ARTICLE

Optimization of Elastin-Like Polypeptide Fusionsfor Expression and Purification of RecombinantProteins in Plants

Andrew J. Conley,1,2 Jussi J. Joensuu,2 Anthony M. Jevnikar,3 Rima Menassa,2

Jim E. Brandle2,4

1Department of Biology, University of Western Ontario, London,

Ontario, Canada2Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada,

London, Ontario, Canada N5V 4T3; telephone: 519-457-1470; fax: 519-457-3997;

e-mail: menassar@agr.gc.ca3Transplantation Immunology Group, Lawson Health Research Institute, London,

Ontario, Canada4Vineland Research and Innovation Centre, Vineland Station, ON, Canada

Received 10 October 2008; revision received 22 January 2009; accepted 28 January 2009

Published online 3 February 2009 in Wiley InterScience (www.interscience.wiley.com

). DOI 10.1002/bit.22278

ABSTRACT: The demand for recombinant proteins formedical and industrial use is expanding rapidly and plantsare now recognized as an efficient, inexpensive means ofproduction. Although the accumulation of recombinantproteins in transgenic plants can be low, we have previouslydemonstrated that fusions with an elastin-like polypeptide(ELP) tag can significantly enhance the production yield of arange of different recombinant proteins in plant leaves. ELPsare biopolymers with a repeating pentapeptide sequence(VGVPG)n that are valuable for bioseparation, acting asthermally responsive tags for the non-chromatographicpurification of recombinant proteins. To determine theoptimal ELP size for the accumulation of recombinantproteins and their subsequent purification, various ELP tagswere fused to green fluorescent protein, interleukin-10,erythropoietin and a single chain antibody fragment andthen transiently expressed in tobacco leaves. Our resultsindicated that ELP tags with 30 pentapeptide repeats pro-vided the best compromise between the positive effects ofsmall ELP tags (n¼ 5–40) on recombinant protein accu-mulation and the beneficial effects of larger ELP tags(n¼ 80–160) on recombinant protein recovery duringinverse transition cycling (ITC) purification. In addition,the C-terminal orientation of ELP fusion tags producedhigher levels of target proteins, relative to N-terminal ELPfusions. Importantly, the ELP tags had no adverse effect onthe receptor binding affinity of erythropoietin, demonstrat-ing the inert nature of these tags. The use of ELP fusion tagsprovides an approach for enhancing the production ofrecombinant proteins in plants, while simultaneously assist-ing in their purification.

Correspondence to: R. Menassa

� 2009 Wiley Periodicals, Inc.

Biotechnol. Bioeng. 2009;xxx: xxx–xxx.

� 2009 Wiley Periodicals, Inc.

KEYWORDS: elastin-like polypeptides; ELP; inverse-transi-tion cycling; molecular farming; transgenic tobacco;recombinant protein production

Introduction

Transgenic plants have great potential as bioreactors for thelarge-scale production of various recombinant proteins,such as vaccines, antibodies, biopharmaceuticals andindustrial enzymes (Giddings et al., 2000; Ma et al.,2005). Plants offer many advantages over conventionalexpression systems including potentially low productioncosts, rapid scalability, the absence of human pathogens andthe ability to correctly fold and assemble complex multi-meric proteins (Twyman et al., 2003). In particular, tobaccois a well-established model system for recombinant proteinproduction because it is readily amendable to geneticengineering, it has a high biomass yield and the platform isbased on leaves, which removes the need for flowering; thusminimizing the risk of gene leakage into the environmentthrough pollen or seed dispersal (Rymerson et al., 2002).Importantly, tobacco addresses many regulatory barriersbecause it is a non-food, non-feed crop, thereby eliminatingthe risk of plant-made recombinant proteins entering thefood supply (Menassa et al., 2001). Although tobacco isinherently biosafe, the low yield of some recombinant

Biotechnology and Bioengineering, Vol. xxx, No. xxx, 2009 1

proteins in tobacco leaves often prevents this host systemfrom being economically feasible (Doran, 2006). Further-more, the presence of phenolics and toxic alkaloids intobacco may preclude it from oral delivery, so the targetprotein must be purified prior to administration, which cancontribute to greater than 80% of the product cost (Kusnadiet al., 1997). Therefore, a strategy is needed for increasingthe accumulation of recombinant proteins in plants, whilealso assisting in their subsequent purification.

Elastin-like polypeptides (ELPs) are synthetic biopoly-mers composed of the repeating pentapeptide sequence Val-Pro-Gly-Xaa-Gly, where the guest residue Xaa can be anyamino acid except proline (Urry, 1988). In an aqueoussolution, ELPs undergo a reversible inverse phase transitionfrom soluble protein into insoluble hydrophobic aggregateswhen heated above their transition temperature (Tt) (Urry,1997). This thermally responsive property of ELP is alsotransferred to fusion partners, enabling a simple non-chromatographic method for protein purification called‘inverse transition cycling’ (ITC) (Meyer and Chilkoti,1999).

