported byelementary flux analysisAna Teixeira1, Carlos Alves1, Paula Alves2,Manuel Carrondo1,2 and Rui Oliveira11REQUIMTE, Departamento de Quımica, Faculdade deCiencias e Tecnologia, Universidade Nova de Lisboa, P-2829-516 Caparica, Portugal2IBET/ITQB Instituto de Biologia Experimental e Tecnologia/Instituto de Tecnologia Quımica e Biologica, Apartado 12, P-2781-901 Oeiras, Portugal


Background: The central metabolic pathways of manybiological systems with industrial interest are currently known.Knowledge of intracellular fluxes is crucial to understand cellmetabolism. Bioreactor dynamic optimisation schemes couldprofit from the incorporation of this knowledge [1, 2]. Anumber of methods have been developed to study the structureof biochemical networks. The elementary flux modes (EFMs)method is particularly attractive since it allows to reducenetwork complexity to a minimal set of reactions [3].In previous studies [4], a bioprocess batch-to-batch optimisationscheme supported by a hybrid model was developed and appliedto the optimization of a BHK culture expressing the fusionglycoprotein IgG1-IL2. The main contribution of the presentstudy is to improve the previous method by incorporating theknowledge of the metabolic network. The incorporation of themetabolic network in the form of EFMs, may increase thegeneralization properties of the model and may thus contributeto the increase of the rate of success of the optimizationmethod.

Results: The proposed methodology is based on the premisethat the biological system under consideration is only partiallyknown in a mechanistic sense. Following this principle, a hybridparametric/nonparametric representation of the biologicalsystem was adopted to support a batch-to-batch optimizationscheme (Figure 1).


Variation of threshold cycle numbers using plasmid-free E. coli (left) and CHO cells (right) spiked with pDNA. In E. coli, 5 104 (), 2.5 105 () and 3.5 106

() cells were spiked with 8.5 10-4 to 8.5 ng of pDNA. With CHO, 1.2 104 cells were spiked with pDNA masses ranging from 5 pg to 100 ng. A linearworking range was obtained from 5 pg to 2.5 ng and was subsequently used for quantitation.


Proposed optimisation scheme.



In the first step, the metabolic network structure of thebiological system under study is analyzed using the elementaryflux modes technique. Elementary flux modes are the simplestpaths within a network that connect substrates with end-products [3], thus they define the minimum set of n species thatmust be considered for modelling and how they are connectedin a simplified reaction mechanism. The EFM analysis of a givenbiosystem results in m elementary flux modes and thecorresponding n � m stoichiometric matrix K, with n thenumber of compounds that must be considered for modelling.The BHK metabolic network analyzed in this work considers themost relevant pathways involving the two main nutrients(glucose and glutamine) within the central metabolism of BHKcells. The FluxAnalyzer software [3] was used to determine theEFMs of BHK metabolic network. There are seven EFMs describingthe BHK metabolic network. Assuming the balanced growthcondition it is possible to eliminate the intermediate metabolitesfrom each EFM resulting in a set of simplified reactions connectingextracellular substrates (glucose and glutamine) with end-products(lactate, ammonia, alanine, carbon dioxide, purine and pyrimidine).Furthermore, some assumptions concerning the fluxes were madebased on literature, resulting in five EFMs. The followingstoichiometric matrix was obtained.

μ

− − −− − −

−

=

k r r r r r r

K

1 0 0 0 0 0 0

0 1 1 0 0 2 0

0 0 0 1 1 5 0

0 2 0 0 0 0 0

0 0 0 1

d IgG1 2 3 4 5

22 2 0

0 0 0 1 0 0 0

0 0 0 0 0 0 1

X

Glc

Gln

Lac

Amm

Ala

I

V⎡

⎣

⎢⎢⎢⎢⎢⎢⎢⎢⎢

⎤

⎦

⎥⎥⎥⎥⎥⎥⎥⎥⎥ ggG

(1)

Note that K also accounts for cell growth and productformation as completely independent fluxes since the stoichio-metry of theses reactions is not accurately known.The state space vector is formed by the n concentrations ofcompounds of the final reactions set and additionally, theconcentrations of viable cells, Xv, and product, IgG:

c = [Xv, Glc, Gln, Lac, Amm, Ala, IgG]T. (2)

Once a reaction mechanism has been established using the EFMmethod, the next step is the identification of the EFM kineticsfrom data. Here we adopted a hybrid parametric/nonparametricmodel structure assuming that that reaction kinetics of EFM arepartially known or even completely unknown. This modelstructure can be formulated mathematically by the following twoequations [4]:

ddt

D ac

r c w c u= ( ) − + ( ), 3

r(c, w) = K<’j(c) � �j(c, w)>j = 1, ...,m (3b)

with r a vector of n volumetric reaction rates, K a n � mcoefficients matrix obtained from the elementary flux modesanalysis, ’,(c) are m kinetic functions established from mechanisticknowledge, �j(c,w) are m unknown kinetic functions, w a vector ofparameters that must be estimated from data, D is the dilution rate,u is a vector of n volumetric input rates (control inputs).For the system under study the vector of known kineticfunctions is given by:

’(c)= [Xv XvGlc XvGlc XvGln XvGln XvGlnGln Xv]T, (4)

whereas the vector of unknown kinetics is given by:� = [� - kd r1 r2 r3 r4 r5 rIgG]

T = �(Glc, Gln, Amm, w). (5)A backpropagation neural network with a single hidden layerwas used for the identification of �i(c, w):

�(c, w) = �maxs(w2s(w1c+b1)+b2) (6)

with �max a vector of scaling factors with dim(�max) = m, w1, b1,w2, b2 are parameter matrices associated with connectionsbetween the nodes of the network, w is a vectored form of w1,b1, w2, b2 and s(.) the sigmoid activation function defined asfollows:

s xe x( ) =

+( )−

1

17

Finally, the last term in eq. (3a), the control input vector is u =[0 FGlc FGln 0 0 0 0] with FGlc and FGln the volumetric feeding ratesof glucose and glutamine respectively.Off-line measurements of the seven state variables from fiveexperiments were used for model training and validation. Theneural network had three inputs: glucose and glutamine, themain limiting nutrients, and ammonia, the main toxic by-product.The output vector was formed by the seven unknown specifickinetics: �-kd, r1, r2, r3, r4, r5, rIgG. The criterion to stop thetraining was the minimum modelling error of the validation dataset. The best result was obtained with five hidden nodes.Figure 2 presents the hybrid modelling results for one of thetraining and one of the validation data sets. A relevant result isthe fact that the hybrid model was able to describe simulta-neously all five batches with high accuracy.


Hybrid model results for a training data set (a) and a validation data set (b).



Optimisation results: With the hybrid model just developed,the process performance (described as the glycoprotein quantityat the end of the bioreaction) is optimized with respect to controlinputs FGlc and FGln, using a micro-genetic algorithm [5]

maxu

IgG IL f fJ C t V t= ( ) ( ) ( )−1 2 8

The optimization (8) is constrained by the hybrid dynamicalmodel and by the risk of ANN inputs being outside the trustregion. The optimisation results are presented in Figure 3showing the optimal trajectories of viable cells, glucose,glutamine and product concentrations. The final product titreis 25 mg/l representing a 67% improvement of performanceobtained in the fed-batch experiments so far.According to the iterative batch-to-batch optimisation schemeshown in Fig. 1, the next step is to perform a new experiment tovalidate this optimization results. If measured data and predictedoptimal process trajectories deviate considerably, additionaliterations are performed until convergence of model andprocess performance is achieved.Conclusion: A bioinformatic tool was developed that integratesclassical optimal control and elementary flux analysis tools. A hybridparametric/nonparametric modelling framework was adopted thatdoes not require detailed knowledge of intracellular kinetics. Adynamic optimisationmethod is employed constrained by the risk ofnonparametric components unreliability. The method was appliedto a recombinant BHK-21 cell line expressing the fusionglycoprotein IgG2-IL1. The final hybrid model was then used tooptimise conditions that favour product formation showing that highproductivity increments are likely for the process at hand.AcknowledgementsThe authors acknowledge the financial support provided by theFundacao para a Ciencia e Tecnologia through project POCTI/BIO/57927/2004 and PhD grant SFRH/BD/13712/2003.References1. Provost A and Bastin G: Dynamic metabolic modeling

under balanced growth condition. J Process Control2004, 14:717–728.

2. Mahadevan R, Burgard A, Famili I, Van Dien S andSchilling C: Applications of metabolic modeling todrive bioprocess development for the production ofvalue-added chemicals. Biotechnol Bioprocess 2005,10:408–417.

3. Klamt S, Stelling J, Ginkel M and Gilles E: FluxAnalyser:exploring structure, pathways, and flux distributionsin metabolic networks on interactive flux maps.Bioinformatics 2003, 19:261–269.

4. Teixeira A, Cunha A, Clemente J, Moreira J, Cruz H,Alves P, Carrondo M and Oliveira R: Modelling andoptimisation of a recombinant BHK-21 cultivationprocess using hybrid grey-box systems. J Biotechnol2005, 118:290–303.

5. Krishnakumar K: Micro-Genetic Algorithms for Sta-tionary and Non-Stationary Function Optimization.SPIE: Intelligent Control and Adaptive Systems Philadelphia, PA.;1989, 1196.

P52Application of a genome-scale metabolic model tothe inference of nutritional requirements andmetabolic bottlenecks during recombinantprotein production in Escherichia coliSonia Carneiro, Isabel Rocha and Eugenio FerreiraCentro de Engenharia Biologica, Universidade do Minho,4710-057 Braga, PORTUGAL


Background: Escherichia coli has been the organism of choicefor the production of many recombinant proteins with hightherapeutic value. However, while the research on molecularbiology has allowed the development of very strong promoters,there are still some phenomena associated with this processthat hamper the full use of those technologies like the so-calledstringent response, caused by an imbalance in intracellular aminoacid uptake and the endogenous amino acid synthesis rates. Itoccurs due to high demands of certain amino acids forrecombinant protein production [1], causing several cellularresponses, from the inhibition of the synthesis of rRNA to theinduction of proteases. The main consequence of this responseis a decrease in process productivity due to the lower specificgrowth and production rates observed.An important research topic is the identification of themetabolic fluxes that suffer major changes during proteinproduction, giving information that can be used for theformulation of new strategies for improving the processperformance either by genetic or operational manipulations.In this work, a recombinant protein production process wasanalysed using genome-scale models and Flux Balance Analysis(FBA) [2] in order to identify potential sources of metabolicbottlenecks that can trigger the stringent response.Results: The existing genome-scale metabolic model of E. coli[3] was modified by including an equation for protein production(the eYFP – enhanced Yellow Fluorescent Protein), based on itsamino acids content. Additionally, equations that represent themetabolic burden caused by the presence of plasmids wereadded, based on knowledge about the precursor balances andenergetic requirements for plasmid replication and markerprotein expression [4]. This modified metabolic model was used




on simulations using the FBA approach [2], optimizing forbiomass and for recombinant protein synthesis.When comparing the distribution of internal fluxes for bothcases, it was observed that most fluxes over amino acidbiosynthetic reactions suffered an increase in the recombinantprotein production experiment (flux variation (a) in Table 1).Differences in amino acid composition between averageproteins in E. coli and eYFP. The variation of fluxes acrossmajor amino acid biosynthetic routes is also shown when onlyrecombinant protein is produced (a) and when both biomass andrecombinant proteins are being formed (b)Interestingly, there is not a clear correspondence betweenthose differences and the ones found in the relative amino acidcomposition of biomass and eYFP. This demonstrates that it isvery difficult to predict the consequences of the production ofrecombinant proteins in amino acid biosynthetic pathways.In a second experiment (Flux variation (b) in Table 1), the solutionspace of the genome-scale metabolic model was constrained byimposing limits on the protein production flux according to whatwas observed experimentally – 0.06 g protein.g-1 biomass. h-1 - whileoptimizing for biomass, forcing the model to represent both growthand protein production simultaneously. Taking again the optimiza-tion of biomass growth with no restrictions as reference, a trendsimilar as before was observed, although the variations are lower.Finally, cellular requirements represented by input fluxes to thecell of relevant nutrients were compared. It can be seen thatrecombinant protein production imposes additional demandsregarding ammonium and sulphates, while both oxygen require-ments and carbon dioxide excretions are lower (see Table 2).Conclusion: The application of Flux Balance Analysis to amodified genome-scale model of E. coli allowed the identificationof the metabolic fluxes for which there is a predicted increasewhen recombinant proteins are being expressed, elucidating theshift in metabolism that is necessary to occur upon induction.This insight can help to identify the main sources for theobserved stringent response and to design strategies to avoidthe occurrence of that phenomenon.

The need of high amino acid content for recombinant proteinproduction also affected the ammonium, sulphates and oxygenrequirements, giving useful indications for medium design.However, for the clarification of these phenomena associatedwith recombinant protein production it is now important toproceed with the integration of the information obtained fromgenome-scale models with experimental data obtained fromtranscriptomics, proteomics and metabolomics.AcknowledgementsThis work was supported by FCT – Portuguese ScienceFoundation under the scope of the recSysBio Project (POCI/BIO/60139/2004) and the PhD grant SFRH/BD/22863/2005 (S.Carneiro)References1. Chang DE, Smalley DJ and Conway T: Gene expression

profiling of Escherichia coli growth transitions: anexpanded stringent response model. Mol Microbiol2002, 45:289–306.

2. Edwards JS, Covert M and Palsson B: Metabolic model-ling of microbes: the flux-balance approach. EnvironMicrobiol 2002, 4:133–140.

3. Reed L, Vo TD, Schilling CH and Palsson B: An expandedgenome-scale model of Escherichia coli K-12 (iJR904GSM/GPR). Genome Biol 2003, 4:R54.

4. Ozkan P, Sariyar B, Utkur FO, Akman U and Hortacsu A:Metabolic flux analysis of recombinant proteinoverproduction in Escherichia coli. Biochem Eng J 2005,22:167–195.

P53Analysis of bottlenecks in Rhizopus oryzae lipaseproduction in Pichia pastoris using the nitrogensource-regulated formaldehyde dehydrogenasepromoter (PFLD1)David Resina1, Oriol Cos1, Brigitte Gasser2,Michael Mauer2, Hans Marx2, Michael Sauer2,Francisco Valero1, Diethard Mattanovich2

and Pau Ferrer11Departament d’Enginyeria Quımica, Escola Tecnica Superiord’Enginyeria, Universitat Autonoma de Barcelona, 08193-Bellaterra (Cerdanyola del Valles), Spain2Institute of Applied Microbiology, Department ofBiotechnology, University of Natural Resources and AppliedLife Sciences, Muthgasse 18, A-1190 Vienna, Austria


Background: Methanol-free high cell density fed-batch culti-vation strategies for the P. pastoris expression system have been

Table 2 (abstract P52) Differences in carbon dioxide secretionrate and in requirements regarding ammonium, sulphates andoxygen when the metabolism is optimized for recombinantprotein production as opposed to biomass production.

Flux Variation (a) Flux Variation (b)

NH4 requirements 16% 9%O2 requirements -27% -15%SO4 requirements 31% 17%CO2 secretion rate -23% -13%

Table 1 (abstract P52)Amino acids Biomass eYFP Flux variation (a) Flux variation (b)

L-ala 9,6 3,8 -38% 1%L-arg 5,5 2,5 -19% -11%L-asn 4,5 5,5 122% 69%L-asp 4,5 7,6 23% 13%L-cys 1,7 0,8 31% 17%L-gln 4,9 3,4 -59% -33%L-glu 4,9 6,7 38% 22%Gly 11,0 9,2 21% 12%L-his 1,8 4,2 351% 199%L-ile 5,4 5,0 75% 42%L-leu 8,4 8,4 84% 48%L-lys 6,4 8,4 142% 80%L-met 2,9 2,5 57% 32%L-phe 3,5 5,5 189% 107%L-pro 4,1 4,6 106% 60%L-ser 4,0 3,8 20% 12%L-thr 4,7 5,9 101% 57%L-trp 1,1 0,4 -6% -3%L-tyr 2,6 5,0 268% 152%L-val 7,9 6,7 58% 33%



recently developed by expressing a Rhizopus oryzae lipase (ROL)under the transcriptional control of the PFLD1 [1]. Thesecultivation strategies were based on the use of sorbitol andmethylamine as carbon and nitrogen source, respectively, duringthe induction phase of the cultivation process. Fed-batchfermentations were performed at three different specific growthrates and showed that productivities were strongly correlatedwith this parameter (i.e. with the cell’s physiological state).Moreover, intracellular active product accumulation and adecrease in the specific product secretion rate were observedalong the induction phase of the fermentation process. Theseresults suggested the presence of a bottleneck(s) throughoutthe synthesis and secretion process of the heterologous lipase.In this context, several studies have pointed out the presence ofan effect produced by the accumulation of misfolded proteins inthe endoplasmatic reticulum, namely the unfolded proteinresponse (UPR) of yeast, filamentous fungi and higher eukar-yotes. In particular, heterologous overexpression of someproteins in P. pastoris has been reported to provoke animportant accumulation of misfolded proteins in the endoplas-matic reticulum causing the activation of the unfolded proteinresponse (UPR) [2]. Some approaches have been made in orderto relax this bottleneck. For instance, the constitutive over-expression of the Aspergillus niger unfolded protein responsetranscription factor HacA in this host has been shown to enablean important increase of the secretion of several heterologousproteins in this host [3].Another potential bottleneck in recombinant protein secretionis the passage of secreted proteins through the yeast cell wall[4]. Some strategies have been attempted in order to increaseyeast cell wall porosity. For instance, inactivation of the Gas1pgene in S. cerevisiae may lead to a supersecretory phenotypeyielding a considerable increase in secreted protein production[5]. The Gas1p, a glycoprotein anchored to the outer leaflet ofthe plasma membrane through a glycosylphosphatidylinositol,plays a key role in yeast cell wall assembly.In this study, we report the application of flow cytometrytechniques to the analysis of molecular bottlenecks during theextracellular production of the Rhizopus oryzae lipase (ROL) inP. pastoris under the transcriptional control of PFLD1. More-over, these studies were carried out in a series of high celldensity cultivation fed-batch experiments.Results: Fed-batch fermentations were performed at twospecific growth rates, namely at a low specific growth rate ofabout 0.005 h-1 (20% of �max) and a high specific growth rate ofabout 0.02 h-1 (near �max). These cultivations were performedwith three different strains: i) a P. pastoris strain expressing ROLunder the PFLD1 control, ii) a P. pastoris strain co-expressing theinduced form of the S. cerevisiae’s UPR transcription factorHac1p gene under the control of the constitutive PGAPpromoter and, the ROL gene under the PFLD1 control, andiii) a P. pastoris strain with its Gas1p gene knocked outexpressing ROL under the PFLD1 control.Cell viability, the content of BiP, a chaperone of the HSP70 class thatplays an important role in the UPR, and intracellular ROL levelswere monitored throughout the fed-batch phase of the cultivationsset by immunofluorescent techniques using flow cytometry.The results obtained with the first strain demonstrated that i)cell viability is not significantly affected by ROL overexpression

in cells growing on methylamine as a sole nitrogen source, ii)ROL overexpression in P. pastoris under the control of thePFLD1 leads to intracellular accumulation of this protein andincreased BiP levels, i.e. triggering the UPR.The use of the engineered strain constitutively expressing theactivated Hac1 resulted in a 2-fold increase in the specific ROLproductivity in both cells growing at the lower and higherspecific growth rates. Remarkably, the maximum qp valuesobtained using the Hac1p-engineered strain were 3 and 1.7-foldhigher for cells growing at a � of 0.005 h-1 and 0.02 h-1,respectively, than those obtained in the corresponding cultiva-tions carried out with original host strain. However, theintracellular ROL and secretion rate (qp) profiles still followedthe same pattern as in the non-engineered strain, i.e. theysuffered an exponential decrease after the sharp increase duringthe initial stages of the induction phase.Finally, overexpression of ROL in the Gas1p knock out strainalso yielded a significant increase in extracellular lipase levels.Conclusion: The combined use of flow cytometry, classicanalytical techniques and a reduced set of engineered strains hasallowed us to assess and monitor the state of the ROL secretionprocess under bioprocess-relevant cultivation conditions.Overall, constitutive up-regulation of the UPR has a positiveeffect on ROL productivities of the process. However, resultsstrongly suggested that ROL overexpression may be downregulated at the transcriptional level as a result of the stressresponse (repression under secretion stress).In order to confirm this hypothesis, further quantitative analysesof the transcriptional levels of ROL and other key genes areunder way.Besides, the cell wall appears to pose an additional bottleneck tothe excretion of ROL to the extracellular medium, i.e. that thereare bottlenecks in different stages along the ROL synthesis,processing and secretion pathways. Moreover, the extent ofthese bottlenecks is dependent on growth conditions.AcknowledgementsThis work was supported by a grant from the Spanish Programon Chemical Processes Technologies (CTQ2004-00300).References1. Resina D, Cos O, Ferrer P and Valero F: Developing high

cell density fed-batch cultivation strategies forheterologous protein production in Pichia pastorisusing the nitrogen source-regulated FLD1 promo-ter. Biotechnol Bioeng 2005, 91:760–767.

2. Hohenblum H, Gasser B, Maurer M, Borth N andMattanovich D: Effects of gene dosage, promoters,and substrates on unfolded protein stress of recom-binant Pichia pastoris. Biotechnol Bioeng 2004,85:367–375.

3. Valkonen V, Ward M, Wang HM, Penttila M andSaloheimo M: Improvement of foreign-protein pro-duction in Aspergillus niger var. awamori by constitu-tive induction of the unfolded-protein response. ApplEnviron Microbiol 2003, 69:6979–6986.

4. Ferrer P, Diers I, Asenjo JA and Andrews BA: Yeast cellpermeabilizing �-1,3-glucanases: A tool for theintegration of downstream processes and metabolicengineering applications to yeast. Biotechnol Bioeng1998, 58:321–324.



5. Vai M, Brambilla L, Orlandi I, Rota N, Ranzi BM,Alberghina L and Porro D: Improved secretion ofnative human insulin-like growth factor 1 fromgas1 mutant Saccharomyces cerevisiae cells. ApplEnviron Microbiol 2000, 66:5477–5479.

P54Increasing the quality of recombinantproducts – Higher attraction of ribosomesleads to suppression of secondary ribosomebinding sitesUlf Liebal1, Olli Niemitalo1, Anu Mursula1, Andre Juffer2

and Peter Neubauer11Bioprocess Engineering Laboratory, Dept. Process &Environm. Engin. and Biocenter Oulu, P.O.Box 4300, Universityof Oulu, FIN-90014 Oulu, Finland; www.oulu.fi/bioprocess2Triacle Biocomputing, www.triacle-bc.com, FIN-90540 Oulu,Finland


Background: In translation initiation the 3’ end of the 16s rRNAbinds to the complementary Shine Dalgarno (SD) sequence.Together with bound initiation factors translation can subsequentlybegin at the AUG start codon. However, there may be SD relatedsequences throughout the coding region of the mRNA. Thesesecondary SD sequences can recruit ribosomes as well, in particularif they are embedded in purine rich regions [1]. The existence ofsuch secondary ribosome binding sites can greatly reduce theexpression efficiency since ribosomes recruited to the secondarySD site hinder elongating ribosomes in their progression. If a startcodon is nearby a secondary SD site even truncated protein couldbuild up in expense of full length protein [2].Results: In our attempts to increase the production ofrecombinant Wnt4 protein in E. coli BL21(DE3) we optimisedthe 5’ coding sequence by secondary structure modelling withsilent mutations to promote the single stranded nature of thetranslation initiation region of the mRNA. Interestingly, a majorresult of this optimisation, which was performed to providehigher ribosome loading to the wnt mRNA, was the disap-pearance of a shorter variant of Wnt4, which was formed due asecond internal ribosome binding site (nucleotides 90 to 97).Conclusion: As no other properties of the expression systemand conditions were changed we argue that a higher ribosomeloading from the regular SD site raises the ribosome coverage of themRNA such that the secondary ribosome binding site is obscured bytranslating ribosomes. As a result the production of truncatedprotein is reduced.AcknowledgementsThis study was supported by the TEKES "Neobio" programmeand a grant to AM by the Academy of Finland.References1. Ivanov I, Alexandrova R, Dragulev B, Saraffova A and

AbouHaidar MG: Effect of tandemly repeated AGGtriplets on the translation of CAT-mRNA in E. coli.FEBS Lett 1992, 307:173–176.

2. Ozin AJ, Costa T, Henriques AO and Moran CP Jr:Alternative translation initiation produces a shortform of a spore coat proteinin Bacillus subtilis.J Bacteriol 2001, 183:2032–2040.