Many fusion tags have been developed to facilitate thepurification of recombinant proteins using affinity chro-matography techniques (Lichty et al., 2005; Terpe, 2003),but these methods are costly and difficult to scale-up toindustrial levels of protein purification (Waugh, 2005). Asan early step in any purification system, ITC offers severalbenefits as a simple, rapid, scalable and inexpensive non-chromatographic means of purifying recombinant proteinsfrom transgenic tobacco (Menkhaus et al., 2004; Meyer andChilkoti, 1999).

A synthetic (ELP)121 gene has been successfully expressedin the nucleus (Zhang et al., 1996) and chloroplasts (Gudaet al., 2000) of transgenic tobacco plants, but accumulationof the ELP protein was relatively low. On the other hand,protein fusions to ELP tags have recently been shown tosignificantly enhance the accumulation of a range ofdifferent recombinant proteins in transgenic tobacco leaves(Floss et al., 2008; Patel et al., 2007) and seeds (Scheller et al.,2006). Furthermore, ITC was used to purify cytokines (Linet al., 2006) and spider silk proteins (Scheller et al., 2004)from transgenic plants.

Although ELP fusions have been shown to increase theaccumulation of recombinant proteins in plants and aid intheir purification, very little work has been done to optimizeELP tag size for protein yield and recovery. The Tt of an ELPfusion varies with the sequence, size and concentration ofELP, as well as the hydrophobicity of the fusion partner andthe ionic strength of the aqueous solution (Meyer andChilkoti, 2004; Trabbic-Carlson et al., 2004b; Urry et al.,1991). To keep the Tt between 30 and 408C, most proteinpurification studies have used large ELP tags in the 35–75kDa range (i.e. 90–180 pentapeptides) (Banki et al., 2005;Meyer and Chilkoti, 1999; Shimazu et al., 2003; Stiborovaet al., 2003). However, the concentration of recombinantproteins in E. coli was found to be inversely proportional tothe size of ELP tag (Meyer and Chilkoti, 1999), with the yield

2 Biotechnology and Bioengineering, Vol. xxx, No. xxx, 2009

of thioredoxin increasing fourfold when the size of ELP tagwas reduced from 36 to 9 kDa (Meyer et al., 2001).Moreover, large ELP tags make up a greater proportion ofthe total recombinant protein fusion, thus reducing theamount of target protein.

This work provides the first thorough investigation inplants evaluating the effect of ELP size, orientation andamino acid sequence composition on the accumulationof four different recombinant proteins, green fluorescentprotein (GFP), interleukin-10 (IL10), erythropoietin (EPO),and a single chain antibody fragment (scFv). In addition, theeffect of ELP size on the efficiency of ITC purification fromplant extracts and the biological activity of EPO wasexamined. This comprehensive approach facilitates bio-technological applications by establishing an optimal size ofELP for both the accumulation of a range of recombinantproteins in plants and their subsequent purification.

Materials and Methods

Construction of Plant Expression Vectors

The complete genetic sequence of green fluorescent protein(GFP), interleukin-10 (IL10) and erythropoietin (EPO)were constructed using a combined ligase chain reaction(LCR)/PCR approach (Au et al., 1998), which utilizes a set ofoverlapping oligonucleotides designed by the web-basedprogram Gene2Oligo (Rouillard et al., 2004) to assemblesynthetic genes. This technique was also used to create thesingle chain variable fragment (scFv) antibody, elastin-likepolypeptide (ELP), random ELP (rELP), sizeELP, ELP-5 andELP-10 fragments with their additional 50 or 30 tags. TherELP sequence was designed by randomly rearranging all thecodons that make up ELP. A KasI restriction site was fusedto the 30-end of GFP, IL10 and EPO to allow for in-frameligation with various C-terminal tags. The N-terminal ELPfusion constructs were sequentially assembled by inter-changing previously synthesized construct componentsusing homology overlap PCR combined with restrictionenzyme digestion and in-frame ligation. The PR1b secretorysignal peptide from tobacco (Cutt et al., 1988) was placedupstream of all coding sequences to target the proteins intothe secretory pathway.

The ELP-10 monomer was sequentially oligomerized inpBlueScript by recursive directional ligation (Meyer andChilkoti, 2002) to generate a library of ELP tags of increasingsize. Flanking KasI restriction sites were introduced to theGFP, IL10, EPO and scFv genes by extension PCR and thencloned into the sizeELP expression vector. The library of ELPtags were digested with PflMI and BglI and then inserted intothe SfiI restriction site of each modified sizeELP expressionvector as described previously (Meyer and Chilkoti, 1999).The number of VGVPG pentapeptides for each ELP tag ofthe size library is given by n, where a 10-pentapeptide ELPhas a molecular weight of 4.11 kDa.