P55Transcriptional profiling of recombinant CHOcells by a novel inter-species analysis strategyWolfgang Ernst, Evelyn Trummer, Hermann Katinger,Friedemann Hesse and Dethardt MullerDepartment of Biotechnology, University of Natural Resourcesand Applied Life Sciences, Vienna, Austria


Background: Despite the widespread use of CHO cells forthe production of many industrially relevant biopharmaceuticals,this system is poorly understood on the genetic level and mainlyrelies on empirical procedures, due to the lack of adequatesequence information. In order to advance its overall perfor-mance, we successfully tested the applicability of a cross-speciesmicroarray approach, for investigating CHO specific transcrip-tion profiles [1]. In the present study we show expressionsignatures of individual recombinant CHO clones whichcorrelate with their associated phenotype.Results: Clones were cultivated in repeated batch mode(5 batches) in Sixfors bioreactors. Several CHO model cloneswere analyzed on an Agilent genomics platform using 60-meroligonucleotide mouse microarrays. We could detect distinctivetarget genes between different model clones, all characterizedby high production levels of the recombinant protein. Whatwe also found were commonly enriched Gene Ontology (GO)categories present in all the clones with this particularproperty. In addition, a group of similar clones with improvedsialylation capabilities was studied, since the protein used inthese experiments is heavily glycosilated, and found to sharecommon signature genes and enriched GO terms. In a furthermodel we tried to identify predictive genes for stresssusceptibility under bioreactor conditions. We compared aresistant and a susceptible clone in spinner cultures and duringbioreactor cultivation. There was a striking similarity both in theexpression profile and the level of expression when early andlate growth stages of this clone were compared. Hence, theseprognostic signatures could be used as a selection tool andfurther help to understand some of the factors involved in stressresponse under altered growth conditions.Conclusion: Transcriptome analysis has the potential toefficiently identify parameters for cell line improvement. Inparticular, cross-species analysis can be a useful tool to studygene expression profiles of related organisms for which species-specific microarrays are not available. The signature genes wewere able to detect correlated with the phenotypic propertiesof the investigated clones, like high production levels, improvedcapabilities of product glycosilation, and stress resistance underbioreactor growth conditions. This knowledge could be helpfulin understanding cellular mechanisms and could further serveto develop a robust host cell line for effective processperformance.Reference1. Ernst W, Trummer E, Mead J, Bessant C, Strelec H,

Katinger H and Hesse F: Evaluation of a GenomicsPlatform for Cross-Species Transcriptome Analysisof Recombinant CHO Cells. Biotechnol J 2006,1:639–650.



P56Limitations using GFP as a protein expressionreporter in Pichia pastorisAnna Surribas, David Resina, Pau Ferrerand Francisco ValeroDepartament d’Enginyeria Quımica, Universitat Autonoma deBarcelona, Bellaterra, Barcelona, Spain


Background: The development of fluorimetric sensors duringthe last decade and the advantages of fluorimetry as a noninvasive, highly specific and sensitive technique have favouredthe utilization of this signal not only in biology but also inbioprocesses. In this context, the Aequoria victoria greenfluorescent protein (GFP) has appeared as a popular reporterprotein to study both prokaryotic and eukaryotic systems. Itdoes not demand any cofactors to fluoresce, is a small moleculeand requires no fixation techniques. Among different applica-tions, GFP has been used as a reporter in gene delivery, as atracer in subcellular trafficking and as a fusion partner tomonitor protein location. For bioprocess development, GFP hasbeen used as a protein fusion partner to monitor and optimiserecombinant protein production [1]. However, production ofsoluble, secreted GFP or protein-GFP fusions in P. pastoris hasproved to be a difficult task. Also, decrease of the target proteinproduction levels when fused to GFP is case dependent.In this work, GFP (S65T) has been fused to a Rhizopus oryzaelipase (ROL) produced in P. pastoris to study its applicability inprocess monitoring.Results: In the present work, wild type X-33 P. pastoris strainwas used for the extracellular expression of ROL fused to GFP(S65T), either at its N- or C- terminal end. The nitrogen sourceregulated formaldehyde dehydrogenase promoter, PFLD, wasutilized to drive recombinant protein expression and, theSaccharomyces cerevisiae a-factor signal peptide was selectedfor secretion. Both constructions were tested for extracellularexpression. Only when GFP was fused to the ROL N- terminal,extracellular lipolytic activity was detected. Therefore, thisconstruction was selected for further expression studies.A batch culture using sorbitol and methylamine as carbon andnitrogen sources, respectively, was carried out in order to testthe expression levels and growth performance of the strainexpressing the GFP-ROL fusion protein in bioreactor controlledconditions.Notably, the culture producing the GFP-ROL fusion proteinachieved a lower extracellular expression level compared tothat obtained with the ROL expressing strain under the sameculture conditions. Western Blot analysis of culture super-natants confirmed that the fusion protein was properlyprocessed and secreted when using the a-factor signalpeptide.Confocal microscopy images of the cells showed two GFPfluorescence distribution patterns. In some of them, GFP wasfound in the periphery of the cells, while other cells showedGFP located in large compartments such as vacuoles. Similarresults have been reported by Lenassi et al. when expressingGFP intracellularly. A possible explanation for this fact couldbe that a bottleneck might exist in the fusion protein foldingand secretion pathway, resulting in protein intracellularaccumulation.

Flow cytometry analyses confirmed this hypothesis. GFPintracellular fluorescence from batch samples was measuredand normalized by cell size. Consistently with the formerresults, the normalized intracellular fluorescence increased withthe culture time.Although lipolytic activity was detected in culture supernatantswith the GFP-ROL expressing strain, measured extracellular GFPfluorescence levels were essentially identical to those obtainedwith the control strain, i.e. expressing only the fungal lipase.The GFP variant used in this work (S65T), has an excitation andemission spectra close to that of riboflavin, which is a biogenicfluorophore of P. pastoris. Consequently, when measuringfluorescence from culture supernatants riboflavin signal overlapsGFP emission signal and the fusion protein can not be opticallydetected by means of GFP fluorescence analysis.Interestingly, no riboflavin intracellular fluorescence could bemeasured spectrophotometrically. Moreover, intracellular GFPcould be detected both by spectrofluorometry and by flowcytometry.To assess that the GFP fusion protein was fluorescent aftersecretion, riboflavin was removed from culture supernatant byultrafiltration. Thereafter, GFP fluorescence could be detectedat its emission maximum at 510 nm, as expected for this mutant.Conclusion: In this study, GFP was used as a fusion partner tomonitor protein expression and secretion, as well as subcellularlocalization. As GFP fusions are being increasingly used for thiskind of study, the results from our work reveal that someconsiderations must be taken into account when using thisstrategy in P. pastoris:Firstly, the expression levels of the GFP fusion protein should becompared to those with the strain expressing the not fusedprotein. This goal is especially critical when attempting to useGFP fusions for bioprocess development. In this sense, both thefusion construction and the secretion signal play an importantrole. In our work, results point out a possible bottleneck in thesecretion process.Secondly, care must be taken when selecting the optimal GFPmutant. In our experiments, no riboflavin fluorescence wasobserved intracellularly. Therefore GFP mutants with anexcitation and emission spectra close to those for riboflavinmight be useful to monitor intracellular events. Nevertheless, ifGFP fusions are desired to monitor protein secretion orextracellular/total fluorescence for bioprocess monitoring andoptimization, GFP mutants with spectral characteristics close tothose of riboflavin should be avoided. Red shifted mutants orthose with excitation wavelengths around 395 nm, such asGFPuv, might be a better option to avoid possible interferences[3]. Care should also be taken when using mutants withexcitation wavelengths around 360 nm, where NADH and othermedia components are also excited [4].References1. Jones JJ, Bridges AM, Fosberry AP, Gardner S, Lowers RR,

Newby RR, James PJ, Hall RM and Jenkins O: Potential ofreal-time measurement of GFP-fusion proteins.J Biotechnol 2004, 109(1–2):201–211.

2. Lenassi A, Trobec S, Gaberc-Porekar V and Menart V: Highexpression of green fluorescent protein in Pichiapastoris leads to formation of fluorescent particles.J Biotechnol 2004, 109(1–2):115–122.

3. Cha HJ, Shin HS, Lim HJ, Cho HS, Dalal NN, Pham MQ andBentley WE: Comparative production of human



interleukin-2 fused with green fluorescent protein inseveral recombinant expression systems. Biochem EngJ 2005, 24:225–233.

4. Surribas A, Montesinos JL and Valero F: Biomass estimationusing fluorescence measurements in Pichia pastorisbioprocess. J Chem Technol Biot 2006, 81(1):23–28.

P57Cell culture efforts to reduce glycation inrecombinant humanized antibodyInn H Yuk1, Hung Huynh1, Kimberly Leach1, Amy Shen1,Boyan Zhang2, George Dutina1, Patrick McKay3,Amy Lim3 and Brad Snedecor11Early Stage Cell Culture Process Development, Genentech,Inc., South San Francisco, CA 94080, USA2Early Stage Analytical Development, Genentech, Inc., SouthSan Francisco, CA 94080, USA3Early Stage Purification Development, Genentech, Inc., SouthSan Francisco, CA 94080, USA


Background: Glycation is a common post-translational modifi-cation of proteins, resulting from the chemical reaction betweenreducing sugars such as glucose and the primary amino groups onprotein [1]. This non-enzymatic glycosylation reaction generatesstructural heterogeneity in recombinant IgG1 antibodies producedby cell culture processes [2]. Recent analytical characterization of afull-length humanized antibody secreted by Chinese HamsterOvary (CHO) cells revealed that glycation of this protein occurspredominantly at lysine 49 on the light chain of the antibody [3].This finding contrasts with historical data that have suggested thatglycation sites are typically located randomly at all accessible lysineresidues distributed over the entire molecule [2, 3].The glycated species accounted for 40–50% of the total antibodyproduced by transient CHO cell transfections in bioreactors. Bycontrast, other recombinant antibody molecules produced byCHO cultures generally showed only ~5% glycation [3]. Thiswork documents cell culture process development efforts takento reduce glycation of this antibody in stably expressing CHOcell lines.Results: Early stable antibody expression by different CHOclones in 60 mm plates demonstrated no significant differencesin glycation (see Table 1). This supports the expectation thatglycation of this antibody is an extracellular event such thatglycation levels should depend on cell culture conditions andshould not vary from clone to clone.Antibody glycation was ~40–60% in 40L bioreactors and ~10–15%in 1L spinners at the time of harvests (see Table 2). Since theextent of this chemical modification should increase with reactiontime and substrate availability, the considerable disparity inglycation between bioreactor and spinner samples is attributedpartly to the differences in cultivation time and glucoseconcentrations.

The results from plate and spinner experiments show thatthe glycation of this antibody can be reduced to below 40%.Hence, 2L bioreactor experiments were conducted to testthe feasibility of lowering glycation by reducing the glucoseconcentration in the culture medium. The pH, dissolvedoxygen, and temperature profiles were controlled identically in allthe 2L bioreactors. The extent of glycation in the bioreactor samplescollected at the end of culture (day 14) was determined using aboronate affinity chromatographic method previously described [3].In the first series of bioreactor experiments, the glucoseconcentration in the batch feed was lowered by ~67%, and theamount of supplemental glucose added was reduced by ~50%.These modifications lowered glycation to 14–20% for each ofthe three antibody stably-expressing clones tested (see Table 3).Subsequent bioreactor experiments employed clone 2 exclu-sively, and this reduced glucose feed process was used as thecontrol.In the second set of bioreactor experiments, antibody glycationwas further reduced to ~10% (see Table 3) by eliminatingglucose from the batch feed and replacing it with a continuousglucose feed. In the final round of bioreactor experiments, byusing glucose-free inoculation medium and batch feed, and byimplementing a continuous glucose feed strategy to maintainglucose at even lower concentrations throughout the culture,the glycated species was minimized to 6% (see Table 3). Despitethe variation in antibody glycation, product titers and cell-specific productivities were comparable in all the 2L bioreactorexperiments.Conclusion: The percentage of glycated antibody was reducedby lowering the glucose concentration in the culture medium.The extent to which glucose concentration in the cultures wascontrolled directly impacted the antibody glycation level.AcknowledgementsWe thank AORS and Media Preparation department forproviding assay and media preparation support, respectively.We also thank Lily Chu, John Joly, Cindy Quan, Stacey Ma, RonTaticek, Bob Kiss, Dana Andersen, Martin Gawlitzek, RobbShawley and Reed Harris for helpful discussions.References1. Baynes JW: The Maillard hypothesis on aging: time to

focus on DNA. Ann N Y Acad Sci 2002, 959:360–367.2. Harris RJ: Heterogeneity of recombinant antibodies:

linking structure to function. Dev Biol (Basel) 2005,122:117–127.

3. Zhang B, Yang Y and Champion K: Revealing an unusualpattern of antibody glycation. WCBP 2006: 10thSymposium on the interface of regulatory and analytical sciencesfor biotechnology health productsSan Francisco, CA, USA; , Jan24–27, 2006.

Table 1 (abstract P57) Glycation of antibody produced bydifferent CHO clones in 60 mm plates.

Clone 2 25 92 131 169 191 260 274 277

Glycation (%) 19 21 20 18 19 17 18 18 20

Table 2 (abstract P57) Glycation of antibody produced in 40Lbioreactors and 1L spinners.

Vessel Volume Clone Harvest Time Final [Glucose] Glycation

Bioreactor 2 x 40L 2 Day 11 and Day 14 > 9 g/L 42%Bioreactor 40L 2 Day 14 > 10 g/L 58%Bioreactor 40L 2 Day 14 > 7 g/L 42%Spinner 1L 2 Day 5 < 6 g/L 13%Spinner 1L 191 Day 5 < 6 g/L 11%



P58Biochemical assay development for drugdiscovery: a sequential optimization fromprotein expression to enzymatic activityCristina Sidoli, Angela Molteni, Beatrice Bellanti,Loredana Redaelli, Lucia Iuzzolino,Mariantonietta Rubino, Patrizia Arioli, Vanessa Nardeseand Daniele CarettoniBiochemistry, AXXAM srl, San Raffaele Biochemical SciencePark, Milan, Italy


Background: The drug discovery process based on high-throughput screening (HTS) requires highly demanding effortsto comply with the strict criteria of homogeneity, sensitivity,processivity, reproducibility and miniaturization. For enzymatictargets, the ability to screen in parallel multiple parameters is aprerequisite to identify the permissive conditions to developfunctional assays and to upscale the protein production toentirely support an HTS campaign, which generally requiresseveral milligrams of pure protein.Results: To fulfill the requirements of the enzymatic assayconfiguration for HTS, we have developed a streamlined processfor the sequential optimization of recombinant expression,purification and activity of therapeutically-relevant enzymes,applying automation and miniaturization.Concerning recombinant expression, we routinely challenge inparallel three to five chimerical tagged versions of the targetenzyme in 4 different insect cell lines. Insect cell cultures havebeen optimized to effectively support recombinant proteinexpression in 24-deep-well format, allowing the simultaneousscreening of 36 different conditions for each version of thetarget.Tag-based purification by affinity chromatography undergoes arobot-assisted optimization in 96-well format, where 8–16different buffer conditions are screened in parallel. Selection ofthe best-supporting purification conditions is determined byintegrating data on specific activity of the enzyme, degree ofpurity and final production yield. The identified parameters forexpression and purification are applied to a large-scale FPLC-based production, which usually provide the entire amount ofenzyme requested for the assay development.The enzymatic activity is optimized by screening in 384-wellformat pre-assembled plates covering over 250 differentconditions using HTS-compatible fluorescent or luminescentreadout. In particular, 18 buffers ranging from pH 4.5 to 9.5, 20monovalent and divalent cations, and 40 additives, including

detergents, reducing agents, chelators, organic solvents, stabi-lizers, and phospholipids, are challenged in a dose-dependentmanner, to determine the best working conditions for the targetenzyme.Conclusion: The overall optimization on protein expression,purification and enzymatic activity for potential drug targetstests over 800 different conditions, with dramatic impact on thefinal assay configuration. We can demonstrate that the specificactivity of the enzyme is increased on average by 200-fold at theend of the process, with cases reaching up to 600-fold. Thisresult has two direct valuable consequences. First, the assay isconFigured with maximally active enzyme preparations, which isconsidered a hallmark for potentially successful screenings.Second, the enzyme concentration in the reaction is reduced atits minimal amount, keeping unaltered the robustness of theassay. This implies that the sensitivity to potential inhibitors issignificantly increased, and screening of large libraries withdifficult-to-express enzymes is made realistic.

P59Evaluation of the baculovirus and E.coli-expressednon-structural (NS) proteins of bluetongue virus(BTV) as antigen in an indirect or competitionELISA to differentiate infected from vaccinatedanimalsLissette Lopez1, Angel Venteo1, Miguel Angel Jimenez-Clavero2, Juan Luis Carrasco1, Marıa Jose Cano1,Elena Soria1, Concepcion Gomez-Tejedor2,Antonio Sanz1, Carmen Vela1 and Paloma Rueda11INGENASA, C/Hnos. Garcıa Noblejas, 41. 28037 Madrid,Spain2Laboratorio Central de Veterinaria. Ctra. de Algete, Km 8.28110 Madrid, Spain


Background: BTV is an important disease of wild anddomestic animals (sheep, goats and cattle) both from the aspectof animal health as well as economical impact. BTV is theprototype of the Orbivirus genus in the Reoviridae family,possesses 10 double-stranded RNA segments enclosed bythree consecutive capsid layers of multiple proteins. In additionto the seven structural proteins, there are three non-structuralpolypeptides (NS1, NS2 and NS3). Vaccination is the mosteffective mean to prevent the infection; however it was notroutinely carried out. At present, it is widely discussed the useof inactivated vaccines. This vaccine offers significant advantagesover attenuated vaccines because of the absence of replicating

Table 3 (abstract P57) Glycation of antibody produced in the first, second, and third series of 2L bioreactor experiments. Eachcondition was evaluated in duplicate bioreactors. Data represents average ± standard deviation obtained from duplicate cultures.

Experiment Clone Condition Glycation Final [Glucose]

1 2 Control 19 ± 2 % 2.9 ± 1.1 g/L1 191 Control 17 ± 2 % 1.9 ± 0.4 g/L1 274 Control 15 ± 1 % 1.4 ± 0.5 g/L2 2 Control 16 ± 2% 1.8 ± 0.1 g/L2 2 Partial Continuous Glucose Feed 10 ± 1% 0.8 ± 0.1 g/L3 2 Control 19 ± 1% 2.2 ± 0.2 g/L3 2 Full Continuous Glucose Feed 6 ± 0 % 0.5 ± 0.4 g/L



virus, furthermore, if commercial vaccines are purified, it willopen the possibility of developing diagnostic based on non-structural proteins to differentiate infected from vaccinatedanimals.Results: In this study, the production of NS3 protein in twodifferent expression systems, baculovirus and E. coli wascompared. NS3-coding sequence was amplified by PCR fromBTV infected cells. In E. coli system, NS3 gene was cloned intothe pET28 vector that carries an N-terminal Histidine tag fusedto the expressed protein. The protein was purified by guanidinechloride 6 M and a further step by immobilized metal affinitychromatography (IMAC) (Ni2+).In the case of the baculovirus expression system, the gene wascloned into the pAcHLT-A transfer vector which also added apoly-His tag at the N-terminus. Routinely, the infected insectcells were harvested and lysed by osmotic shock in 25 mMsodium bicarbonate solution. After centrifugation, the presenceof the NS3 protein was analysed in the soluble and insolublefractions. NS3 protein was only detected in the insoluble cellularpellet. In view of these results, the purification of NS3 proteinwas improved with the lysis of insect cells with the followingbuffer: 25 mM sodium bicarbonate containing 6 M guanidinechloride and 0.5 M NaCl. After sonication (5 � 20s), cells werecentrifuged, and NS3 protein was finally purified by IMAC fromthe soluble fraction.The best results in terms of productivity and product qualitywere obtained when NS3 protein was produced on thebaculovirus expression system: 2 mg purified NS3/1 � 108 Sf9cells, for that reason this protein was selected as antigen inimmuno-assays.An indirect enzyme-linked immunosorbent assay (ELISA) wasdeveloped as an attempt to differentiate BTV infected fromvaccinated animals using the purified recombinant NS3 proteinas antigen. A panel of experimental sera from 11 immunizedcows (with inactivated vaccine) collected at different days post-immunization, 4 non immunized sheep as negative control and 1infected sheep as positive control, were evaluated in the newdeveloped ELISA. Specific NS3 antibodies (Ab) were detected inall vaccinated animals approximately two weeks after vaccina-tion. The same results were obtained in infected animals.To determine the immunologic level against BTV, all sera wereassayed by the commercial kit INGEZIM BTV, which detects Absagainst the VP7 structural protein. All vaccinated animalsdeveloped antibodies anti-VP7 Abs as similar level as anti-NS3,however, antibodies anti-NS3 were detected earlier than anti-VP7 in the infected sheep. This result could indicate that NS3antigen is better marker than VP7 on BTV infection.Similar results were also obtained when we performed westernblot using NS3 protein as antigen.NS3 protein was used to immunize Balb/c mice and a panel of 6monoclonal antibodies (MAbs) has been obtained. These MAbswill be used to develop a competition ELISA for detection ofantibodies anti-NS3 and also a double antibody sandwich (DAS)ELISA for detection of NS3 protein in inactivated vaccine batches.At present, NS1 and NS2 recombinant proteins are beingexpressed on both systems (E. coli and baculovirus) in order toselect the best system for each protein. Finally, their ability fordiscrimination between infected and vaccinated animals will beanalysed by ELISA.Conclusion: Non structural proteins of BTV have beenexpressed in two different expression systems.

NS3 was solubilized from the insoluble Sf9 cellular fraction bylysis with 25 mM sodium bicarbonate containing 6 M guanidinechloride and 0.5 M NaCl.An indirect ELISA for detection of antibodies against BTV usingNS3 protein as antigen has been developed.The indirect ELISA based on the NS3 antigen is not able todifferentiate infected animals from vaccinated animals but itcould be an appropriate system to detect earlier infections.

P60Automation for higher throughput in proteinexpression: visions, facts and fictionsMarion Mahnke1, Jean Marc Schlaeppi1, Yann Pouliquen1,Louise Barys2, Catherine Rolvering3, Mario Henke1,Rita Schmitz1, Sabine Geisse1 and Frank Kolbinger11Biomolecules Production Unit, Novartis Institutes forBiomedical Research, Basle, Switzerland2Oncology Department, Novartis Institutes for BiomedicalResearch, Basle, Switzerland3Faculty of Science, Technology and Communication,University of Luxembourg, Luxembourg


Background: Down-scaling, parallelization and automationare new trends in the field of recombinant protein expressionin the post genomic era [1, 2, 3]. During the past years manycompanies and academic institutions have heavily invested inprocess and automation technologies. Does this trend keep itspromise? Can post genomic protein production issues beovercome with few automated processes?This abstract wants to highlight two years of experience inrunning a Protein Production Center in an industrial environ-ment applying the expression systems BEVS, E. coli (and transientHEK.EBNA). We describe the streamlined and partially auto-mated processes, the automation equipment applied; discussresults from the past two years of experience and strategies toeliminate remaining bottlenecks.Results: Proteins expressed in a generic way in the E. coliexpression system are all his-tagged and often N-terminallyfused to thioredoxin or other fusion partners in order toimprove solubility. After small scale expression evaluation in 24-deepwell blocks recombinant proteins are produced in 1-Lfermenter vessels using fully automated and unattendedinductions and temperature shifts. The described method isapplicable to all host strains and induction systems, providing theoptimal induction time point and harvest for each construct.To run the BEVS in an automated high throughput environmentstill represents a major challenge. In order to meet thischallenge, we have developed robust automated protocols atvarious formats for the small scale BEV process on anepMOTION 5070 workstation and semi automated large scaleprotocols for 10-L wave bioreactors and purification on Akta3D and Akta Express. The expression of DUB family and variousother proteins were used as an example to validate theseprocesses (Table 1).During the validation of the technologies described above someweak points of the automated processes became obvious: thetendency of proteins to form soluble or insoluble aggregates atany stage of the process and the presence of proteases, whichrequires close control of the fermentation process andinterferes with fully automated and unattended runs. While



means to control the aggregation phenomenon are still underinvestigation, a generic method has been found to control theproteolysis issue in the BEVS. We demonstrate that the additionof 10 mM of the cysteine protease inhibitor E-64 during thecourse of production effectively reduces/prevents proteolyticdegradation of various recombinant proteins over a minimum of72 hours of culture time, thus improving the quality and yields ofsecreted as well as intracellular recombinant proteins (Figure 1).Conclusion: We have demonstrated that recombinant pro-tein expression can be evaluated, scaled up and proteins bepurified in a parallel and (semi-) automated fashion using theBEVS and E. coli expression systems. Pre-requisites are thatproteins are appropriately tagged and grouped. However, inorder to attain optimal quality and yield of different recombinantproteins by generic processes the influence of proteindestabilizing factors needs to be thoroughly understood andmanaged.AcknowledgementsThe authors want to thank Rene Amstutz and Hans Kocher forsupport of this work and Sabine Deutsch, Eveline Eglin, BrendanKerins, Stefan Dalcher and Sybille Bossart for excellent technicalassistance.References1. Rupp B, Segelke BW, Krupka HI, Lekin T, Schafer J,

Zemla A, Toppani D, Snell G and Earnest T: The TB

structural genomics consortium crystallization facil-ity: towards automation from protein to electrondensity. Acta Crystalogr D Biol Crytaloogr 2002,58:1514–1518.