To assist in subsequent cloning steps, BamHI andEcoRI restriction sites were incorporated at the 50- and 30-end of all completed constructs. The final constructs weremoved into the plant binary expression vector pCaMterX(Laurian Robert, personal communication), where thecoding sequences were placed under the control of thedouble enhancer cauliflower mosaic virus (CaMV) 35Spromoter (Kay et al., 1987), a tCUP translational enhancer(Wu et al., 2001) and the nopaline synthase (nos)terminator. The expression constructs were electroporatedinto Agrobacterium tumefaciens strain EHA105 (Hood et al.,1993) and used for plant transformation.

Agroinfiltration of Tobacco Leaves

For transient expression, the intact leaves of 10- to 14-week-old Nicotiana tabacum (cv. 81V9) (Menassa et al., 2001)or Nicotiana benthamiana plants were infiltrated withAgrobacterium strains as previously described (Kapila et al.,1997; Yang et al., 2000). Briefly, the Agrobacteriumsuspensions were adjusted to a final OD600 of 1.0 and thendirectly injected into the intercellular spaces of leaves using a1-mL syringe with a 29-gauge needle. For the co-infiltrations, the bacterial strains were each adjusted toan OD600 of 1.0, prior to being mixed together in equalamounts. Each biological replicate is represented by anagroinfiltrated leaf panel, where a panel is the area betweenthe midrib and secondary veins. To compensate forvariability between plants, leaves and location on a leaf,comparably sized leaves from ten different plants of similarage were systematically agroinfiltrated for each expressionconstruct. After infiltration, the plants were maintained for4 days in a controlled growth chamber at 228C, under a 16 hphotoperiod. Tissue samples from the infiltrated leaf panelswere analyzed separately, with the average of the ten panelsused to represent the concentration of a given recombinantprotein.

Plant Protein Extraction

Leaf samples, consisting of four 7 mm leaf discs(approximate fresh weight of 25 mg), were frozen in liquidnitrogen and homogenized using 2.3 mm ceramic beads(BioSpec Products, 11079125z, Bartlesville, USA) in a MixerMill MM 300 (Retsch, Haan, Germany). The resultingpowder was then resuspended with 12 volumes (v/w) of ice-cold extraction buffer (phosphate-buffered saline (PBS), pH7.4, 0.1% Tween-20, 2% PVPP, 1 mM EDTA, 100 mMascorbic acid, 1 mM PMSF and 1 mg/mL leupeptin). Thehomogenate was clarified twice by centrifugation at 20,000gfor 10 min at 48C. The total soluble protein (TSP)concentration was measured using the Bradford assay(1976) with Bio-Rad reagent (Bio-Rad, Hercules, USA) andbovine serum albumin as a standard.

Inverse Transition Cycling (ITC) Purification

Individual leaf panels from 10 agroinfiltrated tobacco plantswere pooled together and frozen in liquid nitrogen, followedby homogenization with a pre-chilled mortar and pestle.For protein extraction, three volumes (v/w) of ice-coldextraction buffer were added and the homogenate clarifiedby centrifugation at 20,000g for 10 min at 48C. To trigger theinverse phase transition of ELP, the cleared lysate was pre-warmed to 378C and NaCl was added to 3 M, followed bycentrifugation at 20,000g for 10 min at 378C. The pellet wasresuspended in 1/10 of the original volume with coldextraction buffer and the suspension clarified again bycentrifugation at 20,000g for 10 min at 48C.

Quantification of EPO, IL10 and GFP Protein Levels

The concentration of EPO in tobacco leaf extracts wasdetermined by a sandwich enzyme-linked immunosorbantassay (ELISA). Nunc-Immuno MaxiSorp surface plates(Nalge Nunc, Rochester, USA) were coated withmouse anti-EPO monoclonal antibody (Stem Cell Technologies, 01350,Vancouver, Canada) diluted in disodium phosphate buffer(0.1 M, pH 9.0) to a concentration of 2 mg/mL andincubated overnight at 48C. The wells were blocked for 1 h atroom temperature with 2.7% ELISA Blocking Reagent(Roche, Mannheim, Germany) in PBS. To lie within thedetection range of the assay, plant extracts were seriallydiluted in dilution buffer (PBS containing 2.7% ELISABlocking Reagent and 0.05% Tween-20) and incubated onthe plate overnight at 48C. The plate was then incubated atroom temperature with 4 mg/mL of rabbit anti-EPOantibody (Sigma, E2531, St. Louis, USA) for 1.5 h and a1:1000 dilution of horseradish peroxidase-conjugated goatanti-rabbit IgG (Bio-Rad, 170-6515) for 1 h. Betweenincubation steps, the plates were washed five times withPBS-0.05% Tween-20. The plates were developed with anABTS substrate solution (Sigma, A-1888) and the opticaldensity was measured at 405 nm on a Bio-Rad 550microplate reader. The assay was calibrated using knownamounts of recombinant human EPO (rhEPO) (CellSciences, CRE600B, Canton, USA).

The concentration of plant recombinant IL10 wasdetermined by human IL10 ELISA using antibodies andstandard protein according to the manufacturer’s instruc-tions (BD Biosciences, Mississauga, Canada).