2. Lesley SA: High-throughput proteomics: proteinexpression and purification in the postgenomicworld. Protein Expr Purif 2001, 22:159–64.

3. Scheich C, Sievert V and Bussow K: An automatedmethod for high-throughput protein purificationapplied to a comparison of His-tag and GST-tagaffinity chromatography. BMC Biotechnol 2003, 3:12.

P61Potentials and limitations of prokaryotic andeukaryotic expression systems for recombinantprotein production – a comparative viewDethardt Muller, Karl Bayer and Diethard MattanovichInstitute of Applied Microbiology, Department ofBiotechnology, University of Natural Resources and AppliedLife Sciences (BOKU), Muthgasse 18, A-1190 Vienna, Austria


Background: Within the last recent years biopharmaceuticalsales have reached 30% of all new pharmaceutical sales in theUnited States expecting an increase from 30 billion USD (2003)

Table 1 (abstract P60) Expression of 30 DUB proteins in the BEVS at 10-L scale and processed in a semi-automated mode.

Analysis Method AVERAGE RANGE Av. Yield [%]

Small Scale Expression [mg/L] ELISA 27 3 – 140Large Scale Expression [mg/l] ELISA 43 0 – 270Large Scale Expression [mg] ELISA 448 0 – 1400 100 %Cross-flow yields [mg] ELISA 169 0 – 825 41 %Pure protein yields [mg] HPLC 21 0 – 103 (16%)*

* different analysis method


Effect of the addition of E-64 (cysteine protease inhibitor) and Pepstatin A (aspartic acid protease inhibitor) on the expression of a secreted wntantagonist in Sf21 infected insect cells.(A): Western Blot (-His) of cell lysates and medium supernatants taken at various time points (48, 67, 73, 90, 97hours) post infection (expected size of protein: 28.1 kDa).



to almost 60 billion USD until 2010 [1]. One major industry ofthe fast growing biopharmaceutical market is the manufacture ofrecombinant proteins for therapeutic and diagnostic use. Hence,the rising demand for new biopharmaceuticals requiresincreased production capacities as well as new productionprocesses that exhibit increased space-time yields and shor-tened development times which also implies the use of suitableexpression systems.In the 1970s, when recombinant DNA technology found its wayinto molecular biology laboratories, the bacterial cell wasprevalently presented as a universal host for heterologousprotein expression. However, due to their inability toadequately process complex proteins and due to theirinsufficient protein secretion capabilities prokaryotic expressionsystems nowadays are mainly used for the production of ratheruncomplex proteins and peptides. In order to produce complexhuman recombinant proteins a reinforced development ofeukaryotic expression systems has proceeded in the lastdecades, which was mainly based on yeast cells and mammaliancells. As a result, mammalian cell-based biopharmaceuticalsaccount for almost 60% of today’s biopharmaceutical market.But, what do we really know about the production capacity of aparticular host cell related to cell mass and cell volume? Whatcan we learn in order to ease the choice of a suitable expressionsystem for a particular protein? How can we use this knowledgeto optimize the product yield related to process time and space?We have evaluated different expression systems which havebeen technologically used for recombinant protein production:an inducible prokaryotic expression system (Escherichia coli) forintracellular human superoxide dismutase [2], an E. coli secretionsystem for antibody Fab fragments [3], a constitutive eukaryoticsecretion system (Pichia pastoris) for human trypsinogen andantibody Fab fragments [4, 5] as well as an intracellular P. pastorissystem [6], and a constitutive eukaryotic expression system(CHO) for an EPO/Fc fusion protein and human monoclonalantibodies [7]. We developed production scenarios for eachexpression system and compared specific growth and produc-tivity as well as product secretion rates to determine the fullpotential in a bioprocess.Results: Based on own experience in our labs, as well as onliterature data, we have developed three main scenarios forprotein production, based on E. coli, P. pastoris and CHO cells asalternative host systems. Maximum and average values forspecific growth rates, specific product formation (secretion)rates, and space time yields (volumetric productivities) aresummarized in Table 1.

Relating these data to total protein synthesis rates enables theestimation of the upper limits of production capacities, while thevolumetric productivities inform about the economic efficienciesof the different scenarios.Conclusion: Up to now it was not usual practice to comparemammalian cell culture processes on a common basis withmicrobial processes. The presented data provide a clear basis tojudge both the economic capabilities of present processes andthe potential and constraints of the different host systems,pointing the attention to the most severe bottlenecks that limitthe economic feasibility of the respective production systems.These data enable us to generalize the understanding of thebiological limitations of protein synthesis and secretion, thusobviating the major potentials for the optimization of currentlyavailable expression systems.References1. Birch J: Mammalian cell culture: current status,

future prospects. Oral presentation. Cell Culture andUpstream Processing, Berlin 2004.

2. Kramer W, Elmecker G, Weik R, Mattanovich D andBayer K: Kinetic studies for the optimization ofrecombinant protein formation. Ann NY Acad Sci1996, 782:323–333.

3. Maier T: An E. coli based secretion system for theproduction of proteins and Fabs. Bioprocess Interna-tional, Prague 2006.

4. Hohenblum H, Borth N and Mattanovich D: Assessingviability and cell-associated product of recombinantprotein producing Pichia pastoris with flow cytome-try. J Biotechnol 2003, 102:281–290.

5. Gasser B, Maurer M, Gach J, Kunert R and Mattanovich D:Engineering of Pichia pastoris for improved produc-tion of antibody fragments. Biotechnol Bioeng 2006 inpress.

6. Hasslacher M, Schall M, Hayn M, Bona R, Rumbold K,Luckl J, Griengl H, Kohlwein SD and Schwab H: High-levelintracellular expression of hydroxynitrile lyase fromthe tropical rubber tree Hevea brasiliensis in micro-bial hosts. Protein Expr Purif 1997, 11:61–71.

7. Trummer E, Fauland K, Seidinger S, Schriebl K,Lattenmayer C, Kunert R, Vorauer Uhl-K, Weik R,Borth N, Katinger H and Muller D: Process parametershifting: Part I. Effect of DOT, pH and temperatureon the performance of Epo-Fc expressing CHO cellscultivated in controlled batch bioreactors.2006 inpress.

Table 1 (abstract P61) Comparison of typical max. cell dry masses, achieved product concentrations, specific product formationrates and volumetric productivities of the selected model processes.

CHO cells P. pastoris P. pastoris E. coli E. coli

Destination of product secreted secreted cytoplasm secreted cytoplasm[g·L-1] 0.5 – 5 80 – 150 80 – 150 20 35Typical spec. growth rate [h-1] 0.02 0.02 0.02 0.1 0.1Typical specific product formation rate [mg·g-1·h-1] 1.5 – 4.5 0.05 – 0.5 4 0.25 – 2.5 20Product concentration (per culture volume) [g·L-1] 0.1 – 0.5 0.25 – 2.5 20 0.2 – 2 7Volumetric productivity [mg·L-1·h-1] 1 – 2 (- 25 for perfusion) 1 – 6 (- 25 for continuous processes) 160 4 – 40 200



P62Production of human a1 proteinase inhibitor fromAspergillus nigerLiat Chill1, Loc B Trinh1, Elena Karnaukhova2,Yakir Ophir2, Basil Golding2 and Joseph Shiloach11Biotechnology Unit, NIDDK, NIH, Bethesda, MD 28092, USA2Division of Hematology, CBER, FDA, Bethesda, MD 28095,USA


Background: a1 proteinase inhibitor (a1PI) belongs to the serinprotease inhibitor (serpin) family; it is responsible for 90% of thetrypsin inhibitory capacity of human plasma. Its primary physiologicalrole is to inhibit neutrophil elastase that degrades elastin, collagenand proteoglycan and can cause extensive lung damage. a1PIdeficiency can result in lung emphisema in adults, liver disease inchildren and is also associated with cystic fibrosis, arthritis and othermalignant conditions [1, 2, 3]. Human plasma-derived a1PI is alicensed product used for replacement therapy. Although viralinactivation measures are taken, products derived from humanplasma carry the risk of contamination with blood-borne pathogens,and therefore recombinant is an attractive alternative.a1PI exhibits a molecular mass of approximately 52 kD, has 394amino acids, a single cystein residue and three carbohydrateattachment sites at asparagine residues 46, 83 and 247 [4, 5].Significant research effort was directed to produce recombinanthuman a1PI in E. coli [6], yeast [7], transgenic mice and sheep aswell as plant cells [3]. E-coli produced an unglycosylated product,the yeast produced incorrect glycosylation and the transgenicanimals pose the same threats as plasma derived product.Results: Human a1PI was cloned and expressed in Aspergillusniger, filamentous fungus that can grow in defined media and hadthe ability to perform glycosylation. Submerged culture condi-tions were established: using starch as carbon source, 30%dissolved oxygen concentration, pH 7.0 and 28˚C; 10 mg perliter of active a1PI were secreted to the growth media in 40hours. Controlling the protein proteolysis was found to be animportant factor in the production from the fungus. The effect ofcarbon sources and growth conditions on the production andstability of the protein will be presented and evaluated.References1. Mattes E, Matthiessen HP, Turecek PL and Schwarz HP:

Preparation and properties of an alpha-1-proteaseinhibitor concentrate with high specific activity. VoxSang 2001, 81:29–36.

2. Travis J, Owen M, George P, Carrell R, Rosenberg S,Hallewell RA and Barr PJ: Isolation and properties ofrecombinant DNA produced variants of Human a1-proteinase inhibitor. J Biol Chem 1985, 260:4384–4389.

3. Huang J, Sutliff TD, Wu L, Nandi S, Benge K, Terashima M,Ralston AH, Drohan W, Huang N and Rodriguez RL:Expression and purification of functional human a-1-antitrypsin from cultured plant cells. Biotechnol Prog2001, 17:126–133.

4. Chen SX, Hammond DJ, Klos AM, Wood WD, Wydick JEand Lebing WR: Chromatographic purification ofhuman a1 proteinase inhibitor from dissolved Cohnfraction IV-1 paste. J Chromatogr A 1998, 800:207–218.

5. Carrell RW, Jeppsson JO, Laurell CB, Brennan SO, Owen MC,Vaughan L and Boswell R: Structure and variation ofhuman a1-antytrypsin. Nature 298:329–334.

6. Courtney M, Buchwalder A, Tessier L, Jaye M, Benavente A,Balland A, Kohli V, Lathe R, Tolstoshev P and Lecocq J:High level production of biologically acyive a-1-antitrypsin in Escherichia coli. Proc Natl Acad Sci USA1984, 81:669–673.

7. Kwon KS, Song M and Yu MH: Purification andcharacterization of a1-antitrypsin secreted byrecombinant yeast Saccharomyces diastaticus.J Biotechnol 1995, 42:19.

P63Kluyveromyces lactis SSO1 and SEB1 genes arefunctional in Saccharomyces cerevisiae and enhanceproduction of secreted proteins whenoverexpressedJaana H Toikkanen, Lena Sundqvist and Sirkka KeranenVTT, Industrial Biotechnology, P. O. Box 1000, FI-02044 VTT,Finland


Background: The SEB1/SBH1 and the SSO genes encodecomponents of the protein secretory machinery functioning atthe opposite ends, ER translocation and exocytosis, respec-tively, of the secretory pathway of Saccharomyces cerevisiae.Overexpression of these genes can rescue temperature-sensitive (ts) growth defect of many sec mutants impaired inprotein secretion. Their overexpression in wild-type yeastenhances production of secreted proteins in S. cerevisiae, whichsuggests that may be rate-limiting factors in this process.Results: Kluyveromyces lactis homologs of S. cerevisiae SEB andSSO genes were isolated by multicopy suppression of Sacchar-omyces mutations [1]. KlSSO1 and KlSEB1 are up to 70%identical with the S. cerevisiae homologs at the amino acid leveland can functionally replace them. These single copy genes wereable to complement the ts growth defect of sso2-1 and seb1�seb2� sem1� strains, respectively. In addition, KlSSO1 multi-copy suppressed both sporulation defects of S. cerevisiaemutants sso1�/sso1� or mso1�/mso1� and ts growth defectof exocyst mutant sec15-1. Furthermore, KlSSO1 and KlSEB1enhanced production of a secreted protein (Fig.1) similarly toSSO and SEB1 genes of S. cerevisiae [2, 3] when overexpressed.Conclusion: The single copy genes KlSSO1 and KlSEB1 areboth structurally and functionally conserved in evolution withtheir duplicated S. cerevisiae homologs.References1. Toikkanen JH, Sundqvist L and Keranen S: Kluyveromyces

lactis SSO1 and SEB1 genes are functional inSaccharomyces cerevisiae and enhance production ofsecreted proteins when overexpressed. Yeast 2004,21:1045–1055.

2. Ruohonen L, Toikkanen J, Tieaho V, Outola M, Soderlund Hand Keranen S: Enhancement of protein secretion inSaccharomycescerevisiae by overproduction of Ssoprotein, a late-acting component of the secretorymachinery. Yeast 1997, 13:337–351.

3. Toikkanen JH, Miller KJ, Soderlund H, Jantti J and Keranen S:The � subunit of the Sec61p endoplasmic reticulumtranslocon interacts with the exocyst complex inSaccharomyces cerevisiae. J Biol Chem 2003,278:20946–20953.



P64Antibody production by a protease-deficientstrain of methylotrophic yeast, Ogataea minutaKousuke Kuroda1, Yoshinori Kitagawa1,Kazuo Kobayashi1, Haruhiko Tsumura1,Toshihiro Komeda2, Yasunori Chiba3

and Yoshihumi Jigami31Kirin Brewery Co., Ltd., CMC R&D Lab, Gunma, Japan2Kirin Brewery Co., Ltd., Central Lab for Frontier Technology,Kanagawa, Japan3National Institute Advanced Industrial Science andTechnology (AIST), Ibaragi, Japan


Background: At present the expression system of mammaliancells such as CHO has been adopted as the conventionalmethod to produce antibody for pharmaceuticals. Howevera novel method of producing antibody has been sought afteras a substitute for the costly production of antibody bymammalian cells. Therefore, we tried to construct a novelantibody production system by using methylotrophic yeast,O. minuta.Results: When human antibody genes were introduced in themethylotrophic yeast O. minuta to produce an antibody, theheavy chain of the antibody was partially degraded (see Figure 1,

lane 1). Peptide sequencing revealed that degradation occurredin the CH1 region (see Figure 2). In order to inhibit thisdegradation, the YPS1 gene coding Aspartic protease attached tothe plasma membrane, a homologue of Saccharomyces cerevisiae,was cloned from O. minuta, and we constructed the �yps1strain. As a result, the �yps1 strain repressed the partialdegradation of the antibody (see Figure 1, lane2).Conclusion: We constructed a protease-deficient strain andconfirmed the secretion of full-length antibody by yeast.Improving the production of antibody should be a subject offurther research.AcknowledgementsWe thank N.Kawashima, A. Oonishi, Y. Yamamoto, M. Tezukaand K. Sato for their valuable assistance with this research. Weare also grateful to Dr. M. Tsukahara for helpful suggestions.References1. Bourbonnais Y, Larouche C and Tremblay GM: Production

of full-length human pre-elafin, an elastase specificinhibitor, from yeast requires the absence of afunctional yapsin 1 (Yps1p) endoprotease. ProteinExpr Purif 2000, 20:485–491.

2. Wood CR, Boss MA, Kenten JH, Calvert JE, Roberts NAand Emtage JS: The synthesis and in vivo assembly offunctional antibodies in yeast. Nature 1985,314:446–449.


Increased production of secreted -amylase by overexpression of KlSSO1() and KlSEB1 () in comparison to the control strain carrying emptyvector (). The cell growth is presented with open symbols.


Culture supernatant from the yeast O. minuta was subjected to Westernanalysis with HRP conjugated anti-human Fc antibody. Lane1: heavy chainsecreted by YPS1+ strain. Lane2: heavy chain secreted by yps1 strain.


Position of the partial degradation in the heavy chain on its CH1-hingeregion produced by O. minuta YPS1 + strain. Arrows show the positionof the degradation.



P65Heterologous expression of isotopically labeledTrichoderma reesei tyrosinase 2 in Pichia pastorisAnn Westerholm-Parvinen, Maija-Liisa Mattinen,Emilia Selinheimo and Markku SaloheimoVTT Biotechnology, P.O. Box 1000, FIN-02044 VTT, Finland


Background: Tyrosinase (EC 1.14.18.1) is a copper-containingoxidase that is widely distributed in mammals, invertebrates, plantsand microorganisms. In mammals the enzyme is essential for theformation of melanin pigments, whereas tyrosinases in fruit andvegetables are related to the browning reaction that occurs uponbruising and long term storage. Tyrosinase is of great interest formany applications in the field of medicine, biotechnology and foodengineering. It is a promising target enzyme for prodrug activationsin melanomas and in biotechnological applications including cross-linking of protein matrices. It is of great importance to find ligandsand inhibitors for tyrosinase. Structural studies and screening forligands and inhibitors can be carried out using NMR spectroscopywith isotopically labeled tyrosinase. Therefore, we cloned a noveltyrosinase from Trichoderma reesei and expressed it heterologouslyin the methylotrophic yeast Pichia pastoris.Results: A novel tyrosinase, tyrosinase 2 (TYR2), was cloned fromTrichoderma reesei. The cDNA sequence was expressed under thecontrol of the AOX1 promoter in the Pichia pastorisX-33 strain. TheSaccharomyces cerevisiaea-MFpreprosequencewasused for secretionand anN-terminal His6-tagwas fused to the tyrosinase to facilitate thedetection and purification of the recombinant protein. Heterologousexpression was carried out in shake flask cultivations and theenzymatic activity was measured directly on the culture medium,using L-Dopa as a substrate. A protein of approximately 45 kDa wasdetected byWestern blot with antibodies against the His-tag. The fulllength of TYR2 has a predicted MW of 60.4 kDa. When TYR2 ishomologously over-expressed in Trichoderma reesei, the C-terminal iscleaved and themature protein has aMWof 43.5 kDa. Thus, it seemsprobable that Pichia pastoris processes the C-terminal correctly.Extensive optimisation of the expression in shake flasks was carriedout as the stable isotope labels are costly. Different temperatures,different CuSO4 and NH4SO4 concentrations and different shakeflasks were tested. The expression level of recombinant TYR2 wasincreased tenfold as a result of the optimisation. Metabolic 15N-labeling of TYR2 was carried out with 15NH4SO4 in minimal mediumto assess its suitability for investigations by NMR spectroscopy. Initial3D heteronuclear 1H-15N HSQC NMR spectrum of TYR2 showedsignals with chemical shifts typical of folded proteins.Conclusion: The Trichoderma reesei tyrosinase 2 was success-fully expressed and uniformly 15N-labeled in the yeast Pichiapastoris. This methylotrophic yeast is a suitable expression systemfor the production of recombinant proteins for NMR studies as itis cost-effective and possesses the ability to perform many of theposttranslational modifications of higher eukaryotes.

P66Secretion of a hybrid K. lactis-A. niger �-galactosidaseAngel Pereira, Rafael Fernandez, Marıa Esperanza Cerdan,Marıa Isabel Gonzalez Siso and Manuel BecerraDepartamento de Bioloxıa Celular e Molecular. Facultade deCiencias. Universidade da Coruna. Campus da Zapateira, s/n15071. A Coruna. Spain


Background: The �-galactosidase from Kluyveromyces lactis isa protein with an outstanding biotechnological interest. Themain limitation to its industrial production is the high costassociated with extraction and downstream processing due toits intracellular nature [1].Secretion from yeast is an attractive method for producing manyheterologous proteins both because of the facility with whichgenetic manipulations and fermentation can be carried out andbecause of the fidelity of posttranslational modifications.However, adding a signal sequence is not sufficient to leadrecombinant proteins out of the cell: culture conditions play animportant role, the wall acts as a molecular sieve but, moreover,structural determinants, present in the protein, may be requiredfor targeting a protein to the medium [2].In this work, we have constructed hybrid proteins between K.lactis �-galactosidase and Aspergillus niger �-galactosidase, addeda signal peptide and analyzed the secretion and the properties ofthese new hybrid proteins.Results: The highest levels of extracellular �-galactosidasewere obtained when the segment corresponding to the fivedomain of K. lactis �-galactosidase was replaced by thecorresponding five domain of the A. niger �-galactosidase. Asmedium composition can exert a profound effect on the yield ofheterologous protein secretion in yeast, by influencing both cellgrowth and the specific rate of secretion [3] we examinedhybrid �-galactosidase production and secretion on batch liquidcultures on several different media. Best results were obtainedin a rich medium in which pH was maintained at 7.0, since pHvalues under 6.5 or above 7.5 cause a sharp decrease in K. lactis�-galactosidase activity [4]. In this condition the percentage ofhybrid �-galactosidase secretion was in the exponential phase2.2% and reached 16% of the total activity in the stationaryphase.Conclusion: One strategy for improving the secretion ofheterologous proteins is through introducing structural mod-ifications. A hybrid protein between K. lactis �-galactosidase andA. niger �-galactosidase was constructed that increase thefraction of enzyme reaching the growth medium. Moreover, wehave improved secretion percentages by studying the influenceof the culture conditions on heterologous hybrid �-galactosi-dase secretion.References1. Becerra M, Cerdan ME and Siso MI: Recent progress in

Kluyveromyces lactis �-galactosidase. Recent Res DevelBiochem 2003, 4:549–559.

2. Katakura Y, Ametani A, Totsuka M, Nagafuchi S andKaminogawa S: Accelerated secretion of mutant �-lactoglobulin in Saccharomyces cerevisiae resultingfrom a single amino acid substitution. Biochim BiophysActa 1999, 1432:302–312.

3. Chang CC, Park CS and Ryu DDY: Improvement ofheterologous protein productivity through aselected bioprocess strategy and mediumdesign: A case study for recombinant Yarrowialipolytica fermentation. Appl Biochem Biotechnol 1998,74:173–189.

4. Becerra M, Prado SD, SisoMI and Cerdan ME:New secretorystrategies for Kluyveromyces lactis �-galactosidase.Protein Eng 2001, 14:379–386.



P67The first auxotrophic mutant ofZygosaccharomyces bailii for recombinantproductions: a road to practical applicationsPaola Branduardi, Laura Dato, Luca Riboldiand Danilo PorroDepartment of Biotechnology and Biosciences, University ofMilano-Bicocca, Milano, Italy


Background: The yeast Zygosaccharomyces bailii belongs tothe so-called group of non-conventional yeasts, poorly studiedin the past. For this reason, up to now there is no deepknowledge regarding its physiology, genetics and molecularbiology, and its genetic manipulation is not easy. Only recentlythis yeast attracted the attention of the scientific community dueto its characteristics of stress resistance, particularly to acidicenvironments. Our group is working on the construction andimprovement of molecular tools for an exploitation of Z. bailii asa new host system for biotechnological applications.Results: In spite of the good results already obtained for theproduction and secretion of different proteins, a great limit wasrepresented by the lack of an auxotrophic mutant and of areproducible protocol for targeted gene deletion in this yeast.Here we show the strategy used to obtain the first Z. bailiiauxotrophic mutant (Zbleu2 strain) and the consequentexploitation of said mutant for heterologous protein productionand for metabolic engineering applications. The data obtainedwith the new strain showed a great improvement of production,mainly related to higher plasmid stability, if compared with thewild type strain transformed with similar plasmid but selectingfor an antibiotic resistance. In addition, and once moreexploiting the leu2 auxotrophy, we are developing a strategyfor high copy number replication of an heterologous gene ofinterest.Conclusion: According to our knowledge, this is the firstexample of an auxotrophic Z. bailii strain exploited forrecombinant productions. It represents a small but necessarystep to develop this host for biotechnological applications, thatalready resulted in significant improvement in the productions ofinterest.