The concentration of GFP in the leaf extracts wasdetermined by measuring fluorescence intensity with aMFX Microtiter Plate Fluorometer (Dynex, Chantilly,USA). Briefly, samples were added to Microfluor 2 Blackmicrotiter plates (Thermo Scientific, Calgary, Canada)with the fluorescence detected using excitation and emis-sion wavelengths of 485 and 527 nm, respectively. Toaccount for background fluorescence of the plant tissue,Agrobacterium carrying an empty-vector control wasinfiltrated into leaves and used to normalize the data. All

Conley et al.: ELP Fusion Optimization for Expression and Purification 3

Biotechnology and Bioengineering

sample dilutions were compared to a standard curveconstructed with purified GFP (BioVision, 4999-100,Mountain View, USA).

Western Blots for the Quantification of scFv and ELPProtein Concentration

Extracted plant proteins were separated by sodiumdodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes bysemi-dry electroblotting. To prevent nonspecific binding ofantibodies, the membranes were blocked with 1% WesternBlocking Reagent (Roche) in Tris-buffered saline (TBS,50 mM Tris, 150 mM NaCl, pH 7.5) overnight at 48C. Themembranes were incubated for 1 h at room temperaturewith a 1:2000 dilution of mouse anti-myc monoclonalantibody (Invitrogen, R950-25, Carlsbad, USA). Theprimary antibody was detected with a 1:5000 dilution ofHRP-conjugated goat anti-mouse IgG (Bio-Rad, 170-6516)and visualized using the ECL detection kit (GE Healthcare,Mississauga, Canada) as described by the manufacturer. Themembranes were washed four times between each step withTBS-0.05% Tween-20 and all antibodies were diluted in TBScontaining 0.5% Western Blocking Reagent.

The Western blots were photographed and the concen-tration of recombinant scFv and ELP protein was analyzedby using image densitometry with TotalLab TL100 software(Nonlinear Dynamics, Durham, USA). The band intensitieswere compared to lanes containing known amounts ofPositope control protein (Invitrogen), which contains ac-myc tag.

Figure 1. Schematic representation of the plant expression constructs used for

Agrobacterium-mediated transient expression in tobacco. (A and B) All transgene

EPO Receptor Binding Assay

Plant-derived EPO’s ability to bind the human EPO receptor(EPOR) (Sigma, E0643) was determined by modifying theabove-mentioned quantitative EPO ELISA protocol. Inbrief, microtiter plates were coated overnight at 48C with 4mg/mL of mouse anti-EPOR antibody (R&D Systems,MAB3071, Minneapolis, USA). Next, the wells were blockedfor 1 h at room temperature with PBS containing 2.7%ELISA Blocking Reagent. Soluble plant extracts and EPOstandards were serially diluted in blocking buffer containinga saturating amount of EPOR (200 ng/mL) and incubatedon the plate overnight at 48C. The rest of the procedure wasperformed as for the ELISA described above for EPOquantification.

expression fragments were placed under the control of the cauliflower mosaic virus

35S promoter with dual-transcriptional enhancer (CaMV 35S), a tCUP 50-untranslated

region and the nopaline synthase (nos) terminator. GFP, green fluorescent protein;

IL10, human interleukin-10; EPO, human erythropoietin; scFv, single chain variable

fragment antibody; ELP, elastin-like polypeptide tag (28� VPGVG); rELP, random ELP;

(ELP)n, tags with various numbers (n) of pentapeptide repeats; GOI, gene of interest;

SP, PR1b tobacco secretory signal peptide; TEV, tobacco etch virus protease

recognition site; StrepII, purification tag; KDEL, ER-retention signal; Xpress and c-

myc, detection tags; sizeELP, all recombinant proteins and ELP tags were cloned into

this genetic cassette to create the ELP size libraries.

Results and Discussion

Design of the ELP Fusion Constructs

Although the successful expression and purification of ELPfusion proteins has been demonstrated in plants, very little

4 Biotechnology and Bioengineering, Vol. xxx, No. xxx, 2009

work has been performed on determining themost favorablefusion orientation and optimal size of ELP tag needed formaximum recombinant protein yield and purificationefficiency.

Plant expression vectors were constructed to produceGFP, IL10 and EPO fused with a combination of C-terminal,N-terminal and random ELP (rELP) tags, each with acorresponding length of 28 pentapeptides (Fig. 1A). TherELP fusion partner was created to determine whetherthe overall hydrophobic nature of ELP was responsible forthe enhanced accumulation of recombinant proteins inplants or if the repetitive nature of the ELP protein had a rolein this phenomenon. A tobacco signal peptide directed theproteins to the secretory pathway, while retention in theendoplasmic reticulum (ER) was achieved with a KDEL

motif (Gomord et al., 1997). All proteins were targeted tothe ER lumen because many reports have demonstratedhigher accumulation of secreted recombinant proteins inthis intracellular compartment (Conley et al., 2009; Fiedleret al., 1997; Huang et al., 2001; Ramirez et al., 2002).