P68Direct and indirect approaches for theimprovement of heterologous proteins secretionlevels in Zygosaccharomyces bailiiLuca Riboldi, Danilo Porro, Laura Datoand Paola BranduardiDepartment of Biotechnology and Biosciences, University ofMilano-Bicocca, Milano, Italy


Background: In the market of biochemical products a veryimportant role is played by heterologous proteins production,and despite recent advances in mammalian cells exploitation,yeasts can still present advantages as host systems. Among them,the spoilage yeasts belonging to the Zygosaccharomyces genushave become, due to some peculiar properties, significantlyattractive. In particular, Z. bailii is characterized by acidresistance, osmotolerance to high sugar and ethanol concentra-tion combined with high biomass yield. Despite still little is

known about its genetics and cellular biology, our group isworking on its development and exploitation for recombinantproductions with an integrated approach coupling physiologicalstudy with the creation of molecular tools for heterologousproteins production. We previously described and developedtwo patent applications regarding the first techniques necessaryto transform this yeast and to express and secrete differentproteins derived from different sources.Results: Here we present and discuss the last data related to hostoptimisation. Two parallel strategies were followed, one exploitingthe reproducible strategy for target gene deletion we developed,one exploiting a screening selection method. On one side, weobtained the Zbgas1 mutant strain, thanks to the cloning and thesubsequent disruption of the gene ZbGAS1 (homologous to the S.cerevisiae GAS1) involved in cell wall biosynthesis.With this strain theproduction of a model heterologous protein results to be slightlybut significantly improved. Also the indirect strategy, implying theselection of clones resistant to orthovanadate, allowed to isolatemutants where heterologous protein production resulted improvedin respect to the wild type strain.Conclusion: Here we showed that direct and/or indirectstrain manipulation allowed to improve heterologous proteinproduction in the yeast Z. bailii. Although confirms once morethe potentiality of Z. bailii, further improvements and develop-ment of new molecular tools are necessary to assess if this yeastcould be consider in the array of hosts for industrialproductions.

P69Disruption of the GAS1 gene of Pichia pastorisconfers a supersecretory phenotype for Rhizopusoryzae lipase, but not for human trypsinogenHans Marx1, Michael Sauer1,2, David Resina3, Marina Vai4,Danilo Porro4, Francisco Valero3, Pau Ferrer3

and Diethard Mattanovich1,21Institute of Applied Microbiology, BOKU – University ofNatural Resources and Applied Life Sciences, Muthgasse 18, A1190 Wien, Austria2School of Bioengineering, fh-campus wien – University ofApplied Sciences, Muthgasse 18, A 1190 Wien, Austria3Department of Chemical Engineering, Universitat Autonomade Barcelona, 08193-Bellaterra (Cerdanyola del Valles), Spain4Dipartimento di Biotecnologie e Bioscienze, Universita degliStudi di Milano Bicocca, P.zza della Scienza 2, 20126 Milano,Italy


Background: The methylotrophic yeast Pichia pastoris iswidely used for the heterologous production of proteins dueto an advantageous secretory potential [1]. To further improvethe capacity of this yeast to synthesise and secrete high levels ofprotein the limitations of the host have to be unravelled andovercome. Folding and disulfide bridge formation have beenstudied in detail and some approaches have been described tomodulate these processes. However, after release from thesecretory organelles like endoplasmatic reticulum and the golgiapparatus a protein still has to penetrate and cross the cell wall.This can be a major obstacle for heterologous protein secretion,but these events are less well studied up to now.While for many heterologous proteins the cell wall is apparentlynot a limiting barrier, there are some indications of significant



protein retention in the cell wall of S.cerevisiae [2, 3]. In one caseit was shown that deletion of the cell wall cross linking enzymeglycophospholipid-anchored surface protein 1 (Gas1) leads to analmost 7 fold increase of the secreted level of human insulin likegrowth factor 1 in S. cerevisiae [4].Gas1p is a glycoprotein anchored to the outer layer of theplasma membrane through a glycosylphosphatidylinositol (GPI)anchor. Gas1p shows beta-1,3-glucanosyltransferase activity,required for the cell wall assembly. Disruption of GAS1 resultsin several morphological defects: yeast cells lose their typicalellipsoidal shape, become larger than wild type cells, aredefective in bud maturation and in cell separation. The cellwall of Gas1 null mutants is highly resistant to zymolyase, moresensitive to cell wall perturbing agents such as congo red, andthe cells are less protected against osmotic destabilizing agentsas sodium dodecyl sulphate.In this work we set out to clone and delete the GAS1 homologue ofP. pastoris and to analyse the secretory capacity of the yeast cells onthe basis of two proteins studied in our labs for heterologousprotein production, namely human trypsinogen and Rhizopus oryzaelipase (ROL).Results: The coding sequences of the GAS1 homologues of nineyeast species were aligned to identify regions of high homology.Within these regions PCR primers were designed to amplify apartial sequence of the P. pastoris GAS1 gene. Subsequently, it waspossible to amplify the full length gene from a cDNA library.Expression of the cloned gene in S. cerevisiae fully complements thephenotype of a GAS1 deletion, indicating that the protein has theexpected localization and enzymatic activity. Sequence comparisonrevealed the anticipated similarities with other Gas1 like proteins,such as a cysteine rich domain and a serine rich domain and anumber of conserved amino acid patches within the amino terminalcatalytic domain.Targeted gene disruption of the GAS1 gene in P. pastoris wassuccessful and led to the particular phenotype, e.g. a budding defectresulting in the accumulation of Mickey Mouse like shaped yeastaggregates (see Figure 1) and sensitivity against congo red and SDS.Finally, we tested the secretion of two model proteins (humantrypsinogen and ROL) in the GAS1 deleted background incomparison to the wt P. pastoris strain. Secretion of humantrypsinogen was not altered, while the specific secretion rate ofROL was increased approximately 2 fold.Conclusion: As has been previously shown, different hetero-logous proteins encounter different bottlenecks upon expres-

sion in yeast. In any case we could show that the cell wall shouldnot be neglected for improvement of heterologous proteinsecretion. We could demonstrate for the first time that thedeletion of a cell wall cross linking enzyme can have a beneficialeffect on secretion of a heterologous protein in P. pastoris.Further understanding of cell wall construction and the meansby which secreted proteins can cross the cell wall are thereforehighly desirableReferences1. Cereghino JL and Cregg JM: Heterologous protein

expression in the methylotrophic yeast Pichia pastoris.FEMS Microbiol Rev 2000, 24:45–66.

2. Rossini D, Porro D, Brambilla L, Venturini M, Ranzi BM,Vanoni M and Alberghina L: In Saccharomyces cerevisiae,protein secretion into the growth medium dependson environmental factors. Yeast 1993, 9:77–84.

3. Venturini M, Morrione A, Pisarra P, Martegani E andVanoni M: In Saccharomyces cerevisiae a short aminoacid sequence facilitates excretion in the growthmedium of periplasmic proteins. Mol Microbiol 1997,23:997–1007.

4. Vai M, Brambilla L, Orlandi I, Rota N, Ranzi BM,Alberghina L and Porro D: Improved secretion ofnative human insulin-like growth factor 1 fromgas1 mutant Saccharomyces cerevisiae cells. ApplEnviron Microbiol 2000, 66:5477–5479.



P71

Genomic and proteomics approaches for vaccinedevelopment in Pasteurella multocidaKeith Al-Hasani, Victoria McCarl, Stephen Bottomley,Ben Adler and John BoyceAustralian Research Council Centre of Excellence in Structuraland Functional Microbial Genomics, Monash University,Victoria 3800, Australia


Background: P. multocida is a Gram-negative pathogen respon-sible for causing disease in animals of economic significance tolivestock industries throughout the world. It is the causative agentof numerous diseases in animals including fowl cholera in avianspecies, hemorrhagic septicaemia in ungulates and atrophic rhinitisin swine. Current vaccines include bacterins, which only providelimited protection against homologous serotypes and live attenu-ated strains, which have been observed to revert to virulence.Therefore there is a need for more effective vaccines to controldiseases caused by P. multocida.As a step towards developing protective vaccines against fowlcholera, a genomics based approach was applied to theidentification of putative vaccine antigens. This approach utiliseda bioinformatics analysis of the P. multocida genome sequenceusing several types of algorithms (eg. PSORTB) to selectcandidate genes according to their vaccine potential, based ontheir predicted sub-cellular location (outer membrane orsecreted) and their similarity to other proteins with putativeor confirmed experimental roles in infection and immunity as


A. P. pastoris GS115 wt cells showing the normal budding phenotype. B.P. pastoris GS115 GAS1::kanMX4. The GAS1 deleted P. pastoris strainshows frequently two buds attached to the mother cell (Mickey Mouse-like appearance) as reported for GAS1 deficient S. cerevisiae. The typicalappearance is indicated by red circles.



well as expression analysis based on microarray studies. Thebioinformatics-based predictions were complemented by acomprehensive proteomics analysis of the P. multocida outermembrane to identify highly expressed membrane associatedproteins.Results: Over 130 P. multocida proteins were predicted to besurface exposed and/or secreted extracellularly, thus necessitatingthe adoption of a high-throughput cloning strategy. A GatewayTM(Invitrogen) cloning and expression system was used to clone PCR-amplified P. multocidaORFs. The forward primers were designed toinclude the sequence 5’-CACCATG required for directionaltopoisomerase cloning, as well as the addition of an ATG initiationcodon. PCR primers were designed so as to amplify the region thatencompasses the entire coding sequence for the subunit but doesnot include the sequence encoding the signal peptide. As the geneswere to be expressed in frame with a C-terminal tag the nativestop codon was removed when designing the reverse primers. ThePCR products were cloned into the Gateway entry vector pENTR/SD/D-TOPOâ (Invitrogen). A sample of the PCR products isshown in Figure 1, panel A.After the target genes were verified by sequencing andrestriction digestion analysis, they were transferred by recom-bination (LR Clonase kit – Invitrogen) from the entry clone tothe Invitrogen destination vector pBAD-DEST49TM to allowsubsequent expression of C-terminal fusions of the expressedproteins. A sample of an expression screening of 14 clones isshown in Figure 1, panel B. 80% of target clones showed positiveexpression (target clones are fused to an N-terminal Thior-edoxin His-Patch as well as a C-terminal His-tag).Greater than 95% of the fusion proteins were found to beinsoluble. Expression at different temperatures as well as thetransfer of 20 targets into an N-terminal GST vector did notenhance solubility. These expressed proteins are currently beingpurified as insoluble inclusion bodies (see Figure 1, panel C). Theinduced cultures were chemically lysed by incubation of theculture with PopCulture (Novagen), benzoase and lysozyme for20 min at 20˚C 1 ml of the cell lysate was then added to a 96-well filter plate andthe solution was drawn through the filterunder vacuum. The insoluble inclusion bodies were retainedwhile soluble proteins passed through the filter. The retainedinclusion bodies were washed once to remove any remainingsoluble proteins. The inclusion bodies were then denatured bythe addition of 200 �l of 8 M urea to each corresponding well,incubated for 16 h at 4˚C and collected under vacuum.Approximately 25 �g of each antigen were injected subcutaneouslyinto 5 mice followed by a booster injection using aluminiumhydroxide as an adjuvant. Antisera were raised against 18 antigens.Immunoblot analysis was performed with cell lysates prepared fromdifferent strains of P. multocida and probedwith polyclonal antiserumspecific for PM1578, which recognised the native P. multocidaprotein. PM1578 was absent in strains harbouring a mutation inpm1578 (see Figure 1, panel D). Antisera raised against 8 of the 18antigens injected into mice recognised the corresponding wild-typeproteins (data not shown).To evaluate expression of Gateway cloned antigens from P.multocida X-73 during infection in the chicken host, convales-cent-phase sera from three infected chickens were tested forreactivity against all the recombinant proteins using Westernblot analysis. The results demonstrated that immune seragenerated during P. multocida infection recognised a total of 23recombinant proteins (data not shown).


Different P. multocida strains pm1578 mutant. Shown are (A)PCR amplification of P. multocida vaccine target ORFs for Gatewaycloning. (B) SDS-PAGE and Coomassie Blue staining of crude lysatesexpressing P. multocida X-73 recombinant proteins in E. coli DH5. Allcultures were induced with arabinose at mid-log phase (C) Isolation oftarget proteins from inclusion bodies. Coomassie Blue staining of 14recombinant proteins solubilised in 8 M Urea. (D) Proteins in lysateswere separated by electrophoresis and the immunoblot was probed withmouse antiserum raised against PM1578.



Conclusion and future development: Gateway cloningsystem provides a highly efficient method to generate plasmidsharbouring genes from P. mutlocida X-73 and for the generationof expression clones.In silico analysis identified 130 membrane associated and secretedproteins, 114 of which have been successfully cloned into aGateway entry vector. Out of these 114 entry clones, 92 weretransferred to a Gateway expression vector. Of these, 80% wereshown to express measurable levels of protein in E. coli.Because of the relatively poor efficiency of recovering solubleproteins tagged with the thioredoxin tag, efforts are underwayto express the target proteins in different expression systems.To address this hurdle and as means of preferably obtainingcorrectly folded proteins for vaccine and functional studies,preliminary attempts to express genes as recombinant proteinsin NusA fusion expression constructs resulted in significantimprovement.

P72Stabilization of heterologous transcripts withhrpA, mRNA of a type III secretion systemcomponentElina Hienonen1, Martin Romantschuk2 and Suvi Taira11Department of Biological and Environmental Sciences,Division of General Microbiology, University of Helsinki, Finland2Department of Ecological and Environmental Sciences,University of Helsinki, Finland


Background: Type III secretion systems are used by manyGram negative bacteria pathogenic to plants and animals totransfer virulence factors (effectors) directly into host cells, andto cause disease. The flagellar secretion systems are alsoclassified as type III secretion systems. Both systems are wellstudied, but some elements still remain puzzling. No clearconsensus sequence has been found for the type III secretionsignal, and opinions of its nature vary from an amphipathic aminoacid signal to an mRNA signal. Some type III secretion substratesuse specific chaperones to aid in their secretion, but othersseem to rely completely on another kind of a signal. This signal isfound in the first 10–28 amino acids or codons of these proteins.The possibility of an mRNA secretion signal led us to study thetranscript of one type III secreted protein of the plantpathogenic bacterium Pseudomonas syringae, HrpA. Hrp-pilus, acomponent of the secretion apparatus, is composed of HrpApilin subunits. HrpA is itself secreted by the type III secretionsystem. hrpA forms an operon with hrpZ in P. syringae pathovartomato, but not in pv. phaseolicola. We have shown that thesecretion signal of hrpA from P. syringae pv. tomato is in the first15 codons [1]. The half-life of hrpA mRNA from different plantpathogenic species is exceptionally long, approximately 20–40minutes [2]. This was true under varying temperature conditionsand also when the transcript was produced in E. coli. Thus thestability of the mRNA is a characteristic of the transcript itself,not dependent on extra factors of the Pseudomonads. Nophysiological function has been assigned for the long half-life, butit may be related to the high abundance of HrpA protein or tothe function of the mRNA as a secretion signal.Results: We have used the unusual stability of hrpA mRNA tostabilize heterologous mRNAs fused to it. Heterologous

transcripts originating from various sources were stabilized byhrpA in E. coli and in P. syringae. Their half-lives were increasedfrom a few minutes of the control strains with no stabilizingelements to up to 25 min. The regions needed for the stabilizingeffect were narrowed down. Protein production levels werealso improved in this system. The amounts of heterologousproteins produced from these stabilized constructs were up to 5times that of the control strain.Conclusion: Naturally stable transcripts can be used tostabilize heterologous transcripts. Specific structures inmRNAs, often hairpins in the 5’ or the 3’ regions, have beenshown to protect mRNAs against RNases. These structures areoften conserved in evolution. In the case of hrpA, thehypothetical stabilizing secondary structures are highly con-served, and thus likely to serve an important function.References1. Hienonen E, Roine E, Romantschuk M and Taira S: mRNA

stability and the secretion signal of HrpA, a pilinsecreted by the type III system in Pseudomonassyringae. Mol Genet Genomics 2002, 266:973–978.

2. Hienonen E, Rantakari A, Romantschuk M and Taira S: Thebacterial type III secretion system-associated pilinHrpA has an unusually long mRNA half-life. FEBS Lett2004, 571:217–220.

P73Over-expression and single-step purification ofhuman IFNa8 and human IFNa2b reveals thehighest antiviral activity of human IFNa8Julio Cesar Sanchez Garcıa1, Alejandro Miranda Ariza1,Alexis Musacchio Lassa2, Luis Javier Gonzalez2

and Vladimir Besada Perez21Process Control Department, Center for Genetic Engineeringand Biotechnology, P.O. Box 6162, C.P. 10600, Havana C.,Cuba2Division of Physical-Chemistry, Center for Genetic Engineeringand Biotechnology, P.O. Box 6162, C.P. 10600, Havana C., Cuba


Background: Human alpha interferons (HuIFNa) comprise amultigene family, originally identified as proteins responsible forthe induction of cellular resistance to viral infections, subdividedinto 13 different subtypes (IFNa1, a2, a4, a5, a6, a7, a8, a10, a13, a14,

a16, a17 and a21). The genes that encode these proteins areclustered on chromosome 9 in human [1]. There have beenreports of obvious differences in the relative biologicalsactivities among IFNa subtypes [2, 3] and it was found that theactivity varied greatly depending on the target cells, the IFNa

subtypes and even if the proteins were compared on base ofboth units of biological activity or mass [4]. The HuIFNa2b and

HuIFNa8 are among the IFN subtypes with highest antiviralactivity. The first one was licensed by the Food and DrugAdministration in 1986 (USA) for the treatment of hairy cellleukemia [5]. On the other hand, the HuIFNa8 has shown thehighest biological activity in several in vitro assays [6, 7, 8].Several progresses in the fundamental understanding oftranscription, translation, and protein folding in Escherichia coli,together with the availability of improved genetic tools(including new mutants) are making this bacterium morevaluable than ever for the expression of complex eukaryotic



proteins. Several strategies have been required to enhance rarecodons gene expression [9, 10].Homogenous HuIFNa preparations were originally obtained in1978 for further chemical and physical characterization [11].Thereafter have been introduced new high performancechromatography techniques useful to obtain enough quantityof these proteins for their chemical, biological and immunolo-gical studies [12]. Although high quantities of these proteins

could be obtained, these procedures involve costly andlaborious purification protocols.In our previous report we attempted to over express the HuIFNa8 inE. coli without success [13]. In the present work, the heterologousexpression of both HuIFNa8 and HuIFNa2b was significantly improved.These proteins were purified by one step and low cost SDS-PAGEprocedure and used for detailed chemical and physic characteriza-tion. Both HuIFNa subtypes obtained were used to compare its


Clusters of 30 aminoacid presents in bothHuIFN subtypes. In boxes, the AGA/AGG triplets coding for Arginine. The ATA and CTA triplets coding forIsoleucine and Leucine, respectively, are underlined.


Expression of both HuIFN subtypes in E. coli cells grown in minimum media. (A) 12.5% SDS-PAGE stained with Coomassie blue, and (B)Immunoblotting analysis using a mixture of rabbit polyclonal antibodies specific for both HUIFN2 and HUIFN8. Samples: (1) E. coli BL-21/ pALF8-4, (2) E.coli BL-21CP RIL/ pALF8-4, (3), E. coli BL-21CP RIL/ pAGUA-4, (4) E. coli BL-21/ pAGUA-4, (5) E. coli BL-21CP RIL. The molecular weight markers (M)are indicated (M).



antiviral activity by using an in vitro model involving Hep-2 cellschallenged with Mengo virus [14].Results: Comparing the rare codon compositions of both

HuIFNa subtypes, two clusters of 30 amino acids, containing fiveminor triplets, were found (see Figure 1). For the HuIFNa2b gene,the minor codons, represented only by AGG/AGA (thatencodes for Arg) are present in both clusters. However,besides the minor triplets that encodes for Arg, in the HuIFNa8

gene one minor codon AUA (ATA triplet, that encodes for Ile)is present in the first cluster, while in the second one, the CUA(CTA triplet, that encodes for Leu) exists.Taking into account the minor codon composition in both

HuIFNa genes, E. coli BL21-codonplus-RIL cells (Stratagene, USA)

were used to increase the heterologous polypeptides expres-sion levels. E. coli BL21-codonplus-RIL cells were transformedwith the plasmids pALF8-4 and pAGUA-4 [13] coding for

HuIFNa8 and HuIFNa2b, respectively. This strain carries outadditional copies of the dnaY, ileY and leuW tRNAs in anadditional Cmr pACYC derivative plasmid. The last two tRNAdecode the AUA and CUA minor codons, respectively. Theobtained transformants were grown in M9 minimum medium.For HuIFNa8 transformants, the protein expression levelincreased from 1% (with E. coli BL-21/pALF8-4) up to 25%(with E. coli BL-21 CP RIL/pALF8-4) of total bacterial proteinsbased on densitometric analysis of scanned gels (see Figure 2).Similar results were obtained with the HuIFNa2b Ampr-Cmr


(A). RP-HPLC analysis of purified HUIFN2 (A) and HUIFN8 (B). The molecular weight of markers shown in SDS-PAGE are: 34 kDa, 26 kDa, 20 kDa and7 kDa, respectively. (B). The LC-MS analysis of the tandem digestion tryptic/V8 revealed that cysteine residues of HuIFN8 and HuIFN2b are correctlylinked by disulfide bridges ((Data not show).



transformants, the expression levels increased from 5% (with E. coliBL-21/pAGUA-4) up to around 20% (with E. coli BL-21CPRIL/pAGUA-4) of total host proteins.The purity of purified HuIFNa subtypes was estimated over 98%,based on the homogeneity observed in the silver stained SDS-PAGE and by RP-HPLC analysis (see Figure 3).The antiviral potency of both HuIFNa subtypes was compared inan in vitro assay with one microgram of each protein challengedwith mengo virus [14]. Results of five independent experimentsare shown in the table 1. The HuIFNa8 had 1,46 fold moreantiviral activity than HuIFNa2b (p < 0,05) in this biologicalsystem.Conclusion: In summary, this work highlights several issues toobtain human proteins with high molecular homogeneityavoiding costly and tedious purification procedures. Therefore,we recommend this experimental strategy to obtain humansproteins with high therapeutic potentials. The highest antiviralactivity of HuIFNa8 shown in vitro add new evidences to takeinto account this proteins as strong therapeutic candidate.AcknowledgementsThe authors thank Prof. Jose Luis Fernandez Sierra for criticalreading of the manuscript, and especially to Prof. Magaly GarciaBlanco for her substantial contribution and support to thiswork.References1. Pestka S, Krause CD and Walter MR: Interferons,

interferon-like cytokines, and their receptors.Immunol Rev 2004, 202:8–32.

2. Foster GR, Rodrigues O, Ghouze F, Schulte Frohlinde-E,Testa D, Liao MJ, Stark GR, Leadbeater L and Thomas HC:Different relative activities of human cell-derivedinterferon-alpha subtypes: IFN-alpha 8 has veryhigh antiviral potency. J Interferon Cytokine Res 1996,16:1027–1033.

3. Yanai Y, Sanou O, Kayano T, Ariyasu H, Yamamoto K,Yamauchi H, Ikegami H and Kurimoto M: Analysis of theantiviral activities of natural IFN-alpha prepara-tions and their subtype compositions. J InterferonCytokine Res 2001, 21:835–841.

4. Blatt L, Davis J, Klein S and Taylor M: The biologicactivity and molecular characterization of a novelsynthetic interferon-alpha species, consensus inter-feron. J Interferon Cytokine Res 1996, 16:489–499.

5. Bekisz J, Schmeisser H, Hernandez J, Goldman N andZoon K: Human interferons alpha, beta and omega.Growth Factors 2004, 22:243–251.

6. Yamamoto S, Yano H, Sanou O, Ikegami H, Kurimoto Mand Kojiro M: Different antiviral activities of IFN-alpha subtypes in human liver cell lines: synergismbetween IFN-alpha2 and IFN-alpha8. Hepatol Res2002, 24:99–115.

7. Foster G, Rodrigues O, Ghouze F, Schulte Frohlinde-E,Testa D, Liao M, Stark G, Leadbeater L and Thomas H:Different relative activities of human cell-derivedinterferon-alpha subtypes: IFN-alpha 8 has veryhigh antiviral potency. J Interferon Cytokine Res 1996,16:1027–1033.

8. Yanai Y, Horie S, Yamamoto K, Yamauchi H, Ikegami H,Kurimoto M and Kitamura T: Characterization of theantitumor activities of IFN-alpha8 on renal cellcarcinoma cells in vitro. J Interferon Cytokine Res 2001,21:1129–1136.