The libraries of recombinant protein-ELP fusions werecreated by introducing GFP, IL10, EPO and scFvimmediately upstream of the various sizes of ELP tags inthe sizeELP cassette (Fig. 1B). Xpress and c-myc tags wereincluded for detection of the recombinant proteins byWestern blots. All coding sequences were placed into a plantbinary expression vector, with transcription driven by theconstitutive cauliflower mosaic virus 35S promoter.

Figure 2. Transient expression of recombinant protein-ELP fusion constructs

(Fig. 1A) in Nicotiana tabacum leaves. The concentration of GFP (A), IL10 (B) and EPO

(C) measured from leaf sectors harvested 4 days post-agroinfiltration. Each column

represents the mean value (n¼ 10) and the standard deviation is represented with

error bars. TSP, total soluble protein.

The Effect of ELP Sequence and Orientation onRecombinant Protein Concentration

To date, ELP tags have been exclusively used as C-terminalfusion partners for increasing recombinant protein yield inplants (Floss et al., 2008; Patel et al., 2007; Scheller et al.,2006). To evaluate the general utility of ELP protein fusionsin plants, Agrobacterium-mediated transient expression inN. tabacum leaves was used to determine the effect of C-terminal, N-terminal and rELP tags on the accumulation ofGFP, IL10 and EPO. Agroinfiltration is a simple andpredictive technique for rapidly evaluating the effect of ELPtags on recombinant protein accumulation, since it avoidsthe positional effects normally associated with stabletransgenic plants (Hobbs et al., 1990; Janssen and Gardner,1989; Kapila et al., 1997).

The C-terminal ELP28 fusion tag significantly increasedthe concentration of IL10 and EPO in plant extracts, but didnot alter the GFP concentration (Fig. 2). We speculate thatin contrast to the other recombinant proteins investigated,ELP did not increase the GFP yield in tobacco leaves becauseGFP is already a highly stable and soluble protein (Bokmanand Ward, 1981; Chiang et al., 2001; Tsien, 1998). The N-terminal ELP tag reduced the concentration of all threerecombinant proteins by an average of twofold, relative tothe C-terminal ELP tag. In numerous other cases, theorientation of protein fusions has also been found to alterthe accumulation, localization, folding and biologicalactivity of recombinant proteins (Chong et al., 1998; Huangand Mason, 2004; Palmer and Freeman, 2004; Puig et al.,2001). The rELP tag, derived from the random rearrange-ment of ELP amino acids, showed a significant reduction ofGFP, IL10 and EPO protein yield by an average of fourfold,when compared to the C-terminal ELP tag. This suggeststhat the proper protein sequence and structure of ELP isimportant for its beneficial effects on the accumulation ofrecombinant proteins, but does not completely excludeELP’s hydrophobic nature from playing a role.

Previous reports have shown that C-terminal ELP tags areresponsible for exceptionally high yields of recombinantproteins in bacteria (Chow et al., 2006; Trabbic-Carlsonet al., 2004a), and for substantially increasing the

accumulation (ca. 2- to 100-fold) of several target proteinsin transgenic tobacco leaves (Floss et al., 2008; Patel et al.,2007; Scheller et al., 2004) and seeds (Scheller et al., 2006).ELPs are thought to increase the stability of recombinantproteins because they are not susceptible to hydrolysis(Raucher and Chilkoti, 2001) or protease cleavage (Zhanget al., 1996), thus reducing the level of protein degradation.ELP tags have also been shown to increase the solubility oftarget proteins by protecting against irreversible aggregationand denaturation at high protein concentrations (Trabbic-Carlson et al., 2004a). Furthermore, Floss et al. (2008)demonstrated that ELP fusions have no adverse effectson the protein quality or N-glycan processing of targetglycoproteins.

Conley et al.: ELP Fusion Optimization for Expression and Purification 5

Biotechnology and Bioengineering

Figure 3. Accumulation of recombinant proteins fused with various sizes of ELP

tags (Fig. 1B). N. tabacum leaves were agroinfiltrated and the concentration of GFP

(A), IL10 (B), EPO (C), scFv (D), and ELP (E) was quantified. ND, not detectable.

The Effect of ELP Size on Recombinant ProteinConcentration

Since only two ELP fusion tags (i.e. a 28- and a 100-pentapeptide tag) have been utilized in plants (Patel et al.,2007; Scheller et al., 2004), we decided to thoroughlyexamine the effect of ELP size on the accumulation ofrecombinant proteins. For the recombinant proteinsevaluated (Fig. 3A–D), their concentration peaked withELP fusions of 5–30 pentapeptide repeats. With theexception of the low accumulating EPO protein, the otherrecombinant proteins approached concentrations of 2000–5000 ng/mg TSP (i.e. 0.2–0.5% of TSP). The ELP fusionproteins were quantified relative to unfused standardproteins, thus the amount of total fusion protein is actuallyunderestimated. Although the presence of an ELP tag had nopositive impact on GFP, IL10 or EPO concentration(Fig. 3A–C), it was able to significantly increase the levelsof scFv protein using our sizeELP expression system(Fig. 3D).