9. Foster G, Masri S, David R, Jones M, Datta A, Lombardi G,Runkell L, Sizing I, James M and Marelli Berg-F: IFN-alphasubtypes differentially affect human T cell motility.J Immunol 2004, 173:1663–1670.

10. Zhou Z, Schnake P, Xiao L and Lal A: Enhancedexpression of a recombinant malaria candidatevaccine in Escherichia coli by codon optimization.Protein Expr Purif 2004, 34:87–94.

11. Rubinstein M, Rubinstein S, Familletti PC, Gross MS,Miller RS, Waldman AA and Pestka S: Human leukocyteinterferon purified to homogeneity. Science 1978,202:1289–1290.

12. Pestka S: The human interferon alpha species andreceptors. Biopolymers 2000, 55:254–287.

13. Acosta Rivero-N, Sanchez JC and Morales J: Improve-ment of human interferon HUIFNalpha2 and HCVcore protein expression levels in Escherichia colibut not of HUIFNalpha8 by using the tRNA(AGA/AGG). Biochem Biophys Res Commun 2002, 296:1303–1309.

14. Santana H, Martınez E, Sanchez JC, Moya G, Sosa A,Hardi E, Beldarrain A, Gonzalez LJ, Betancourt L, Besada V,Curras T, Ferrero J, Pujols V, Gil M and Herrera L:Molecular characterization of recombinant humaninterferon alpha-2b produced in Cuba. BiotecnologiaAplicada 1999, 16:154–159.

P74Bacillus megaterium as a recombinant proteinproduction hostYang Yang1, Marco Malten2, Rebekka Biedendieck2,Wei Wang1, Dieter Jahn2 and Wolf-Dieter Deckwer11Biochemical Engineering, GBF, Mascheroder Weg 1, 38124Braunschweig, Germany2Institute of Microbiology, Technical University Braunschweig,Spielmannstrasse 7, D-38106 Braunschweig, Germany


Background: The gram positive soil bacterium Bacillus mega-terium is well known for its industrial utilization for productionof various extracellular enzymes as amylases. Recently produc-tion and secretion of recombinant proteins in B. megaterium wasalso studied [1, 2]. In this contribution a homologous modelprotein (penicillin G amidase (PGA) from B. megateriumATCC14945) and a heterologous protein (a hydrolase from

Table 1 (abstract P73) Antiviral potency of both purified HuIFNsubtypes.

Sample Titer1

Titer2

Titer3

Titer4

Titer5

Titeraverage

HuIFNa8 5.1 4.5 4.8 3.9 4.1 4.48 (3.86-5.09)a

HuIFNa2b 2.3 2.8 2.5 3 2.1 2.54 (2.08-2.99)a

E. coliBL-21CP RIL

nd nd nd nd nd nd

The titer values are expressed in 106 international units. nd, not antiviralactivity detectable.a Values in parenthesis correspond to confidencelimits (p < 0.05).



Thermobifida fusca DSM43793 (TFH)) were used to furtherinvestigate and improve the system. Penicillin amidase is appliedin the synthesis of semisynthetic penicillins. TFH is able todegrade specific polyesters such as poly (ethylene terephthalate)(PET) or poly (butylene terephthalate) (PBT), which are hitherto regarded as ’non-biodegradable’ plastics [3].Results: We are using the plasmid-based xylose-inducible geneexpression system which was optimized via introduction of amultiple cloning site and removing a cre element mediatingglucose-dependent catabolite repression. In order to improvethe induction efficiency a xylose deficient strain was developedby knocking out the xylose isomerase gene (xylA) from MS941(�nprM), which improved the PGA production 2 fold. Using thelipA signal peptide instead of the native signal peptide from PGAincreased the PGA secretion 1.6 fold in shaking flask cultivation(see Figure 1). N-terminal amino acid sequence results of PGAshowed that it has a signal peptide MKTKWLISVIILFV-FIFPQNLVFA, a 27,000 Da a subunit which began withG25EDKNEGVKVVR and a 57,000 Da � subunit began withS266NAAIVGSEKSATGN. They matched perfectly with theamino acid sequence derived from the nucleotide sequence ofthe cloned pac gene of pRB49 which came from B. megateriumATCC14945. Further cultivation optimization showed that earlyinduction strategy was better than later induction. 2.5 mMCalcium was the best concentration for helping PGA bindingprocess (see Figure 2). Currently microtiter plate cultivationwas developed for growth medium optimization. Compared tothe successful expression of PGA the heterologous TFH geneonly expressed after its codon usage was optimized for B.megaterium using JCat [4]. Foreign protein production wassuccessfully upscaled from shaking flask cultivation over batchfermentation with control of pH to high cell density cultivationin a 2 L bioreactor.

Conclusion: In this new host system both proteins weresecreted directly into supernatant and the good productivitywas obtained from fermentation.References1. Malten M, Hollmann R, Deckwer WD and Jahn D:

Production and secretion of recombinant Leuconos-toc mesenteroides dextransucrase DsrS in Bacillusmegaterium. Biotechnol Bioeng 2005, 89:206–218.

2. Malten M, Biedendieck R, Gamer M, Drews AC, Stammen S,Buchholz K, Dijkhuizen L and Jahn D: A Bacillusmegaterium Plasmid System for the Production,Export, and One-Step Purification of Affinity-Tagged Heterologous Levansucrase from GrowthMedium. Appl Environ Microbiol 2006, 72:1677–1679.

3. Muller RJ, Schrader H, Profe J, Dresler K and Deckwer WD:Enzymatic Degradation of Poly(ethylene terephtha-late): Rapid Hydrolyse using a Hydrolase from T.fusca. Macromolecular Rapid Communications 2005,26:1400–1405.

4. Grote A, Hiller K, Scheer M, Munch R, Nortemann B,Hempel DC and Jahn D: JCat: a novel tool to adaptcodon usage of a target gene to its potentialexpression host. Nucleic Acids Res 2005, 33:W526–W531.

P75Design and production of multi-bioactiverecombinant elastin-like polymer: Mimickingthe extracellular matrixAlessandra Girotti, F Javier Arias, Ana M Testeraand J Carlos Rodriguez-CabelloBIOFORGE (Group for Advanced Materials andNanobiotechnology) Universidad de Valladolid, Centro I+D,Paseo de Belen S/N, Campus Miguel Delibes, 47011,Valladolid, Spain


Background: One of the main tasks in the development oftissue engineering is the advance in the design and production ofmaterials designed to act as support for the growing cells andtissues. The evolution in the development of artificial extra-cellular matrices (ECM) began with the use of biotolerated


Specific activity curve of PGA after it was secreted into the growthmedium by B. megaterium MS941 containing pRB23 with native peptidefrom PGA (), MS941 containing pRB49 with signal peptide lipA () andYYBm1 containing pRB23 () in LB complex medium. At OD578 nm of 0.4production of TFH was induced by the addition of 0.5 (w/v) % xylose tothe growth medium. Samples were taken at various time points afterinduction.


Proteins from 1.5 ml growth medium form samples taken at indicatedtime points which showed as the lines at the bottom of the graph,precipitated by ammonium sulfate, analyzed by SDS-PAGE and stained byCoomassie Brilliant Blue. G250. The lines at the top of the graphseparate the cultivation medium with different Ca2+ concentration 0mM, 2.5 mM and 5 mM. Lane M shows Precision Plus Protein Standard(Bio-Rad, Muechen).



synthetic materials, mostly polymers, which showed cellattachment and spreading capabilities of a rather unspecificnature. Soon, these materials were improved. Some macro-molecules of natural origin were used but more important,more specific functionalities were included in their structure,specially peptide cell attachment sequences.More recently, the development of genetic engineering hasallowed the design and bioproduction of protein polymers.These are mainly made from repeating sequences found innatural proteins, such as elastin, silks, etc., and selectedmodifications [1]. One of the most promising family of proteinpolymers is the Elastin-like polymers (ELPs) which have shownan outstanding biocompatibility [2]. These polypeptides arebased on the recurrence of certain short monomers that areconsidered as building blocks in the natural elastin. The mostrepresentative polymer of this family is poly (VPGVG), and thevast majority of the ELPs described in the literature are madefrom selected modifications of this pentamer or its permutation.ELPs are extremely biocompatible and bioprocessable and showand acute smart and self-assembling behavior. Recently, theadaptation of the genetic engineering techniques to this field hasopened the possibility of obtaining these polymers with absolutecontrol and absence of randomness in the primary structure.This has allowed the production of multifunctional polymersthat can combine physical, chemical and biological functions.This works was intended as being one step more towards thedevelopment of a complex and multifunctional artificial ECMthat mimics the versatility and complexity of the natural ECM.We describe herein the use of artificial genes to direct thesynthesis of ELPs containing cell attachment sequences (RGDand REDV) of precisely controlled architecture and itsbioproduction in E. coli [3, 4].Materials and methods: Plasmid Construction: Stan-dard molecular biology techniques were performed to constructtwo polymer genes and their sequences were verified byautomated DNA sequencing. DNA duplex encoding the monomergenes were obtained by Polymerase Chain Reaction (PCR) usingtwo synthetic oligonucleotides. The first monomer gene (A)codifying the amino-acid sequence [(VPGIG)2-(VPGKG)-(VPGIG)2]2-AVTGRGDSPASS- [(VPGIG)2-(VPGKG)-(VPGIG)2]2and the second one (B) (VPGIG)2-(VPGKG)-(VPGIG)2-EEIQIGH-IPREDVDYHLYP-(VPGIG)2-(VPGKG)-(VPGIG)2-(VGVAPG).After gene cloning, the monomer was obtained by digestion,

isolated and subjected to concatenation (controlled ligation). Theconcatenamer mixture was cloned into a modified expressionvector derived from pET-25(+). Plasmids were selected based ontheir insert length by PCR colony screening and transformed intothe E. coli expression strains BL21(DE3) and BLR(DE3). Bacterialgrowth and gene expression were performed following themanufacturer’s instructions. The polymer purification was carriedout by several cycles of cold (4˚C) and warm (50˚C) centrifugation.MALDI-TOF, amino acid analysis and spectroscopic methodsproved final polymers high purity and correct sequence. Turbidityexperiments and Differential Scanning Calorimetry (DSC) wereused to calculate the Transition Temperature (Tt) of the polymers.Results: The bioproduced polymers comprise different buildingblocks, each showing a different functionality (see Figure 1). First, thefinal matrix is to be designed to show a mechanical responsecomparable to the natural extracellular matrix, so they are producedover a base of an elastin-like polymer of the type (VPGIG)n. Alongwith the desired mechanical behaviour, this base has shown anexcellent biocompatibility. The second building block is a variation ofthe first. It has a lysine substituting an isoleucine so the lysine �-aminogroup can be used for cross-linking purposes while retaining theproperties of elastin-like polymers.The third group contains cell attachment sequences. In polymerA, this sequence is based the well known RGD, while in polymerB is the CS5 human fibronectin domain, which has theendothelial REDV cell attachment sequence. In addition,polymer B also contains the elastase target sequence VGVAPGto favour its bioprocesability by natural routes.The polymers and the corresponding matrices (see Figure 2)have been subjected to mechanical tests, physical-chemical,biochemical and "in vitro" analysis. The results clearly confirmthe excellent behaviour of this matrix for the pursued purpose,as expected from the combination of functionalities andproperties of the building blocks used in its molecular design.Conclusion: Different recombinant elastin-like protein poly-mers have been designed and bioproduced in E. coli to createadvanced scaffolds for tissue engineering. All of them contain, atleast, cell adhesion domains of the type RGD or REDV, andspecific protease target domains. They can be easily cross-linkedto obtain elastomeric hydrogels. These new polymers has beensubjected to mechanical tests, physical-chemical and in vitroanalysis. The results confirm the excellent behaviour of thesematrices for this purpose.


Schematic representation of the polymers A B architecture, identifying each building block with its corresponding amino-acid sequence.



AcknowledgementsThis work was supported by the "Junta de Castilla y Leon"(VA002/02), by the MEC (MAT2003-01205 and MAT2004-03484-C02-01) and by the European Commission (BioPolySurfMRTN-CT-2004-005516).References1. Rodrıguez-Cabello JC, Reguera J, Girotti A, Alonso M and

Testera AM: Developing functionality in elastin-likepolymers by increasing their molecular complexity:the power of the genetic engineering approach. ProgPolym Sci 2005, 30:1119–1145.

2. Rodrıguez-Cabello JC, Reguera J, Girotti A, Arias FJ andAlonso M: Genetic engineering of protein-basedpolymers: The example of elastinlike polymers. AdvPolym Sci 2006 in press.

3. Girotti A, Reguera J, Rodrıguez-Cabello JC, Arias FJ,Alonso M and Matestera A: Design and bioproductionof a recombinant multi(bio)functional elastin-likeprotein polymer containing cell adhesion sequencesfor tissue engineering purposes. J Mater Sci Mater Med2004, 15:479–484.

4. Arias FJ, Reboto V, Martın S, Lopez I and Rodrıguez-Cabello JC: Tailored recombinant elastin-like polymersfor advanced biomedical and nano(bio)technologicalapplications. Biotechnol Lett 2006 in press.

P76N-terminally acetylated tropomyosin generated inE. coli by coexpression of the S. cerevisiae NatBacetylation complex shows functional propertiesin vitroRobin Maytum1 and Manfred Konrad21School of Chemical and Biological Sciences, Queen Mary,University of London, London E1 4NS, UK2Max-Planck-Institute for Biophysical Chemistry, D-37070Goettingen, Germany


Background: One of the most significant differences inproteins produced in eukaryotes is their chemical modification,which does not occur in prokaryotes. These post-translationalmodifications of certain amino acid residues are often essentialfor protein function. Therefore, generating systems that allowthem to be made in bacterially expressed proteins is of greatfundamental interest and of potential benefit in biotechnological

applications. We have developed a system that allows theproduction of one kind of protein modification in bacteria, N-terminal acetylation [1], by coexpressing the yeast NatBacetylation complex and the target protein tropomyosin (TM).Results: Tropomyosin is a protein that plays a role both incontrolling the contraction of muscle and also a much moregeneral role as part of the scaffolding matrix within cells, knownas the actin cytoskeleton. Without an essential acetylation ofone of its ends, TM no longer binds to the cytoskeleton and thecells are severely impaired or even die. A pair of proteins, ofwhich similar versions are found in all eukaryotic cells, does thismodification. In yeast, these proteins are Nat3 (195 amino acids)and Mdm20 (796 aa), together making up the NatB acetylationcomplex [2]. Nat3 is the acetylase core, and Mdm20 the TMbinding protein. Yeast and mammalian TM can be easilyoverproduced in bacteria. However, since bacteria do nothave proteins similar to NatB, TM does not work correctlywhen produced in this way. We generated an E. coli coexpres-sion system for TM plus the two NatB components based on theNovagen pET Duet vectors that allow cloning of two genes intandem under identical T7 promoters.The coding regions of the two proteins were cloned directlyfrom S. cerevisiae genomic DNA via PCR. Expression trials ofthese proteins in isolation showed good overproduction ofNat3, with a clear band visible in total cell lysate separated bySDS-PAGE. This band has an apparent size of around 28 kDa,somewhat larger than the predicted molecular mass of 22.9 kDa.However, no band was visible for Mdm20 at around itspredicted 92.8 kDa. The two coding sequences were thentransferred into the Duet expression vector, and the resultingvector was transformed into E. coli BL21(DE3)pLys. For TMcoexpression, the cells were additionally transformed withpJC20 expression vectors containing either yeast TM1 orvertebrate skeletal TM that we had characterized previously[3, 4, 5]. Vectors were maintained by selection by ampicillin(pJC20) plus either kanamycin (pRSFDuet) or spectinomycin(pCDFDuet). TM was purified and its binding to rabbit F-actinmeasured by cosedimentation studies as described [3]. Theyeast TM1 species was found to behave similar to the onecarrying a short N-terminal extension, such as Ala-Ser, or Ala-Gly-Ser-Ser-Ser, to mimic the acetylation present in native TMfrom eukaryotic sources, thus indicating the presence ofN-terminal acetylation as a prerequisite for high-affinity bindingof TM to actin.Conclusion: Our data show that all three proteins can beproduced via coexpression. However, since their relativequantities proved to be very variable, more work is needed tooptimize the expression conditions to produce balancedquantities of the acetylating proteins (Nat3/Mdm20) and alarger amount of the acetylation target (TM).References1. Polevoda B and Sherman F: N-terminal acetyltrans-

ferases and sequence requirements for N-terminalacetylation of eukaryotic proteins. J Mol Biol 2003,325:595–622.

2. Singer JM and Shaw JM: Mdm20 protein functions withNat3 protein to acetylate Tpm1 protein and reg-ulate tropomyosin-actin interactions in buddingyeast. PNAS 2003, 100:7644–7449.

3. Maytum R, Geeves MA and Konrad M: Actomyosinregulatory properties of yeast tropomyosin are


Photograph of a cross-linked matrix of the bioproduced B polymer.



dependent upon N-terminal modification. Biochemis-try 2000, 39:11913–11920.

4. Maytum R, Konrad M, Lehrer SS and Geeves MA:Regulatory properties of tropomyosin: Effects oflength, isoform, and N-terminal sequence. Biochem-istry 2001, 40:7334–7341.

5. Maytum R, Bathe F, Konrad M and Geeves MA: Tropo-myosin exon 6b is troponin-specific and required forcorrect acto-myosin regulation. J Biol Chem 2004,279:18203–18209.

P77Use of the tetA-promoter in fed-batch cultivations:Repeated supply of anhydrotetracycline isnecessary for production of tetrameric collagenprolyl 4-hydroxylase in Escherichia coliA Neubauer1, J Soini2, M Bollok2, M Zenker2, J Sandquist2,J Myllyharju1 and P Neubauer21Dept. Medical Biochem. & Molec. Biology, P. O. Box 5000;Biocenter Oulu, University of Oulu, FIN-90014 Oulu, Finland2Bioprocess Engineering Laboratory, Dept. Process &Environm. Engin., P. O. Box 4300, Biocenter Oulu, University ofOulu, FIN-90014 Oulu, Finland


Background: Human collagen prolyl 4-hydroxylase (C-P4H),an ER lumenal protein, is a key enzyme in the biosynthesis ofcollagens and consists of two different subunits forming an a2�2

tetramer. Heterologous cytoplasmic production of an activeC-P4H in Escherichia coli using a bicistronic vector with the T5-lacand tet promoters and the strain Origamiä as a host wasdescribed earlier [1]. Gene optimisation of the � subunit that isidentical to protein disulfide isomerase and selection of the bestinduction conditions improved the obtained activity of recombi-nant C-P4H in shake flask cultivations further by a factor of 50 [2].Results: Although high amount of active C-P4H was obtainedin shake flask cultures with long-time induction, the amount waslow in fed-batch cultivations using the same induction strategy.Analysis of the mRNAs of the a and � subunits in fed-batchfermentations by Sandwich hybridisation revealed that singleaddition of the inducer anhydrotetracycline (aTc) leads to onlytransient induction of the a subunit, in contrast to IPTG whichleads to a stable level of � mRNA.Surprisingly, repeated induction with aTc led to a stable level ofa mRNA and, consequently, to higher yields of the active C-P4Htetramers. The expression was strongly dependent on the celldensity and the specific growth rate and provided best results ifthe culture was induced during the batch cultivation phase.Conclusion: Our results indicate specific, so far non-described, features of the tet promoter based expressionsystem. The repeated stepwise addition of aTc at higher celldensities might be of particular importance for other expressionsystems involving the tetA promoter and long expression time.References1. Neubauer A, Neubauer P and Myllyharju J: High-level

production of human collagen prolyl 4-hydroxylasetetramer in Escherichia coli. Matrix Biol 2005, 24:59–68.

2. Niemitalo O, Neubauer A, Liebal U, Myllyharju J, Juffer AHand Neubauer P: Modelling of Translation of HumanProtein Disulfide Isomerase in Escherichia coli.J Biotechnol 2005, 120:11–24.

P78Monitoring protein expression levels in E. coliusing a high throughput approachRegis Cebe and Martin GeiserNovartis Institutes for Biomedical Research, 4000 Basel,Switzerland


Background: We have developed a high-throughput methodto rapidly identify the protein constructs which are wellexpressed and find out which experimental factors influencetheir production. From a sparse matrix designed to screenbetween expression strains, culture media, lysis and purificationbuffers for each construct, the interactions among variablesleading to a higher yield of soluble recombinant protein can beeasily identified.This screening is performed by a combination of small scalefermentation in deep-well blocks, cell lysis with a 24 microtipssonicator, Ni-NTA magnetic beads purification, and an auto-mated gel capillary electrophoresis system, which allows a high-throughput and quantitative analysis of the multiple variables inone experiment.This technique allows one to evaluate as early as possible theexpression level of the constructs, narrowing down the numberof constructs subsequently going through the large scalefermentation and purification module.Results: Using a combination of robotic systems like theQIAGEN BioRobot 3000, a microsonification device (20 kHz 24element probe from SONICS) and an Agilent ALP5100, werapidly monitor in parallel and in a microtiter well format thelevel of expression in E. coli of the different protein constructs,in 15 different conditions (see Table 1). We also aim at thedefinition at an early stage the buffer conditions allowing proteinstabilization during cell lysis and purification (Figure 1).

Table 1 (abstract P78)

Expressionstrain

Culturemedium

Lysis andpurification buffer

Condition 1 Strain 2 Medium 2 Buffer 1Condition 2 Strain 3 Medium 1 Buffer 4Condition 3 Strain 3 Medium 2 Buffer 2Condition 4 Strain 1 Medium 1 Buffer 1Condition 5 Strain 2 Medium 3 Buffer 3Condition 6 Strain 1 Medium 3 Buffer 4Condition 7 Strain 2 Medium 2 Buffer 4Condition 8 Strain 1 Medium 2 Buffer 3Condition 9 Strain 3 Medium 2 Buffer 5Condition 10 Strain 1 Medium 1 Buffer 5Condition 11 Strain 2 Medium 3 Buffer 5Condition 12 Strain 3 Medium 1 Buffer 3Condition 13 Strain 3 Medium 3 Buffer 1Condition 14 Strain 2 Medium 1 Buffer 2Condition 15 Strain 1 Medium 3 Buffer 2

Sparse matrix design (http://www.igs.cnrs-mrs.fr/samba/samba.html):Strain 1: BL21(DE3)/Strain 2: C43(DE3)(from OverExpress)/Strain3:BL21(DE3) harboring pG-KJE8 vector from Takara Medium 1: LB/Medium 2: Auto-induction/Medium 3: Turbo broth+ Augmediumfrom AthenaES Buffer 1: 40 mM HEPES;150 mM NaCl;10%glycerol;pH7/Buffer 2: 40 mM HEPES;400 mM NaCl;100 mM urea;pH7/Buffer3: PBS pH8/Buffer 4: 40 mM Tris;150 mM NaCl;10%glycerol;pH8.5/Buffer 5: 40 mM Tris;400 mM NaCl;100 mM urea;pH8.5



P79Recombinant protein production in cell-freesystems: strategies for improving yield andfunctionalityJan Strey1, Michael Gerrits1, Stefan Kubick1,Helmut Merk1, Uritza von Groll2, Frank Schafer2

and Wolfgang Stiege11RiNA GmbH, 14195 Berlin, Germany2QIAGEN GmbH, 40724 Hilden, Germany


Background: One of the major obstacles of the post-genomeera is the efficient production of proteins for structural andfunctional analysis. To solve this problem cell-free proteinbiosynthesis is an attractive tool. It is fast, easy to handle andadaptable to the requirements of the synthesized proteins.Results: Our highly productive cell-free protein synthesissystem based on E. coli lysates yields up to several hundredmicrograms protein per milliliter in a 1 h batch reaction.Recently we developed an advanced version of this system,which we call the "Recycling System". This system comprisestwo or more cycles, each subdivided in a protein synthesis and asubsequent recycling phase. The recycling step saves highmolecular weight components such as template, ribosomesand transcriptional/translational factors, whereas low molecularweight reaction byproducts (e.g. inhibitory inorganic phosphate)are removed.Since this recycling technology leads to an accumulation of thetarget protein in consecutive synthesis cycles, it is well suited forpreparative approaches such as the synthesis of native or labeledproteins for structural research (NMR, X-ray). Furthermorerecycling can be used for lysate programming, i.e. in the firstsynthesis step proteins can be expressed, which are beneficialfor the production of a target protein in the following synthesisstep(s). As we demonstrate here, this strategy could besuccessfully applied to the preparation of chaperone-enrichedlysates for functional protein production.

Finally we present alternative strategies for enhancing function-ality of cell-free synthesized proteins such as the addition offolding catalysts, specific modification of reaction conditions,cotranslational application of a multifactorial matrix andposttranslational reactivation.Conclusion: Our data clearly show, that cell-free proteinbiosynthesis is a versatile platform for production of functionalproteins in analytical and preparative scale.AcknowledgementsThis work was kindly supported by the German Ministry ofEducation and Science (BMBF) and the Senate of Berlin.