Interestingly, as part of the sizeELP expression system, theconcentration of IL10 and EPO protein lacking an ELPfusion was significantly higher (5.5- and 2.5-fold, respec-tively) when compared to similar unfused IL10 and EPOproteins found in Figure 2B and C. The major differencebetween the design of these expression vectors (Fig. 1B) isthe presence of an N-terminal Xpress tag on the sizeELPconstructs, which is absent from the Figure 1A constructs. Infact, additional experiments demonstrated that the N-terminal Xpress tag significantly enhanced the level of IL10protein by threefold (data not shown), suggesting that itis partially responsible for the positive effect on IL10concentration observed for the sizeELP construct. Perhaps,the Xpress tag serves as an N-terminal cap to the IL10 andEPO proteins and protects them against N-terminalproteolytic degradation. In contrast to prior reports (Flosset al., 2008; Patel et al., 2007; Scheller et al., 2006), ELP tagswere unable in this specific case to generally enhance theaccumulation of their recombinant protein fusion partner.We speculate that the new sizeELP expression vector, whichproduced much higher base levels of unfused IL10 andEPO protein may be responsible for the fact that ELP nolonger increased the accumulation of these recombinantproteins.

The concentration of all recombinant proteins wasdrastically reduced when utilizing the ELP80 tag relativeto the ELP40 tag, with this trend continuing for even largerELP tags. Only minute amounts of the recombinant proteinswere detected with the ELP240 tag. Across all sizes of ELPtags, there was a dramatic effect on the level of recombinantprotein accumulation with the range of concentrations forGFP, IL10, EPO and scFv varying by 60, 550, 50, and 450times, respectively. In E. coli., Meyer et al. (2001) showedthat decreasing the ELP length from 90 to 20 pentapeptidesincreased the expression yield of a thioredoxin-ELP fusionprotein by fourfold. On average, our four plant-derivedrecombinant proteins demonstrated the same 4.25-fold

6 Biotechnology and Bioengineering, Vol. xxx, No. xxx, 2009

enhancement when comparing the ELP80 to the ELP20fusion protein levels.

In the absence of a protein fusion partner, a distinctivelydifferent expression profile was observed for the variouslysized ELP proteins (Fig. 3E). ELP40 accumulated to thehighest level, with undetectable amounts of ELP proteinobserved for the 0, 5, 10 and 240 sizes of ELP tags. Similarly,free ELP tags of 8 and 9 pentapeptide repeats were found toexpress poorly in bacteria (Lim et al., 2007).

Figure 4. The effect of ELP size on the purity of fusion proteins following ITC.

The concentration of GFP (A), IL10 (B), EPO (C), scFv (D), and ELP (E) was quantified in

the original soluble leaf extract prior to precipitation (solid bars) and the resolubilized

pellet after a single step of ITC purification (hatched bars).

The Effect of ELP Size on ITC Purification ofRecombinant Proteins

While we have shown that there is an optimal size of ELP tagthat results in higher accumulation levels of the targetprotein, we were also interested in the effect of ELP size onthe purification of recombinant proteins by ITC. Valine waschosen as the guest residue for our ELP sequence because itis a relatively hydrophobic amino acid which depresses theTt (Urry et al., 1991), thus providing for more efficientpurification at lower temperatures. For technical simplicity,all phase transitions were induced by adding 3MNaCl to theextraction buffer and raising the temperature to 378C.

After a single step of ITC purification using theseexperimental conditions, ELP size was shown to have asignificant impact on the purity of the target protein (Fig. 4).Regardless of the recombinant protein fusion partner, aminimum number of 20 pentapeptide repeats was generallynecessary for the ELP tag to selectively enrich for the proteinof interest in the resolubilized ITC pellet, relative to theoriginal soluble leaf extract. For all fused target proteins, thehighest concentration of purified recombinant protein wasobtained when using the ELP20, ELP30, or ELP40 tags.

The degree of target protein precipitation and recoverywas also assessed, which allowed for a comparison of ITCpurification efficiency based on ELP size (Fig. 5). The Tt isinversely proportional to the ELP length (Meyer andChilkoti, 2004; Meyer et al., 2001), so it was expected thatthe small ELP tags (i.e. 0, 5, and 10) would be unable toselectively precipitate the fusion protein, given ourinvariable set of ITC conditions (i.e. ionic strength andprecipitation temperature). In general, the larger ELP80,ELP120, and ELP160 tags resulted in the highest recoveryrates, but the purification efficiency varied betweenrecombinant proteins. To account for this variation, studieshave demonstrated that the target protein’s surfacehydrophobicity can modulate the Tt relative to the unfusedELP protein (Meyer and Chilkoti, 1999; Trabbic-Carlsonet al., 2004b). Moreover, the concentration of ELP also hasan effect on ITC precipitation behavior, with higherconcentrations of ELP protein leading to more efficientrecovery of the fusion protein (Ge and Filipe, 2006; Meyerand Chilkoti, 2004). In our study, the ELP-fusionconcentration was much lower for larger ELP tags, butthey still purified more efficiently than the more abundantsmall ELP-fusion proteins, suggesting that ELP size is more

Conley et al.: ELP Fusion Optimization for Expression and Purification 7

Biotechnology and Bioengineering

Figure 5. The effect of ELP size on the ITC purification efficiency of various

target proteins. The degree of protein recovery was evaluated for GFP (A), IL10 (B),

EPO (C), scFv (D), and ELP (E), when fused to different sizes of ELP tags.