P80A regulatory acceptable alternative to E. coli: highyield recombinant protein production using theLactococcus lactis P170 expression systemcombined with "Reverse electro enhanceddialysis" (REED) for lactate controlSøren M Madsen1, Astrid Vrang1, Lars H Pedersen1,Sean A MacDonald1, Jens-Ulrik Rype2 and Arvid Garde21Bioneer A/S, Kogle Alle 2, DK-2970 Hørsholm, Denmark2JURAG Separation A/S, Gydevang 4, DK-3450 Allerød,Denmark


Background: The increasing demand for production of newrecombinant pharmaceutical proteins constantly challenges ourefforts to improve existing production systems or to developnew systems. So far the most commonly used expressionsystems are based on the prokaryotic E. coli, which is easy togrow and easy to manipulate, or mammalian cells which are ableto produce complex heterologous proteins. However, these aswell as other expression systems have drawbacks and do notexpress all proteins efficiently, which emphasizes the need fordevelopment of alternative systems.Bioneer has developed an alternative recombinant proteinexpression system, the P170 Expression System [1], which isbased on the Gram positive lactic acid bacteria, Lactococcus lactis.This host organism has GRAS status due to its long term use inthe dairy industry, and proteins produced in the system havealready entered clinical trials. The P170 Expression System isinduced when a certain threshold of lactate is reached in theculture. This type of auto-induction eliminates the addition ofexogenous components to induce gene expression. Further-more, the protein of interest is fused to a secretion signalfacilitating secretion of the gene product into the culturesupernatant. The extracellular destination of the proteinproduct reduces the number of steps needed for the subsequentdownstream processing, thus helping to reduce the overall costof the process. Finally, we have composed a fermentationmedium devoid of animal derived components which is a strictdemand for production of pharmaceutical proteins.Despite all of these advantages, the production of lactic acid –the primary end product of glucose metabolism – will have alimiting effect on biomass production in L. lactis. Lactic acidinhibits growth even when the acid is neutralized by addition ofbase to keep pH constant. As a consequence the yield inbiomass production is below that of other expression systemswith cell densities of approximately 20 OD600 units. With thislimited cell density we have still been able to produce up to200–300 mg/L of secreted protein, but although this level is


Quantitative analysis of a human protein purification after cell lysis andpurification with 5 different buffers: The values were obtained using theALP5100 system. The buffers 1 to 5 are described in the table1.



acceptable for some high value proteins, in most cases higherproduction levels are desirable.JURAG has developed the "Reverse Electro Enhanced Dialysis(REED)" process which is a membrane filtration technology thatallows continuous removal of low-molecular weight negativeions like lactate from the fermentation broth [2]. The lactic acidproduced by the lactic acid bacteria dissociate in the growthmedium to form lactate, which is replaced by alkaline hydroxideions during the REED separation process. In this way both pHand lactate content in the culture are kept constant. By applyingthis technology the exponential growth phase could beprolonged resulting in a substantial increase in the final biomassyield. The effect of the REED unit was demonstrated by anincrease in the biomass yield by more than 10-fold whencompared to a standard fermentation.Results: Here we describe the synergistic effects of the REEDtechnology in combination with the P170 Expression System forincreased production and secretion of the model proteinStaphylococcus aureus nuclease (SNase). In the first phase ofthe fermentation the REED unit was used to control the lactateconcentration below 150 mM to sustain rapid exponentialgrowth until the optical density reached 70 OD600 units. Thelactate concentration was then increased to 250 mM and keptbetween 250 and 350 mM for 21 hours. Growth continued at adecreasing rate, while a high specific productivity of SNase was

obtained. The culture was continuously fed with concentratedmedium, while superfluous volume was drained from thefermentor to keep a constant volume. The Figure belowshows the yield of biomass and the yield of SNase obtainedduring the REED fermentation. As can be seen in Figure 1 thecell density reached 180 OD600 units while the concentration ofsecreted SNase exceeded 2 g/L, which is approximately 10 foldhigher compared to a standard fermentation without lactatecontrol.Conclusion: The present data demonstrates the enormouspotential for increasing the yields of recombinant proteinsproduced in L. lactis by combining the advantages of the P170Expression System for production of recombinant proteins withthe power of the REED separation technology for improvingyields. Two other commercially relevant proteins have also beentested in the combined system and 5–10 fold increasedproduction levels were demonstrated.AcknowledgementsWe would like to thank Anne Cathrine Steenbjerg andAnnemette Brix for excellent technical assistance.References1. Bredmose L, Madsen SM, Vrang A, Ravn P, Johnsen MG,

Glenting J, Arnau J and Israelsen H: Development of aHeterologous Gene Expression System for use inLactococcus lactis. Recombinant Protein Production with


Cell density and yield of secreted heterologous protein (SNase) during continuous fermentation with pH controlled by REED, compared to the batchfermentation where pH was controlled by titration with KOH solution.



Prokaryotic and Eukaryotic Cells A Comparative View on HostPhysiology, Kluwer Academic Press, Dordrecht; NL: MertenO-W 2001, 269–275.

2. Garde A Thesis Ph.D.: Production of lactic acidfromrenewable resources using electrodialysis forproduct recovery. 2002, ISBN 87-90142-84-5.

P81A simple emergency procedure to be used ifbiotechnological protein production isendangered by bacteriophage infection ofEscherichia coli cultures: effective inhibition ofbacteriophage lytic development in infectedcultures by removing a carbon source from themediumGrzegorz Wegrzyn1, Marcin Los1 and Peter Neubauer21Department of Molecular Biology, University of Gdansk,Kladki 24, 80-822 Gdansk, Poland2Biocenter Oulu and Department of Process andEnvironmental Engineering, University of Oulu, Finland


Background: Bacteriophage infections cause serious problemsin both research laboratories and large biotechnological compa-nies. Once infected by bacteriophages, bacterial cultures areusually completely destroyed, as phage lytic development in abacterium ends up with cell lysis and liberation of progeny phagesthat infect neighbor bacterial cells. A possibility of spreading ofbacteriophages throughout a laboratory is even more dangerousthan a loss of a single culture. Namely, subsequent cultures maybe infected, which can lead to cultivation problems lasting evenseveral months or longer. Therefore, a method for inhibition ofbacteriophage lytic development in infected cultures would beuseful. Perhaps it is not difficult in small cultures (e.g. flaskcultures), when simple sterilization of the whole material and aflask should be sufficient. However, phage contamination inbioreactors is a serious technical problem indeed.Escherichia coli is one of the most widely used bacterium in geneticengineering and biotechnology. This bacterium is, however, a hostfor many bacteriophages and thus, it is endangered by phageinfections. Bacteriophages have been considered as models ingenetic and biochemical studies for a long time. However, manyphysiological aspects of bacteriophages’ growth were not suffi-ciently investigated relative to extensive molecular biology studies.On the other hand, recent reports indicated that development ofbacteriophages largely depends on the physiology of the host cells.In laboratories, the physiological status of a cell depends, in turn,on cultivation conditions. Therefore, we aimed to find cultivationconditions that may result in inhibition of bacteriophage develop-ment and are not deleterious for bacterial cells. Previous studiesindicated that development of phages T4 and � is significantly lesseffective in slowly growing host cells than in rapidly growingbacteria. Thus, we aimed to test whether induction of starvation,caused be depletion of a carbon source from the culture medium,may inhibit phage development effectively. Growth of bacterio-phages in bacterial cells cultured on solid (agar) media supportingvarious growth rates and at different temperatures was alsoinvestigated.Results: We found that a decrease in temperature of aninfected bacterial culture might impair development of somebacteriophages (e.g. �) but not others (e.g. T4). Therefore,

usefulness of a method of inhibition of phage development ininfected bacterial cultures based on changes of temperaturewould be limited.The presence of various carbon sources in the same kind ofmedium results in different bacterial growth rates. Weinvestigated phage plaque formation on lawns of bacteriagrowing in media containing various carbon sources (glucose,glycerol, succinate or acetate) at the same temperature. Thechanges in plaque morphology of both phages were significant,though more pronounced in �, as this phage was not able toform plaques when host grew on the medium with acetate as acarbon source, i.e. at the lowest growth rate. Removing thecarbon source induces starvation conditions and minimalbacterial growth rate. Therefore, we asked whether removingthe carbon source can lead to inhibition of formation of progenyphages in infected bacterial cultures.We found that formation of phage progeny was completelyinhibited in infected cultures devoid of the carbon source. Thiswas true for all tested bacteriophages (�, P1 and T4). Additionof glucose to infected cultures of starved bacteria resulted inrestoration of phage progeny production, indicating thatdepletion of the carbon source was the sole reason forinhibition of development of phages �, P1 and T4.Conclusion: Development of bacteriophages �, P1 and T4 iscompletely inhibited after removing a carbon source frominfected E. coli cultures. Therefore, to minimize deleteriouseffects of phage contamination, especially in high-cell densityand/or fed-batch cultivations, it may be recommended to stopfeeding bacteria immediately after observation of first signs ofphage infection. Such a procedure should lead to starvation ofbacteria and inhibition of production of phage progeny.Although unambiguous detection of phage contamination atearly stages of infection may be difficult using traditionalmethods, a newly developed technology of electric DNA chipsallows for early detection of phages in bacterial cultures, even afew generations before they cause visible lysis of host cells.An interesting side aspect of the performed study is the reducedability of T4 and the inability of � to form plaques on mediumwith acetate as the only carbon source. Although it remainsunclear whether this effect is due to the very low growth rate ofE. coli under these conditions or to the specific effect of acetateon the �pH and consequently on the proton motive force, orosmotic pressure of the host cell, this effect is interesting inconnection to the proposed strategy to avoid phage propagationby stop of the feeding. Most E. coli fed-batch cultivations, kept atglucose limitation, would immediately stop phage growth, even ifacetate is still available.

P82A counter-selectable marker for BacillusMichael D Rasmussen1, Jan Martinussen2, Els MarieCeline Defoor2 and Gitte Bak Poulsen11Dept. of Bacterial Gene Technologyy, Novozymes, Bagsvaerd,Denmark2Dept of Molecular Physiology and Genetics, Biocentrum-DTU,Lyngby, Denmark


Background: A putative gene, denoted ysbC, was previouslyidentified by genome sequencing of a Lactococcus lactis strain, butthe gene was not annotated in the databases, and no function of



the predicted encoded polypeptide was identified. Studies showthat the ysbC encodes a membrane associated orotatetransporter enabling orotate to be taken up by the cell andused as a pyrimidine precursor. It was further shown thatorotate transporters are quite rare in bacteria and that mostbacillus species do not possess such a function.Results: In order to exploit the functional properties of theysbC gene it was investigated if an orotate analogue fluoro-orotate could be transported by YsbC. Fluoro-orotate is a toxicpyrimidine precursor which will be incorporated in the hostDNA and very efficiently stop further DNA synthesis anddivisions of the cell. Lactococcus strains with or without the ysbCgene was tested for resistance against fluoro-orotate on minimalplates. Only Lactococcus strains without the ysbC plasmid wasable to grow on minimal plates with fluoro-orotate. Theconclusion is that orotate (and fluoro-orotate) can only betransported over the cell membrane when the specific orotatetransporter encoded by ysbC is present.The gene was transferred to Bacillus subtilis to investigate if theorotate transporter would be functional in this background.Experiments in Bacillus subtilis using a plasmids with the ysbCgene show that transformants can not grow on minimal platessupplemented with Fluoro-orotate. Further experiments pre-sented on the poster show that the ysbC gene can be of use ingeneral bacillus recombinant technology as an efficient counterselectable marker.

P83The expression of truncated form of CP4 5-enolpyruvylshikimate-3-phosphate synthase (CP4EPSPS) from genetically modified plant inEscherichia coliStanislav Stuchlık, Satheesh Natarajan and Jan TurnaDepartment of Molecular Biology, Comenius University,Mlynska dolina B-2, 842 15 Bratislava, Slovakia


Background: During the study of horizontal gene transfer ofthe aroA CP4 gene encoding CP4 EPSPS from geneticallymodified feed through gastrointestinal tract to bacteria living inanimal gut we have observed beside full length gene alsofunctional truncated one present in bacteria [1]. The codonusage of both forms was originally optimized for plant [2].Therefore we have used two different E. coli expression systems(with tac promoter and T7 promoter) to compare enzymaticproperties and functional activities of both forms of CP4 EPSPSprotein.Results: For the protein expression we have prepared pKK1and pKK2 plasmids – tac expression vectors derived frompKK233-2 plasmid, and pMD2 and pMD72, T7 expressionvectors derived from pET28a plasmid. Gene aroA CP4 (fulllength as well as truncated one) from its ATG codon wasamplified by PCR from GM plant and cloned into NcoI andHindIII sites of both expression vectors (see Figure 1). We havecompared phenotypic properties of full length and truncatedCP4 EPSPS forms by growth on solid M9 medium. The resultsfrom this experiment are summarized in Table 1. We have alsoinvestigated growth curves of E. coli �aroA strains containingeither pKK1 or pKK2 plasmid on liquid M9 medium withoutaromatic compounds. Received results are shown on Figure 2.We have received suitable expression levels of both proteins

with T7 expression system for further partial protein purifica-tion based on Ni2+ His tag procedure and comparativeenzymatic assay.Conclusion: We can conclude from our results that truncatedform of CP4 EPSPS (shorter to 31 amino acid residues on N-terminal end) can confer full function of native protein and thesefindings also should be taken into account in risk assessment ofpossible HGT from GM food/feed. The enzymatic propertiesand comparison of both EPSPS forms will be subject of furtherstudies.AcknowledgementsThis work was supported by grant of Slovak Grant AgencyAPVT-20-17102.References1. Stuchlık S, Natarajan S and Turna J: Spontaneously

generated truncated forms of CP4 EPSPS protein.FEBS Journal 2005, 272:, Supplement 1. Abstracts 30th FEBSCongress and 9th IUBMB Conference, Budapest, Hungary2–7 July 2005.

2. Harrison LA, Bailey MR, Naylor MW, Ream JE,Hammond BG, Nida DL, Burnette BL, Nickson TE,Mitsky TA, Taylor ML, Fuchs RL and Padgette SR: Theexpressed protein in glyphosate-tolerant soybean,5-enolpyruvylshikimate-3-phosphate synthase fromAgrobacterium sp. strain CP4, is rapidly digested invitro and is not toxic to acutely gavaged mice. J Nutr1996, 126:728–740.

P84Recombinant lipase immobilised in the cell wall ofBacillus halodurans Alk 36 exploiting the FliCproteinMichael Crampton, Erika du Plessis, Santosh Ramchuran,Eldie Berger and Maureen LouwCouncil for Scientific and Industrial Research (CSIR),Biosciences, Pretoria, South Africa


Background: There are a number of methods in whichheterologous peptides and proteins can be displayed on thecell surface of bacteria. The first use of the FliC system fordisplay was carried out by Kuwajima et al. [1] where an elevenamino acid epitope from the egg-white lysozyme was displayedon the surface of E. coli. Ezaki et al. [2] and Tanskanen et al. [3]both demonstrated that large polypeptides could also bedisplayed successfully using E. coli flagellin. These proteinsincluded an alkaline phosphatase (471 aa) and the collagenbinding region of YadA of Yersinia enterolcolitica (302 aa)respectively. To our knowledge, the display of heterologousproteins using the FliC protein from flagella has not previouslybeen demonstrated in Gram-positive bacteria. Bacillus haloduransAlk36 is an alkalophilic, Gram-positive bacterial strain which hasthe inherent capability of over-expressing the FliC protein. Thiswas harnessed as an opportunity for the development of a novelsurface display system using this protein. The B. haloduransflagellin is a lot smaller (30 kDa) than that of E coli (~52 kDa)with a small variable region. A �hag mutant strain of B.halodurans (BhFC04) was developed and two sites wereidentified in the flagellin variable domain for insertion ofheterologous proteins/peptides.




Physical maps of plasmids pKK1, pKK2, pMD2, pMD72.

Table 1 (abstract P83) Complementation test of the CP4 aroAphenotypes.

Plasmids in E. colistrain 2% Glp 3% Glp 4% Glp MM

pKK1 in SV1TcR +++ ++ + +++pKK2 in SV1TcR +++ ++ + +++pKK233-2 in SV1TcR - - - -pKK233-2 in JM106 ND ND ND +++

pKK1 – pKK233-2 with full length cp4epsps, pKK2 – pKK233-2 withtruncated aroA CP4, Glp – glyphosate, + indicates the intensity of thegrowth, – indicates the absences of growth, MM – minimal M9 mediumwithout aromatic amino acid supplements, ND – not done. SV1TcRstrain carries aroA mutation.


Growth curves of E. coli SV1TcR with plasmids pKK1 and/or pKK2 in M9liquid medium. Both strains were grown in concentration of 0.5% glyphosate.



In order to investigate the possibility of using the FliC displaysystem for biotransformation in B. halodurans, constructpSECNLipC habouring the LipA gene from Geobacillusthermoleovorans was designed and evaluated in shake flasksand batch fermentations. Lipases have been well characterisedand play a role in a number of processes which includedetergents, glycerolysis of fats and oils, direct esterification,chiral resolution and acylate synthesis [4]. Bacterial and fungallipases have been immobilized on the cell surface of Bacillussubtilis using the CWBc cell-wall binding domain. However,instability of fusion proteins was found to be a serious problem[5].Results: Zymogram of lipase activity (Figure 1) in theextracellular (EX), cell surface (CS), cell wall (CW), andintracellular (I) fractions of BhFC04 harbouring the pSECNLipCconstruct.Conclusion: Heterologous lipase expression has been suc-cessfully achieved using the Bacillus halodurans host-vectorsystem. Lipase activity was localised primarily in the cell wallfraction. However, whole cell assays showed that the enzymewas exposed to the extracellular environment and thereforeaccessible to the substrate. Lipase production levels attained inshake flasks and batch fermentations exploiting this host-vectorsystem have demonstrated it to be of commercial potential forwhole cell biocatalysis.References1. Kuwajima G, Asaka JI, Fujiwara T, Fujiwara T, Nakano K and

Kondoh K: Presentation of an antigenic determinantfrom hen egg-white lysozyme on the flagellarfilament of Escherichia coli. Bio/Technology 1988,6:1080–1083.

2. Ezaki S, Tsukkio M, Takagi M and Imanaka T: Display ofheterologous gene products on the Eschericia colicell surface as fusion proteins with flagellin. J FermBioeng 1998, 5:500–503.

3. Tanskanen J, Korhonen TK and Westerlund-Wikstrom B:Construction of a multihybrid display system: flagellarfilaments carrying two foreign adhesive peptides. ApplEnviron Microbiol 2000, 66:4152–4156.

4. Litthauer D, Ginster A and van Eeden Skein E: Pseudomo-nas luteo la lipase: A new member of the 320-residuePseudomonas lipase family. Enz Microbiol Technol 2002,30:209–215.

5. Kobayashi G, Fujii K, Serizawa M, Yamamoto H andSekiguchi J: Simultaneous display of bacterial andfungal lipases on the cell surface of Bacillus subtilis.J Biosci Bioeng 2002, 93:15–19.

P85Development of an antibiotic-free plasmidselection system based on glycine auxotrophy forrecombinant protein overproduction inEscherichia coliLuis Vidal1, Josep Lopez-Santın1, Gloria Caminal2

and Pau Ferrer11Grup d’Enginyeria de Bioprocessos i Biocatalisis Aplicada.Departament d’Enginyeria Quımica. Escola Tecnica Superiord’Enginyeria, Universitat Autonoma de Barcelona,08193-Bellaterra, Spain2Unitat de Biocatalisi Aplicada Associada al IIQAB (UAB-CSIC)


Background: Antibiotics and antibiotics resistance genes havebeen traditionally used for the selection and maintenance ofrecombinant plasmids in hosts such as E. coli. Although a powerfulselection tool, their use has been considered unacceptable inmany areas of biotechnology by regulatory authorities. Indeed,there is much international scientific and regulatory focus on thisissue [1]. For instance, the use of selection markers that conferresistance to antibiotics in vaccine plasmids may introduce therisk of transforming the patient’s microflora and spreadresistance genes. Moreover, in recombinant protein productionfor therapeutic use, the antibiotic must be eliminated from thefinal product. Another problem arises from the potential loss ofselective pressure as a result of antibiotic degradation i.e.ampicillin can be degraded by �-lactamases in less than 30minutes in high cell density cultures [2].In this work, an alternative approach to prevent plasmid lossbased on an amino acid auxotrophy complementation has beendeveloped. A glycine-auxotrophic strain of E. coli M15 has beencreated using PCR products [3]. This strain contains an internaldeletion of the glyA gene, which encodes for serine hydro-xymethyl transferase (SHMT), an enzyme involved in the mainglycine biosynthesis pathway in E. coli [4]. As a result, the glyA-strain can not synthesize glycine and, therefore, needs an externalsource of glycine or a genetic source of SHMT for growing on adefined medium. The construction of a plasmid selection systemderived from the commercial vector pQE40 (QIAGEN) with glyAunder the control of the constitutive weak promoter P3 [5], aswell as its evaluation in batch and fed-batch cultures forexpressing ramnulose 1-phophate aldolase (RhuA) as a modelfor recombinant protein production has been evaluated.Results: Disruption of the glyA gene in E. coli M15 wasaccomplished using a directed knockout gene method employingPCR products through homologous recombination [3]. Thephenotype of the new strain M5�glyA was tested on defined


Lane 1-EX fraction.Lane 2 -CS fractionLane 3- CW fractionLane 4- IfractionLane 5 -Molecular weight marker.



medium cultures with and without glycine, confirming theauxotrophy for glycine.By using SOE-PCR [6], the glyA gene (referred here and after as �fragment) from E. coli K-12 was fused to the weak constitutivepromoter P3 (referred here and after as a fragment). The fusion a�productwas cloned intopQErham,a pQE40-derived vector forRhuAoverexpression [7]. The resulting complementation vector, pQEa�r-ham (Figure 1), was used to transform the E. coliM15�glyA host.Erlenmeyers cultures with glycine-auxotrophic strain M15�glyAtransformed with the complementation expression plasmid cangrow in defined medium adding neither glycine nor ampicillin.This indicated that the plasmid is maintained and SHMT is beingexpressed, thereby generating an endogenous source of glycine.SDS-PAGE of soluble cytosolic proteins under non-inducingconditions indicated that SMHT was not being overexpressed.The capacity of the new complementation system for producingthe recombinant RhuA was compared to that of the conven-tional selection system based on antibiotic resistance (E. coli

M15 pQErham). Induction with IPTG resulted in high over-expression of recombinant RhuA levels in E. coli M15�glyA/pQEa�rham. However, such levels were slightly lower thanthose obtained in the conventional system. SDS-PAGE analysesof cell extracts revealed significantly higher expression levels(compared with non-inducing conditions) of a protein of about45 kDa, corresponding to SHMT. This suggested that thedownstream orientation of a� fragment in relation to the strongT5 promoter could cause a read-through of RhuA gene in thepresence of IPTG, i.e. resulting in higher SHMT expressionlevels. This phenomenon could exert a metabolic burden on thehost cell, resulting in lower specific RhuA activity levels.High cell density fed-batch cultures using the novel comple-mentation system were performed using an exponential feedingprofile strategy analogous to that previously developed for theconventional system [7]. In particular, a fed-batch cultivationwith a controlled specific growth rate of 0.2 h-1 (final DO600 nm

= 185) was performed without ampicillin. Induction with IPTG


Diagram of the complementation vector pQErham.



allowed obtaining high RhuA production (28483 U l-1) andproductivity (1636 U l-1 h-1) levels.Conclusion: The new selection maker based on a glycine-auxotrophy strain plus a plasmid harboring the glyA gene underthe P3 weak promoter is a promising tool, not only for recombinantprotein production, but also for vaccine plasmids productionprocesses where antibiotics can not be present in the mediumformulation. Besides, the use of a chemically defined medium avoidsthe risk of employing components of animal origin that may containviruses or prions [1], increasing the safety level of the system forindustrial processes.AcknowledgementsAuthors wish to acknowledge Dr. Wanner for providing theknockout system. This work was support from MEC CTQ2005-01706. PPQ. The Department of Chemical Engineering is theUnit of Biochemical Engineering of the Centre de Referencia enBiotecnologia de la Generalitat de Catalunya (CeRBa).References1. Glenting J and Wessels S: Ensuring safety of DNA

vaccines. Microbial Cell Factories 2005, 4:26.2. Jung G, Denefle P, Becquart J and Mayaux JF: High-cell

density fermentation studies of recombinant Escher-ichia coli strains expressing human interleukin-1-beta. Ann Inst Pasteur-Microbiol 1988, 139:129–146.

3. Datsenko KA and Wanner BL: One-step inactivation ofchromosomal genes in Escherichia coli K-12 usingPCR products. Proc Natl Acad Sci USA 2000, 97:6640–6645.

4. Stauffer LT, Plamann MD and Stauffer GV: Cloning andcharacterization of the glycine-cleavage enzyme-system of Escherichia coli. Gene 1986, 44:219–226.

5. Lartigue MF, Leflon-Guibout W, Poirel L, Nordmann P andNicolas-Chanoine MH: Promoters P3, Pa/Pb, P4, andP5 upstream from bla(TEM) genes and their rela-tionship to beta-lactam resistance. Antimicrob AgentsChemother 2002, 46:4035–4037.

6. Sambrook J, Maniatis T and Fritsch EF: Molecular cloning:A Laboratory manual. Cold Spring Harbor, NY 1989.

7. Vidal L, Ferrer P, Alvaro G, Benaiges MD and Caminal G:Influence of induction and operation mode onrecombinant rhamnulose 1-phosphate aldolase pro-duction by Escherichia coli using the T5 promoter.J Biotechnol 2005, 118:75–87.

P86Development of extracellular production systemof recombinant proteins in recombinantEscherichia coliJong Hyun Choi1, Zhi Gang Qian1 and Sang Yup Lee1,21Metabolic and Biomolecular Engineering National ResearchLaboratory, Department of Chemical & BiomolecularEngineering, Bioinformatics Research Center and BioProcessEngineering Research Center2Department of BioSystems, Korea Advanced Institute ofScience and Technology, 373-1 Guseong-dong, Yuseong-gu,Daejeon 305-701, Republic of Korea


Background: Escherichia coli has been the workhorse for theproduction of various recombinant proteins and metabolitesbecause of the availability of well established technologies forgenetic manipulation and cultivation. Various strategies have been

employed for the development of E. coli strains which are able toefficiently produce recombinant proteins [1, 2]. Extracellularproduction of recombinant proteins has advantages over secretioninto the periplasm [2]. Extracellular production does not requireouter membrane disruption to recover target proteins, andtherefore, it avoids intracellular proteolysis by periplasmic proteasesand allows continuous production of recombinant proteins, and newapproaches for the extracellular production of recombinantproteins in E coli are discussed.Results: E. coli BL21 strains showed the high accumulation ofOmpF protein in culture medium during high cell densitycultivation (see Figure 1). From this interesting phenomenon, anew and efficient method for the extracellular production ofrecombinant protein in E coli was developed. Using this newdeveloped extracellular production system, various recombinantproteins could be efficiently produced into culture medium.Conclusion: We developed new approaches for the extra-cellular production of recombinant proteins in E. coli by usingone of the outer membrane proteins as a partner.AcknowledgementsThis work was supported by the Korean Systems BiologyResearch Grant (M10309020000-03B5002-00000) from theMinistry of Science and Technology, Regional Innovation System(RIS) from the Korean Ministry of Commerce, Industry, andEnergy, the Brain Korea 21 program of the Ministry of Educationand LG Chem. Chair ProfessorshipReferences1. Choi JH, Keum KC and Lee SY: Production of recombi-

nant proteins by high cell density culture ofEscherichia coli. Chem Eng Sci 2006, 66:876–885.

2. Choi JH and Lee SY: Secretory and extracellularproduction of recombinant proteins using Escher-ichia coli. Appl Microbiol Biotechnol 2004, 64:625–635.

P87ColE1 derived RNA I as a key molecule in a novelantibiotic free plasmid addiction systemIrene Pfaffenzeller, Gerald Striedner, Karl Bayerand Reingard GrabherrDepartment of Biotechnology, University of Natural Resourcesand Applied Life Sciences, Muthgasse 18, 1190 Vienna,Austria


SDS-PAGE analysis of culture supernatant from the high cell densityculture of E. coli BL21(DE3).




Background: Today there are various plasmid selectionsystems available, whereas the most common approachcomprises the use of antibiotic resistance genes and the additionof the corresponding drug into the medium. Bacterial vectorscarrying a ColE1-type origin, e.g. pBR322, the pET-series [1] orthe pUC-vectors are the most commonly used plasmids forrecombinant protein production. For the application of plasmidDNA in gene therapy or DNA vaccination antibiotic resistancegenes on the plasmid backbone are highly unwanted. To avoidthese resistance genes and other additional sequences that canbe used for plasmid maintenance, we intend to exploit theplasmid replication machinery in a novel plasmid addictionsystem. The mechanism is based on RNA/RNA antisenseinteraction involving the naturally occurring RNA I derivedfrom the plasmid’s origin of replication (see Figure 1). Theplasmid replicational regulatory network consisting of RNA I/RNA II and their impact on Plasmid Copy Number (PCN) waslinked to the transcription of an regulatory target gene, presenton the bacterial chromosome.Consequently, the strategy presented in this work can beapplied for conventional protein expression vectors as well asfor therapeutical plasmids.Results: In our experiments we found that RNA I was capableof silencing engineered target genes by RNA antisense reactioncompletely. On the mRNA transcript of the reporter gene gfp

(green fluorescent protein) RNA II-like sequences were addednear the ribosomal binding site. Thus, suppression of achromosomally inserted gfp gene could be achieved by merepresence of the ColE1-type plasmid pBR322 in shaker flaskexperiments and fed-batch fermentation processes.

Conditionally lethal bacterial hosts were created by inserting aninducible promoter on the genome in front of an essential gene, e.g. murA [2] by homologous recombination [3]. By integration ofan IPTG inducible expression cassette on the bacterial genomecontaining the tet-repressor gene (tetR), we were able to inhibitgrowth upon addition of IPTG, when the essential gene was setunder control of the corresponding promoter, pLtetO [4].

For the design of engineered hosts depending on a ColE1-typeplasmid in the cell the tetR transcript was combined with RNA Icomplementary sequences. Thus, the plasmid’s replicationmachinery provides an essential advantage to the host cells.

Conclusion: Basically, the discrete reactions of the plasmidaddiction system were proven to be functional. We demon-strated that the regulatory mechanism of ColE1 plasmidreplication is a useful tool for gene silencing of a designedtarget gene. Moreover, the essential gene murA was shown to bean efficient target for the selection system.

The strategy of regulating gene expression by plasmid replica-tion implicates a novel strategy for plasmid selection inrecombinant protein production processes and for genetherapeutic applications.


Concept of RNA I mediated plasmid addiction system: The ColE1-type plasmid indicated in Figure 1 can be a common protein expression vector or atherapeutical plasmid.



AcknowledgementsThis work was funded by Boehringer Ingelheim Austria.References1. Studier W and Moffatt BA: Use of the bacteriophage T7

RNA polymerase to direct selective high levelexpression of cloned genes. J Mol Biol 1986, 189:113–130.

2. Herring D and Blattner FR: Conditional lethal ambermutations in essential Escherichia coli genes. J Bacteriol2004, 186(9):2673–2681.

3. Datsenko A and Wanner BL: One-step inactivation ofchromosomal genes in Escherichia coli K-12 usingPCR products. PNAS 2000, 97(12):6640–6645.

4. Lutz R and Bujard H: Independent and tight regulationof transcriptional units in Escherichia coli via theLacR/O, the TetR/O and AraC/I1-I2 regulatorysystems. Nucleic Acids Res 1997, 25(6):1203–1210.

P88Expression of trehalose-6-phosphate synthasegene from Arabidopsis thaliana in transgenictobacco: A strategy to increase temperaturestress toleranceAndre de Almeida1, Enrique Villalobos2, Susana Araujo1,Luıs A Cardoso3, Dulce Santos1, Jose M Torne2

and Pedro S Fevereiro1,41Laboratorio Biotecnologia Celulas Vegetais, ITQB-UNL,Oeiras, Portugal2Institut de Biologia Molecular de Barcelona, Barcelona, Spain3Instituto de Investigacao Cientıfica Tropical, Lisboa, Portugal4Departamento de Biologia Vegetal, Faculdade de Ciencias,Universidade de Lisboa, Portugal


Background: Genetic engineering of plants towards osmo-protectant accumulation is gaining increased importance withinthe broad context of abiotic stress tolerance [1]. An enzyme,trehalose-6-phosphate synthase, is believed to play a key role inthe synthesis of the disaccharide trehalose and hence on theimprovement of abiotic stress tolerance [2]. We usedAgrobacterium to transform tobacco plants to express thetrehalose-6-phosphate synthase gene from Arabidopsis thaliana,under the control of CaMV 35S promoter and using the vectorpGreen 0229 [3]. Transgenic T2 plants were evaluated for geneexpression by northern and western blots. Seeds were sown inmedia germinated at: 15, 25 and 35˚C for evaluating germinationrates under high and low temperatures.Results: Three of the transgenic lines obtained (B5A, B5H andB1F) have distinct levels of gene expression: B5H and B5A arehigh expressing lines while B1F is a low expressing one. In non-transgenic controls no expression was detected (Figure 1).Transgenic lines were shown to have significantly highergermination rates under low and high temperatures (respec-tively, 15 and 35˚C) than wild type plants (Table 1).Conclusion: Our results demonstrate that transgenic plantsaccumulating trehalose-6-phosphate synthase have an alteredphenotype that includes temperature stress tolerance upongermination. We suggest that AtTPS1 can be used to engineerimportant crop plants such as maize, wheat or rice to withstanddifferent environmental stresses.

AcknowledgementsTo Fundacao para a Ciencia e a Tecnologia for funding this research.References1. Nuccio ML, Rhodes D, Mcneil SD and Hanson AD:

Metabolic engineering of plants for osmotic stressresistance. Curr Opin Plant Biol 1999, 2:128–134.

2. Romero C, Belles JM, Vaya JL, Serrano R and Culianez-Macia FA: Expression of the yeast trehalose-6-phos-phate synthase gene in transgenic tobacco plants:pleiotropic phenotypes include drought tolerance.Planta 1997, 201:293–297.

3. Almeida AM, Villalobos E, Araujo SS, Leyman B, van Dijck P,Cardoso LA, Fevereiro PS, Torne JM and Santos DM:Transformation of tobacco with an Arabidopsisthaliana gene involved in trehalose biosynthesisincreases tolerance to several abiotic stresses.Euphytica 2005, 146:165–176.

P89Expression of functional recombinant rabies virusglycoprotein in Drosophila melanogaster S2 cellsAdriana Y Yokomizo1, Soraia AC Jorge1,Renato M Astray1, Mariza AG Santos1, Irene Fernandes2,Orlando G Ribeiro3, Denise SPQ Horton4, Aldo Tonso5

and Carlos A Pereira11Laboratorio de Imunologia Viral, Instituto Butantan, 05503-900Sao Paulo, Brasil2Laboratorio de Imunopatologia, Instituto Butantan, 05503-900Sao Paulo, Brasil3Laboratorio de Imunogenetica, Instituto Butantan, 05503-900Sao Paulo, Brasil4Servico de Controle de Qualidade, Instituto Butantan, 05503-900Sao Paulo, Brasil5Departamento de Engenharia Quımica, Escola Politecnica,Universidade de Sao Paulo, Brasil


Background: The rabies virus belongs to the genus Lyssavirusfrom the Rhabdoviridae family and is widely distributed in natureinfecting mammals. Upon infection it can be transmitted toanimals or humans and leads to a fatal disease that nowadays hasno treatment. Vaccines are commercially available and preventthe disease in animals and humans. Protocols for human orveterinarian vaccine manufacturing evolved from animal tissuehomogenates to cell culture technology and today recombinantviral proteins and DNA vaccines are under investigation. Theevidence that rabies virus infects and can cause disease inanimals and humans, being neutralized by an immune responsemounted by very similar vaccines opens a great possibility oftesting new vaccines first in experimental animals prior to use inhumans [1].Results: Recombinant rabies vırus glycoprotein (rGPV) wasexpressed in Drosophila melanogaster Schneider 2 (S2) cells. ThecDNA encoding the GPV gene was cloned in expression plasmidsunder the control of the inducible metallothionein promoter(Mt) or the constitutive actin promoter (Ac). These werealternatively co-transfected into S2 cells together with ahygromycin selection plasmid. Selected S2 cell populations(S2MtGPV or S2AcGPV) had a decreased ability to grow andconsume substrates, when compared to the non transfectedcells (S2). They were shown, by PCR, to express the GPV gene



and mRNA as well as, by immunoblotting, to synthesize therGPV in its expected molecular weight of 65 kDa. ELISA kineticstudies showed the rGPV expression in cell lysates andsupernatants attaining concentrations ranging from 150 to 300�g of rGPV/L. By flow cytometry analysis, about 30% of the cellsin these populations were shown to express the rGPV in theirmembrane. Cell populations selected by limiting dilutionexpressed higher rGPV yields. Mice immunized with S2MtGPVor S2AcGPV derived rGPV were capable of mounting aprotective immune response characterized by the synthesis ofantibodies reacting against the rabies virus. Immunization led toprotection against rabies virus experimental infection inchallenge studies.

Conclusion: The data presented here show that S2 cells aresuitable hosts for the rGPV expression allowing its synthesis in ahigh degree of physical and biological integrity.AcknowledgementsThis work was supported in part by grants and scholarshipsfrom the FAPESP, CNPq, CAPES and Fundacao Butantan. C. A.Pereira is recipient of CNPq research fellowships. We thank M.J.S. Leme for technical assistance performing "in vivo" challengeassays.Reference1. Warrel MJ and Warrel DA: Rabies and other lyssavirus

diseases. Lancet 2004, 363:959–969.

P90Effects of overexpression of X-box binding protein1 on recombinant protein production inmammalian cellsSebastian Ku1,3, Grace Chong1, Maybelline Giam1,Miranda GS Yap1,2,3 and Sheng-Hao Chao11Bioprocessing Technology Institute, Biomedical SciencesInstitutes, 20 Biopolis Way, #06-01, Singapore 1386682Department of Chemical & Biomolecular Engineering,National University of Singapore, 10 Kent Ridge Crescent,Singapore 119220


AtTPS1 gene expression analysis in WT and transgenic tobacco lines. A – Northern blot B – Western blot. In both cases, no AtTPS1 proteinproduction was detected in control wild type plants while transgenic lines showed accumulation of AtTPS1 transcripts and enzyme. WT – Wild Type;B5A, B5H and B1F – Transgenic line. MW – Molecular weight markers (kDa).

Table 1 (abstract P88) Germination rates (Number of seedsgerminated per 100 seeds placed on germination medium) ofthree transgenic lines at three different temperatures.

Temperature WT B5A B5H B1F

24˚C 99.0a(1.0) 99.0a(1.50) 99.0a(2.00) 100.0a(0.00)15˚C 32.3a(4.1) 95.0b(3.97) 97.4b(2.95) 97.0b(2.80)35˚C 18.0a(3.8) 88.0b(7.9) 85.2b(6.35) 83.2b(5.9)

a, bPercentages with different superscripts indicate statistical significance(p < 0.05); Standard deviations are shown between parenthesis; WT –Wild Type plants; B5A, B5H and B1F – Transgenic lines



3NUS Graduate School for Integrative Sciences andEngineering, National University of Singapore, 10 Kent RidgeCrescent, Singapore 119220


Background: X-box binding protein 1 (XBP-1), a key regulator forthe cellular secretory pathway, is essential for the differentiation ofplasma cells and the unfolded protein response. In the XBP-1 knock-out B primary cells, a profound depression in synthesis and secretionof immunoglobulin M was observed, clearly demonstrating theimportance of XBP-1 in protein secretion. There are two proteinisoforms of XBP-1, XBP-1S and XBP-1U. The spliced form of XBP-1,XBP-1S, functions as a transcription activator and upregulates manygenes associated with protein secretion and biosynthesis ofendoplasmic reticula (ER), whereas the unspliced XBP-1U istranscriptionally inactive. Since the production of some recombinantproteins is widely believed to be limited by the secretory capacity ofthe host cell, we reason that an increase in protein productivity maybe achieved by overexpressing XBP-1S in cells. However, XBP-1S isonly synthesized when UPR is initiated. To constitutively expressXBP-1S in cells, but not XBP-1U, we generated a specific expressionplasmid which contains the spliced XBP-1S cDNA. Effects ofoverexpression of XBP-1S on the productivity of human erythro-poietin (hEPO) in CHO-K1 cells were examined.Results: We hypothesized that protein secretion may become adeterminative factor when the production of recombinant proteinsexceeds the secretory capacity of host cells. To simulate thesaturated condition, CHO-K1 cells were transiently transfectedwith a hEPO expression vector. 2- to 3-fold increase in hEPO titrewas observed when XBP-1S was ectopically expressed in the hEPO-saturated cells. Our findings suggest that the putative saturation ofsecretory capacity can be alleviated and protein production can befurther enhanced by overexpression of XBP-1S.Conclusion: XBP-1S could be an ideal gene target to improveproductivity of recombinant proteins by modulating cellularsecretory pathways.

P91Analysis and characterization of differentpreparations of recombinant human folliclestimulating hormone (hFSH) and of its subunitsMaria Teresa CP Ribela, Renan F Loureiro,Joao E Oliveira, Cristiane M Carvalho, Cibele N Peroniand Paolo BartoliniBiotechnology Department, IPEN-CNEN, 05508-900,Sao Paulo, Brazil


Background: Human follicle stimulating hormone (hFSH),synthesized by the human pituitary gland, is a biologically activeglycoprotein composed of two noncovalently bound a- and�-subunits and is critically involved in the maturation of ovarianfollicles and in spermatogenesis. Considerable heterogeneityassociated with different hFSH preparations has been reported,mainly related to the presence of different glycoforms [1]. Thecharacterization of preparations of hFSH utilized as a therapeu-tic agent in reproductive medicine is therefore very important,especially considering that no specific monography has yet beenpublished by the main pharmacopoeias.In this work four hFSH preparations were analyzed, two of thembeing natural (pituitary- and urinary-derived) and the other two

recombinant (CHO-derived). Studies were conducted to assessand compare hydrophobicity, molecular weight, charge hetero-geneity and purity of the natural and recombinant heterodimericpreparations. These characteristics were examined by reversed-phase high performance liquid chromatography (RP-HPLC),matrix-assisted laser desorption ionization time of flight massspectrometry (MALDI-TOF), isoelectric focusing and size-exclusion high performance liquid chromatography (HPSEC).Results: RP-HPLC analysis indicated a significant difference(p < 0.005) between the retention time (tR) of the pituitary and ofthe two recombinant FSH preparations. Urinary-derived hFSHwas found more heterogeneous than the other three prepara-tions. HPSEC analysis showed a significant difference (p < 0.001)between the tR of the urinary preparation and that of the pituitaryor of the recombinant preparations. Urinary-derived hFSHpresented the lowest HPSEC tR, in agreement with the highestmolecular mass more accurately determined by MALDI-TOFmass spectrometry. The relative molecular mass (Mr) for theheterodimeric form of urinary, pituitary and recombinant hFSHpreparations was 32527, 29176 and 28536 respectively.An efficient subunit dissociation process (dissociation yield of95%) was also set up by incubating pituitary- and CHO-derivedFSH preparations with 3 M acetic acid, overnight, at 37˚C. Yieldsof approximately 52% and 48% for the � and a subunitrespectively were obtained via RP-HPLC, in agreement withtheoretical yields based on the mass determined in this work viaMALDI-TOF mass spectrometry (53% and 47%). The Mr of theindividual subunits determined by this methodology forpituitary- and CHO-derived hFSH were respectively 14467and 14082 for the a subunit and 16509 and 16067 for the �subunit. The urinary preparation presented a Mr of 15139 forthe a subunit and of 17196 for the � subunit (see Table 1). Allsubunits, when analyzed on RP-HPLC, presented retentiontimes significantly different from the retention time of theheterodimer (p < 0.01) and between them (p < 0.001). Themean relative retention times (tRR = tR subunit/tR heterodimer),though, were found highly constant, 1.100 ± 0.004 (CV = 0.4%)and 1.517 ± 0.023 (CV = 1.5%), respectively for the �- anda-subunit of the three preparations (see Table 2)Conclusion: Different isoforms were observed, by RP-HPLC, inthe analysis of hFSH preparations of different origins (CHO, urinaryand pituitary-derived). While the recombinant and pituitary hFSHpreparations presented one main peak, the urinary-derived hFSHpresented two major isoforms, one of which was equivalent to themajor form of the other preparations. The other form could be anoxidized form of FSH present in this urinary preparation in highamount, as reported [2]. The RP-HPLC characterization of the hFSHheterodimer and of individual subunits revealed differences inhydrophobicity in the following order: a-subunit > �-subunit >

Table 1 (abstract P91) Relative molecular mass (Mr) of theheterodimer (+) and related subunits of different hFSHpreparations, determined by Maldi-Tof mass spectrometry

Heterodimer

Preparation -subunit -subunit Experimental Calculated + Calc/Exp

p-hFSH 14467 16509 29176 30976 1.06r-hFSH 14082 16067 28536 30149 1.06u-hFSH 15139 17196 32527 32335 0.99



heterodimer. For the first time a quite satisfactory separation of theheterodimer from the dissociated �-subunit was attained.Urinary-derived hFSH showed a higher Mr (11–14%) whencompared with pituitary and recombinant hFSH, while pituitaryhFSH showed a slightly higher Mr (~ 2%) in comparison with therecombinant preparation.AcknowledgementsSupported by FAPESP and CNPq.References1. Loumaye E, Dreano M, Galazka A, Howles C, Ham L,

Munafo A, Eshkol A, Giudice E, De Luca E, Sirna A,Antonetti F, Giartosio CE, Scaglia L, Kelton C, Campbell R,Chappel S, Duthu B, Cymbalista S and Lepage P: Recombi-nant follicle stimulating hormone: development ofthe first biotechnology product for the treatment ofinfertility. Hum Reprod Update 1998, 4:862–881.

2. Bergh C, Howles CM, Borg K, Hamberger L, Josefsson B,Nilsson L and Wikland M: Recombinant human folliclestimulating hormone (r-hFSH; Gonal-F) versushighly purified urinary FSH (Metrodin HP): resultsof a randomized comparative study in womenundergoing assisted reproductive techniques. HumReprod 1997, 12:2133–2139.

P92Characterization of Medicago truncatula cellsuspension cultures producing valuablerecombinant proteinsGuadalupe Cabral1, Ana Sofia Pires2,Pablo Gonzalez-Melendi3 and Rita Abranches11Plant Cell Biology Laboratory, Instituto de Tecnologia Quimicae Biologica – UNL. Av. Republica, Apartado 127, 2781-901Oeiras, Portugal2Plant Cell Biotechnology Laboratory, Instituto de TecnologiaQuimica e Biologica – UNL. Av. Republica, Apartado 127,2781-901 Oeiras, Portugal3Department of Plant Biology, Centro de InvestigacionesBiologicas, CSIC. Ramiro de Maeztu 9, 28040 Madrid, Spain


Background: Nowadays, the use of plants for large-scaleproduction of recombinant proteins is gaining wider acceptancebecause of their many practical, economic and safety advantages,compared with traditional microbial and animal production systems.However, production systems that use whole plants to expressrecombinant proteins may lack several of the intrinsic benefits ofcultured cells, such as the precise control over growthconditions. Plant cell cultures may combine the merits of plantsystems with those of microbial and animal cell cultures [1].Optimization of environmental conditions in culture could be

used to enhance foreign protein synthesis and stability, reducingthe total cost of protein production. Moreover, a greatadvantage of synthesizing recombinant proteins in plant cellcultures is the very simple procedure of product purification,especially when the product is secreted into the liquid culturemedium. As well as the potential commercial benefits, in vitrosystems also represent an important tool for studying theprocess of foreign protein synthesis, assembly, secretion andturnover in plant cells and tissue.Results: Recently, we have proposed the legume model plantMedicago tuncatula as a promising production system [2]. In thiswork, specifically, several suspension cell lines were establishedfrom transformed M. truncatula plants expressing two valuablerecombinant proteins from different sources, human Erythropoie-tin (EPO) and fungal phytase. For both proteins two versions wereavailable, one where the protein is secreted and the other where itis retained in the Endoplasmic Reticulum (ER).Callus induction was achieved through incisions using a sharprazor blade (perpendicular to the mid-vein of the folioles) andthe abaxial side of the folioles was maintained in contact withthe medium with appropriate growth regulators. Calli were keptat 23˚C in the dark on solid media for approximately twomonths. When calli reached the appropriate size, cells weretransferred to Erlenmeyer flasks with shosen medium, kept withagitation in the dark at 24˚C, and subcultured to fresh mediumevery week (A.S. Pires, unpublished results).Identification of recombinant EPO or phytase was performed byWestern blot analysis, showing that different cell suspension linesexhibit different expression levels of recombinant protein. Inter-estingly, we observed that in some cell lines with higher expressionlevels of the secreted version of recombinant protein, part of it isnot being secreted to themedium.On the other hand, in suspensioncells generated from transgenic plants engineered to producerecombinant protein targeted to the ER, the proteins are not beingtotally retained. These results are probably related with stressimposed to the cell by high expression levels of a foreign protein.Preliminary immunolocalization analysis, using electron microscopy,revealed that in those cell lines with different expression levels, thesubcellular localization of the recombinant protein is significantlydifferent as well as the subcellular structure itself. As we can see inFigure 1, in phytase higher expressor cells, anti-phytase signal islocated on unidentified structures under the cell wall but in thelower expressor cells the labelling is concentrated in apparently lyticvacuoles.We hope these studies will help us to answer the followingquestion: is it the cell morphology that determines the subcellularfate of the product or the opposite?In order to compare the obtained results with other plant basedsystems, leaves from transgenic M. truncatula original plants andtransgenic BY2 Tobacco Suspension Culture expressing thesame recombinant proteins were also analysed.