8 Biotechnology and Bioengineering, Vol. xxx, No. xxx, 2009

important than ELP concentration for effective ITCpurification.

Under optimized ITC purification conditions, recoveryrates of 70–95% have been obtained with bacterialexpression systems (Ge et al., 2006; Shimazu et al., 2003;Trabbic-Carlson et al., 2004a). We suspect that our reducedrecovery rates are due to the much lower expression levelsobtained for plant-made recombinant proteins (i.e. 0.01–0.5% of TSP), relative to bacteria (i.e. 0.1–1.6 g/L) (Chowet al., 2006; Meyer et al., 2001; Trabbic-Carlson et al., 2004a),along with the fact that we used a single set of ITC conditionsfor all recombinant proteins and all sizes of ELP. Moreover,the rate of recovery was higher for all detectable unfused ELPproteins (Fig. 5E) when compared to the same-sized ELPfusions (Fig. 5A–D), suggesting that the recombinantprotein fusion partners have a detrimental effect on theITC purification behavior of these ELP fusion proteins.

As a first step in our purification scheme, one cycle of ITCallowed for a simple and effective means for removing alldetectable amounts of toxic water-soluble alkaloids fromthe tobacco leaf extracts (data not shown). ITC can alsosimultaneously purify and concentrate the protein ofinterest, prior to any costly downstream processing steps,such as affinity chromatography. As well, aggregated ELP-fusion proteins have been shown to possess improvedstability relative to the soluble fusion protein, allowing forbetter long-term storage and application of the protein(Shamji et al., 2007; Shimazu et al., 2003).

The optimization of ITC conditions for combinations ofrecombinant proteins fused to smaller ELP tags would likelybe advantageous as they accumulate to much higher levels inplanta than those with larger ELP tags. Another strategywould be to use very small cationic ELP tags containingionizable Lys residues (i.e. eight pentapeptides), whichpossess enhanced salt sensitivity and have been utilized toefficiently purify bacterial-derived thioredoxin by ITC (Limet al., 2007).

Enhanced Transient Expression of ELP Fusions inN. benthamiana

To demonstrate the utility of the sizeELP expression vectorand ITC purification system, it was also tested in the mostroutinely used plant system for transient recombinantprotein production. That is, N. benthamiana plants wereagroinfiltrated with the GFP- and IL10-ELP size librariesalong with the p19 suppressor of gene silencing, which hasbeen found to significantly increase the production levels ofrecombinant proteins in plant leaves (Silhavy et al., 2002;Sudarshana et al., 2006; Voinnet et al., 2003). As waspreviously shown in N. tabacum (Fig. 3), the accumulationof recombinant protein is inversely proportional to the sizeof ELP tag in N. benthamiana (Fig. 6). However, in thisN. benthamiana expression system, the majority of thesefusion proteins are clearly visible on a commassie-stainedSDS-PAGE gel because their concentration is much higher

Figure 6. Co-expression of the recombinant protein-ELP fusions and p19 suppressor of gene silencing in the leaves of N. benthamiana plants. The GFP-ELP (A) and IL10-ELP

(B) fusion protein libraries were evaluated before (i.e. soluble leaf extract) and after (i.e. resolubilized pellet) a single step of ITC purification. Total soluble protein from the soluble

leaf extract (10 mg/lane) and resolubilized pellet (7 mg/lane) were resolved by 10% SDS-PAGE and the proteins visualized by commassie-staining of the gels. Arrowheads indicate

the visible target proteins, which were further validated by Western blot analysis (data not shown). C, non-transgenic leaf extract.

with the addition of the gene silencing suppressor p19. Onaverage, the levels of GFP and IL10 fusion proteins wereenhanced by 50- and 15-fold, respectively, in the presence ofp19. In the N. benthamiana leaf extracts, the maximum levelof GFP reached 21% of TSP with the ELP10 tag whenexamined by fluorometry while the maximum level of IL10reached 4.5% of TSP with the ELP5 tag when determined byELISA (data not shown). In fact, we are now able to produceplant-derived IL10 at a concentration that is approximately850 times higher than our IL10 transgenic tobacco plantsused as a therapeutic treatment in a mouse model ofcolitis (Menassa et al., 2007). These new advances in theproduction yields of plant recombinant proteins wouldallow for higher dosages of plant-derived IL10 to beadministered to the mice, likely leading to a more effectivetherapeutic response.