Table 2 (abstract P91) Retention times of heterodimeric hFSH before dissociation, of -and -subunits after dissociation and relativeretention times (tRR) of the and subunits with basis on heterodimeric hFSH, determined on RP-HPLC (n = 2).

SAMPLE heterodimer tR -subunit tR -subunit tR -subunit tRRa -subunit tRR

a

p-hFSH 24.43 ± 0.156 26.98 ± 0.160 36.63 ± 0.198 1.104 1.499r-hFSH Gonal F 25.19 ± 0.129 27.62 ± 0.235 38.86 ± 0.214 1.096 1.543r-hFSH Puregon 25.29 ± 0.070 27.85 ± 0.131 38.16 ± 0.127 1.101 1.509

atRR, (relative retention time) = tRsubunit/tRheterodimer



Conclusion: Similarly to M. truncatula leaves [2], resultsobtained for cell suspension cultures established from thisplant also indicate that this alternative could be highly suited forthe production of recombinant proteins, with all the advantageswidely recognized for plant cell cultures. Further studies,including the systematic analysis of pos-translational modifica-tions of the recombinant proteins produced by this system, arestill required to improve its potential to compete with otherexpression platforms for production of valuable recombinantproteins.AcknowledgementsFundacao para a Ciencia e Tecnologia (Pos-doctoral fellowshipSFRH/BPD/21619/2005 and Project POCI/BIA-BCM/55762/2004)Conselho de Reitores das Universidades Portuguesas (CRUP,Portugal) for travel fundingReferences1. Hellwig S, Drossard J, Twyman RM and Fischer R: Plant

cell cultures for the production of recombinantproteins. Nat Biotechnol 2004, 22:1415–1422.

2. Abranches R, Marcel S, Arcalis E, Altmann F, Fevereiro Pand Stoger E: Plants as bioreactors: A comparativestudy suggests that Medicago truncatula is a promis-ing production system. J Biotechnol 2005, 120:121–134.

P93A novel in vitro translation system basedon insect cellsStefan Kubick1, Helmut Merk1, Michael Gerrits1,Jan Strey1, Uritza von Groll2, Frank Schafer2

and Wolfgang Stiege11RiNA GmbH, 14195 Berlin, Germany2QIAGEN GmbH, 40724 Hilden, Germany


Background: Various genome sequencing projects are identi-fying many new protein sequences but it is key to attributefunctions to these proteins. A huge number of proteins havebeen expressed in vivo to date, most of them being functionally,antigenically and immunogenically similar to their authenticcounterparts. This is mainly due to the properties of culturedeukaryotic cells, which are able to carry out many types of

posttranslational modifications such as addition of N- andO-linked oligosaccharides, but also palmitoylation, myristylationand phosphorylation.

Results: Based on the versatile properties of cultured celllines, e.g. insect cells, we have developed a novel eukaryotic invitro translation system [1]. Our homogenization proceduremaintains the functional integrity of subcellular components,thus allowing the cell-free synthesis of membrane proteins andposttranslationally modified proteins, e.g. glycoproteins. Anindispensable prerequisite for the reliable high-throughputexpression of different proteins in cell-free systems is thegeneration of efficient templates. Therefore, we have developeda PCR-based methodology which allows the user to introduceregulatory elements as well as tags for protein purification intothe desired gene. Additionally, we have shown that recombinantviral mRNA is a suitable template in this system and we havealso demonstrated activity of the Rhopalosiphum padi virus(RhPV) 5’-UTR IRES in our lysates [2].

Conclusion: The standardized large-scale production oflysates from various cell-lines is a powerful tool for thedevelopment of novel eukaryotic in vitro translation systemswith individual cell type- and tissue-specific properties.

AcknowledgementsThis work was kindly supported by the German Ministry ofEducation and Science (BMBF) and the Senate of Berlin.

References1. Kubick S, Schacherl J, Fleischer-Notter H, Royall E,

Roberts LO and Stiege W: In vitro translation in aninsect-based cell-free system. Cell-free protein expressionBerlin, Heidelberg, New York: Springer: Swartz JR 2003,209–217.

2. Royall E, Woolaway KE, Schacherl J and Kubick S: TheRhopalosiphum padi virus 5’-IRES is functional inSpodoptera frugiperda 21 cells and in their cell-freelysates: implications for the baculovirus expressionsystem. J Gen Virol 2004, 85:1565–1569.




Electron microscope images of subcellular localization of recombinant phytase in plant cells harvested from three different suspension cell linesestablished from M. truncatula plants: a wild type, a higher expressor and a lower expressor of this recombinant protein.



P95Purification of a chimeric virus-like particlefrom a complex culture mediumLuısa Pedro and Guilherme NM FerreiraCentro de Biomedicina Molecular e Estrutural (CBME),Universidade do Algarve, Faro, Portugal


Background: Virus-like particles are promising delivery vec-tors for molecular therapy since they combine the majoradvantages of viral vectors with minimal, or even with completedepletion of the viral vectors disadvantages. A chimeric Simian –Human Immunodeficiency virus-like particle (VLP) have beenconstructed by fusion of Simian matrix protein (p17) and HIV-1p6 protein which assemble as viral-like particles and whenproduced in HEK 293T cells are released to the culture medium[1]. The purification of these VLPs from a complex culturemedium involves an ultrafiltration/dialysis step, using a mem-brane which excludes proteins above 300kDa, and an anionicchromatography in which the particle is eluted at high saltconcentrations.Results: 293 cells were transfected with the vectors encodingthe chimeric protein SIV p17-HIV p6. The chimeric protein has amolecular weight of about 24kDa that assemble in sphericalstructures of about 80 nm. Preliminary studies with analytical

ultracentrifugation indicated that this spherical structure has amolecular weight above 1000 kDa. Therefore we expect particleretention in 300 kDa cut-off membranes, which were thus usedas primary purification/concentration step.Figure 1 shows a diagram of the concentration and dialysis of45 mL supernatant of transfected 293T cells using a 300 kDa cut-off ultrafiltration membrane, and Figure 2 shows the correspon-dent denaturing 12% poliacrilamide gel and western blot analysis.As shown our VLP was successfully retained and concentratedwith the 300 kDA cut off membrane. To further remove residualimpurity compounds we optimize an anion exchange (AEX)chromatography, which yielded a pure VLPs solution containingno proteinaceous compounds (Figure 3).Conclusion: Purified virus-like particles were obtained from acomplex mammalian culture medium using a simple two-steppurification process: a concentration/dialysis step, using anultrafiltration membrane, and an anionic chromatography.AcknowledgementsFinancial support from Fundacao para a Ciencia e a Tecnologia,project number POCI/BIO/62476/2004, is acknowledged.Reference1. Costa MJL, Pedro L, Matos APA, Aires-Barros MR, Belo JA,

Goncalves J and Ferreira GNM: Construction of chi-meric Simian – Human Immunodeficiency virus-likeparticles. in press.


Variation of the absorbance during supernatant concentration and dialysis. Firstly all supernatant were concentrated to 5 mL and then diluted with 15mL of 20 mM phosphate buffer at pH 8 and then concentrated again to 5 mL (this as been repeated five times). After each dilution it is visible proteinconcentration.



P96Identifying key signatures of highly productiveCHO cells from transcriptome and proteomeprofilesArleen Sanny1, Yee Jiun Kok1, Robin Philip1,Song Hui Chuah1, Sze Wai Ng1, Kher Shing Tan1,Lee Yih Yean1, Kathy Wong1, Hu Weishou3,Miranda Yap1,2 and Peter Morin Nissom1

1Bioprocessing Technology Institute, Biomedical Sciences Institutes,20 Biopolis Way, #06-01 Centros, Singapore 1386682Department of Chemical & Biomolecular Engineering, NationalUniversity of Singapore, 10 Kent Ridge Crescent, Singapore1192603Department of Chemical Engineering and Materials Science,University of Minnesota, 421 Washington Avenue SE,Minneapolis, Minnesota 55455-0132, USA


Background: One of the key challenges in biotherapeuticsproduction is the selection of a high-producing animal cell line to

maximize protein yield in cell culture. Clone selection is often atedious process, involving rounds of selection and single cellcloning which is costly in both money and time. In an effort toincrease the throughput of clone selection, we seek to identifykey signatures of a highly productive cell line using an integratedgenomic and proteomic platform. In our study, we analysedmicroarray and proteomics data generated from a characteriza-tion of two populations of CHO cells stably expressing high andlow levels of green fluorescent protein (GFP). The highproducer cells (HP) make 6x more GFP than the low producercells (LP) as determined by ELISA. Comparison of transcriptlevels between HP and LP in the mid-exponential phase wasperformed using a proprietary 15k CHO cDNA microarraychip, of which 7559 genes are unique [1], while proteomicanalysis on samples in the mid-exponential and stationary phaseswas performed using iTRAQ quantitative protein profilingtechnique [2]. Although there was a general lack of correlationbetween mRNA levels and quantitated protein abundance,results from both datasets concurred on groups of proteins/genes based on functional categorization.


Silver Staining of a denaturing 12% poliacrilamide gel presenting the protein pattern during centration/dialysis in a 300 kDa ultrafiltration membrane.Western blot were performed using a conjugated antibody, anti-HA HRP Roche). Molecular Weight markers were marked in kDa. Lane A.Supernatant of transfected 293T prior to concentration B. First permeate C. Second permeate D. Third permeate E. Fourth permeate F. Fifthpermeate G. Sixth permeate H. Concentrated supernatant.



Results: From microarray analysis, 84 genes had a change inrelative abundance of� 1.5-fold, either up or down, with p-value of� 0.05. A significant number (23%) was involved in proteinmetabolism, transcription and RNA processing. Other majorgroups of genes include cell cycle regulation, signal transductionand transport. 50% of the genes had unknown functions and thiscould serve as a source of discovery for new and novel genes.Proteomic analysis gave 20 and 26 proteins that satisfied the cut-offcriteria (� 1.2-fold change, 95% confidence) for the mid-exponential and stationary phase respectively. Proteins identifiedwere mainly involved in protein metabolism, carbohydratemetabolism and transport (Figure 1). Proteome and transcriptomeprofiles of HP showed an up-regulation of biological processesrelated to protein metabolism such as protein folding (PPIB andHyou1) and translation (Eef1a1, EIF2S3). With more proteinproduction, genes involved in ubiquitylation (Arih1, Nedd4, Psma4,Psmc5 and Usp10) were also up-regulated to regulate misfoldedproteins. Interestingly, a few of the identified genes involved inubiquitylation have also been implicated in transcription. Inparticular Psmc5, a subunit of the 19S proteasome, interacts withTADs (Transcriptional Activation Domain) and general transcrip-tion factors TBP and TFIIH [3]. Key molecular chaperone genes of

the UPR (unfolded protein response) pathway did not showsignificant differential expression, except for GRP78, an endoplas-mic reticulum molecular chaperone gene implicated in ER overloadresponse, which was down-regulated in HP. We also founddifferential expression in transcription and splicing factors, whichgive rise to a more active transcription and more efficient mRNAprocessing. Enzymes responsible for opening up chromatin, Hmgn3and Hmgb1, were up-regulated while enzymes that condensechromatin, histone H1.2, were down-regulated. Both Hmgn3 andHmgb1 bind to nucleosomes and reduce the compactness of thechromatin fiber, thus enhancing transcription from chromatintemplates [4, 5]. Genes and proteins that promote cell growth(Igfbp4, Ptma, S100a6 and Lgals3) were down-regulated while thosethat deter cell growth (Ccng2, Gsg2 and S100a11) were up-regulated, in agreement with the growth kinetics of HP comparedto LP in our study. Mitochondrial and mitochondrial biogenesisgenes and proteins (Cox7a2, Hspd1 and Mdh2) were up-regulated,indicating perhaps, more mitochondria. There was also a generalup-regulation of proteins involved in carbohydrate metabolism(Pkm2, Gpd2, Idh1 and Gapd). This seems to point towards moreenergy generation in HP and hence a higher capacity for proteinbiosynthesis.


Silver Staining of a denaturing 12% poliacrilamide gel presenting the protein pattern during the q-sepharose chromatography. Western blot wereperformed using a conjugated antibody, anti-HA HRP (Roche). Molecular Weight markers were marked in kDa. Lane A. Concentrated supernatant;lanes B to H – Unretained and washed material. Lanes I and J – Eluted fractions from the AEX column



Conclusion: Our results show that an integrated approach usingmicroarray and proteomics platform can be effectively utilized astools to monitor transcriptional and post-transcriptional events ofmammalian cells in culture, enabling us to identify distinctivechanges in cells caused by recombinant protein expression. Thisinformation, together with changes in other important cellularprocesses, would be valuable in a rational approach for engineeringcell-lines as well as for the designing of media and cell cultureparameters to enhance product yield in CHO cells.AcknowledgementsWe thank the support of A*Star, Agency for Science,Technology and Research, Singapore for funding the project.Angie Chang, Lu Wei Da, Toh Poh Choo and Wong Chun Loongand members of the proteomics group for their excellenttechnical assistance.References1. Wlaschin KF, Nissom PM, Gatti ML, Ong PF, Arleen S,

Tan KS, Rink A, Cham B and Wong K, et al: ESTsequencing for gene discovery in Chinese hamsterovary cells. Biotechnol Bioeng 2005, 91:592–606.

2. Schneider LV and Hall MP: Stable isotope methods forhigh precision proteomics. DDT 2005, 10:353–363.

3. Muratani M and Tanse WP: How the ubiquitin-protea-some system controls transcription. Nat Rev Mol CellBiol 2003, 4:192–201.

4. Ito Y and Bustin M: Immunohistochemical localizationof the nucleosome-binding protein HMGN3 inmouse brain. J Histochem Cytochem 2002, 50:1273–1275.

5. Travers AA: Priming the nucleosome: a role forHMGB proteins? EMBO Rep 2003, 4:131–136.

P97Kinetics of aggregation and structural propertiesof proteins in inclusion bodies studied by Fouriertransform infrared spectroscopyAntonino Natalello1, Diletta Ami1, Pietro Gatti-Lafranconi1, Ario de Marco2, Marina Lotti1

and Silvia Maria Doglia11Department of Biotechnology and Biosciences, University ofMilano-Bicocca, Piazza della Scienza 2, 20126 Milano, Italy2EMBL Scientific Core Facilities, Mayerhofstr. 1, D-69117,Heidelberg, Germany


Background: Protein aggregation plays a crucial role inmedical sciences and in biotechnology, as it occurs in severaldiseases and in the expression of recombinant proteins inbacterial cells in the form of inclusion bodies (IBs). Interestingly,it has been suggested that the presence of native-like structureswithin IBs [1, 2, 3, 4] improves the efficiency of refolding


Genomic and proteomic profiles of differentially expressed genes in high producing cell line.



protocols that employ mild solubilization methods [5]. Thisproperty could also explain the residual enzymatic activity ofrecombinant proteins in IBs, with possible applications inbiocatalysis [6].As recombinant protein production in bacteria is a central issuein biotechnology, it would be instructive to monitor the kineticsof protein aggregation and the extent of native-like secondarystructures within IBs.Results: We will present a new Fourier transform infraredspectroscopy (FT-IR) approach to study the aggregation ofrecombinant proteins in E. coli in the form of aggregates ofincreasing complexity. The method enables to monitor thekinetics of aggregate formation within intact cells in a rapid andnon invasive way and to obtain structural information onproteins within IBs. We will report results on four recombinantproteins: human growth hormone (h-GH), human interferon-alpha-2b (IFN-alpha-2b) [4, 7], Pseudomonas fragi lipase [3], andgreen fluorescent protein – glutathione S-transferase fusionprotein (GFP-GST) [8].Kinetics of aggregate formation was investigated at differentproduction temperatures. The rate of protein aggregation,monitored by the marker band of aggregation in the FT-IRabsorption spectrum (Amide I band), was found to increase withthe raising of production temperature. Furthermore, the proteinexpression in its soluble and insoluble fraction was alsoevaluated by the analysis of the FT-IR spectrum, in excellentagreement with SDS-PAGE analysis.To obtain structural information on protein aggregates,extracted IBs were also studied in the Amide I absorptionregion. Two structural features were observed, namely thepresence of native-like residual structures and the intermole-cular �-sheet interaction of proteins within IBs.Interestingly, for the same protein the residual native-likestructures in IBs were found to change with the level ofexpression. Therefore, by modulating the culture conditions,the extent of native-like structures in IBs can be optimised withuseful applications in biotechnology.Furthermore, additional structural features were obtained bythe comparison of the FT-IR spectra of the native form, IBs andthermal aggregates for the same protein.Conclusion: This FT-IR analysis offers a simple and rapidmethod to monitor in vivo the development of aggregatesformed by heterologous proteins and the effect of culturecondition modification on the process. Furthermore, themethod indicates that aggregating proteins modify at differentextent their secondary structures from native a-helices andintramolecular �-sheets to intermolecular �-sheets typical ofamorphous aggregates and fibrils.AcknowledgementsThis work was supported by INFM (Istituto Nazionale Fisicadella Materia) grant to S.M.D. The support of F.A.R. (Fondo diAteneo per la Ricerca) grants to S.M.D. and M.L. is alsoacknowledged.References1. Oberg K, Chrunyk BA, Wetzel R and Fink AL: Native-like

secondary structure in interleukin-1 beta inclusionbodies by attenuated total reflectance FTIR.Biochemistry 1994, 33:2628–2634.

2. Przybycien TM, Dunn JP, Valax P and Georgiou G:Secondary structure characterization of beta-lacta-mase inclusion bodies. Protein Eng 1994, 7:131–136.


4. Ami D, Natalello A, Taylor G, Tonon G and Doglia SM:Structural analysis of protein inclusion bodies byFourier transform infrared microspectroscopy.Biochim Biophys Acta 2006 in press.

5. Patra AK, Mukhopadhyay R, Mukhija R, Krishnan A, Garg LCand Panda AK: Optimization of inclusion body solubi-lization and renaturation of recombinant humangrowth hormone from Escherichia coli. Protein ExprPurif 2000, 18:182–192.


7. Ami D, Bonecchi L, CalI S, Orsini G, Tonon G andDoglia SM: FT-IR study of heterologous proteinexpression in recombinant Escherichia coli strains.Biochim Biophys Acta 2003, 1624:6–10.

8. Schrodel A and de Marco A: Characterization of theaggregates formed during recombinant proteinexpression in bacteria. BMC Biochemistry 2005, 6:10.

P98Production and purification of high molecularweight oligomers of Yersinia pestis F1 capsularantigen released by high cell density culture ofrecombinant Escherichia coli cells carrying thecaf1 operonTzvi Holtzman1, Yinon Levy2, Dino Marcus1,Yehuda Flashner2, Emanuelle Mamroud2, Sara Cohen2

and Rephael Fass11Department of Biotechnology, Israel Institute for BiologicalResearch, P. O. Box 19, Ness Ziona, 74100, Israel2Department of Biochemistry and Molecular Genetics, IsraelInstitute for Biological Research, P. O. Box 19, Ness Ziona,74100, Israel


Background: Yersinia pestis fraction 1 antigen (F1) is the majorcomponent of the pathogen capsule. F1, a 15.5 kDa monomerthat forms high molecular oligomers (>1000 kDa), is a highlyprotective antigen and is considered as a key constituent of asubunit anti-plague vaccine.F1 antigen is encoded on the caf1 operon together with atranscriptional regulator (caf1R), a chaperone (caf1M) and anusher protein (caf1A) and its expression is induced by atemperature shift to 37˚C. Production and purification of F1protein from Y. pestis cells is a tedious and time consumingprocedure. Use of recombinant Escherichia coli for the produc-tion of F1 was reported [1, 3].Here, we describe an efficient procedure for production andpurification of F1 released from the cell surface of E. coli cells,carrying the caf1 operon, grown to a high cell density.Results: The caf1 operon, derived from Y. pestis Kimberley53virulent strain [2], was cloned into the medium copy-numberplasmid pBR322-Kanamycin (to yield pBRK-F1), and theexpression plasmid was introduced into Escherichia coli



MC1060. Under expression conditions the cells were found toproduce high levels of F1 as a capsule (Y.L., E.M., S.C., Y.F.,unpublished data).Fermentation: Expression of F1 by E. coli MC1060 (pBRK-F1)was studied in a 4-liter computer-controlled fermentor using arich medium devoid of animal products: soy protein extract,trace elements and glycerol as carbon source (SY). This mediumenabled efficient bacterial growth to a high cell density (60OD600, Fig 1A). No loss of the expression plasmid was observedunder these conditions. Unexpectedly, high and constant level ofF1 expression was observed throughout the fermentation (up to24 hours) even at 28˚C (no temperature shift to 37˚C wasrequired, Fig. 1B). Moreover, at a high cell density (>10 OD600),F1 was released from the encapsulated E. coli cells andaccumulated up to 0.9 g/L in the culture medium (Fig. 1C).Purification: Following centrifugation, the supernatant con-taining F1 was micro-filtered through 0.2 �m tangential flowcartridges with no apparent yield loss. Native PAGE and sizeexclusion chromatography analyses indicated a high molecularweight of F1 oligomers (>1000 kDa). Despite the oligomers size,F1 passed through ultra-filtration tangential flow cartridgeshaving nominal pore size of 300 and 100 kDa and could be finallyconcentrated using ultra-filtration cartridges of 50 kDa (Fig. 2).

Further purification steps were as follows: (a) 33% saturationammonium sulfate precipitation, (b) size exclusion chromato-graphy and (c) endotoxin removal resulting in at least 3-logreduction of endotoxin content, to ~200 EU/ml. The highlypurified F1 antigen (>90%) consisted of high molecularoligomers with final recovery yield of 14% (125 mg F1/L culture).Conclusion:1. E. coli cells expressing F1 could be grown to a high cell densityin SY medium at 28˚C, using a computer-controlled fermentor.2. Continuous high level expression of F1 protein byrecombinant E. coli was achieved during fermentation with noplasmid loss.3. F1 antigen was massively released from the encapsulated cellsat high cell density cultures.4. High molecular weight oligomers of F1 were purified to >90%following few purification steps. This preparation was used forvaccination experiments in animal models.References1. Andrews GP, Heath DG, Anderson GW, Welkos SL and

Friedlander AM: Fraction1 capsular antigen (F1)purification from Yersinia pestis CO92 and from anEscherichia coli recombinant strain and efficacyagainst lethal plague challenge. Infect Immun 1996, 64(6):2180–2187.

2. Flashner Y, Mamroud E, Tidhar A, Ber R, Aftalyon M, Gur D,Lazar S, Zvi A, Bino T, Ariel N, Velan B and Shafferman A:Generation of Yersinia pestis attenuated strains bysignature-tagged mutagenesis in search of novelvaccine candidates. Infect Immun 2004, 72(2):908–915.

3. Miller J, Williamson ED, Lakey JH, Pearce MJ, Jones SM andTitball RW: Macromolecular organization of recom-binant Y. pestis F1 antigen and the effect of structureon immunogenicity. FEMS Immunol Med Microbial 1998,21:213–221.


Expression and release of F1 protein by recombinant E. coli MC1060(pBRK-F1) during fermentation. Culture growth was followed (A), andSDS-PAGE and Western blot analysis were performed on bacterial celllysates (B, 0.05 OD600/lane), and culture supernatant (C, 15 �l/lane).


Purification process of rF1 antigen. (1) Fermentation culture supernatant.(2) 0.2 �m micro-filtration filtrate. (3) 50 kDa ultra-filtration retentate.(4) 33% ammonium sulfate precipitate. (5) Flow-through of Superdex-200 size-exclusion chromatography. (6) Y. pestis F1 standard.



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