Similar to our results with N. tabacum (Fig. 4), wedemonstrate here that intermediate sizes of ELP tags (i.e. 20,30, 40, and 80) result in the highest purities of target proteinafter a single step of ITC purification. The maximum purityof GFP reached 66% of TSP with the ELP20 tag while themaximum purity of IL10 reached 30% of TSP with the ELP30tag (data not shown). To further enhance the purity of ELPfusion proteins, a pretreatment or post-treatment step couldbe employed prior to or after the ITC step to remove

the rubisco protein (ca. 46 kDa), which is the majorcontaminating protein.

The Effect of ELP Size on EPO’s Binding Affinity

To investigate the effect of ELP fusion tags on the biologicalfunction of recombinant proteins, the EPO-ELP size librarywas tested for its ability to bind the human EPO receptor(EPOR) via an indirect ELISA. The EPOR-binding assaydetected plant recombinant EPO (prEPO) with the sameELP-size dependent expression trend (data not shown)observed in Figure 3C, but the absolute values of EPOaccumulation varied between the two assays. Recombinanthuman EPO (rhEPO) was used to create both standardcurves and served as the common reference point allowingfor comparison between the EPOR-binding assay and thequantitative EPO ELISA, suggesting that prEPO has adifferent binding affinity for the human EPOR than rhEPO.Taking the ratio of the absolute values determined by thetwo assays showed that prEPO interacts with the EPOR witha uniformly higher binding affinity than the rhEPO,regardless of the size of ELP tag (Fig. 7). Therefore, thepresence of variously-sized ELP tags appears to have noadverse effect on the receptor binding capacity of prEPO.Our results are supported by other studies demonstrating

Conley et al.: ELP Fusion Optimization for Expression and Purification 9

Biotechnology and Bioengineering

Figure 7. Binding of plant-derived EPO-ELP fusions to the human EPO receptor

(EPOR). For each size of EPO-ELP fusion, the relative difference in binding affinity of

plant recombinant EPO (prEPO) and recombinant human EPO (rhEPO) to the EPOR was

determined by comparing the ratio of EPO accumulation detected in the EPOR-binding

assay (data not shown) to the quantitative ELISA results (Fig. 3C). In both assays, the

standard curve was comprised of rhEPO, which served as the common reference point

allowing for comparison between the two assays. A value of 1.0 represented an equal

ability of prEPO and rhEPO to bind the EPOR.

that ELP fusions generally do not negatively impact thefunctionality of the target protein (Scheller et al., 2006;Shamji et al., 2007; Trabbic-Carlson et al., 2004a). Thebioactivity of the EPO-ELP fusions was also preserved afterITC purification (data not shown), which has also beenshown for other recombinant proteins (Meyer and Chilkoti,1999; Shimazu et al., 2003). These are important stepstowards demonstrating that ELP will not modify thebiological activity of recombinant proteins needed forclinical or industrial applications.

Conclusions

For the first time, this study demonstrated the effect of ELPsize, orientation and sequence on the level of recombinantprotein accumulation in plant leaves, along with the effectof ELP size on the ITC purification efficiency. Our dataindicated that C-terminal ELP fusion tags with theappropriate sequence (i.e. VPGXG) are most advantageousfor increasing the concentration of target proteins. As well, anovel utilization of the Xpress epitope tag as an N-terminalfusion was shown to enhance recombinant protein yields inplants. Generally, small ELP tags (i.e. 5–40 pentapeptides)provided the highest yield of recombinant proteins. On theother hand, relatively large ELP tags (i.e. 80–160 pentapep-tides) resulted in the highest recovery rate of target proteinsduring ITC, which provides a simple, rapid, scalable andinexpensive means of protein purification. Importantly, ELPtags of any size were not found to have an effect on thereceptor binding activity of EPO or the fluorescence capacityof GFP, suggesting that ELP will generally have no effect onthe biological activities of their respective fusion partners.Our data illustrates that ELP tags with approximately 30

10 Biotechnology and Bioengineering, Vol. xxx, No. xxx, 2009

pentapeptide repeats provide the best compromise betweenthe positive effects of small ELP tags on recombinant proteinaccumulation and the beneficial effects of larger ELP tags onpurification efficiency during ITC purification. ELP fusiontechnology provides a general method for enhancing theyield of recombinant proteins in plants, while also providinga simple and scalable scheme for their subsequentpurification.

The authors wish to thank Linda Le for technical assistance and Alex

Molnar for his assistance with the preparation of the figures. Thanks

to Dr. Brian McGarvey and Bob Pocs for the tobacco alkaloid analysis

and Alex Richman for critical comments on the manuscript and

helpful discussions. The p19 vector was gratefully provided by Jozsef

Burgyan. This research was supported by the Agriculture and Agri-

Food Canada Matching Investment Initiative Programme. The Acad-

emy of Finland is acknowledged for providing a fellowship to J.J.J.

We thank the Natural Sciences and Engineering Research Council

(NSERC) Postgraduate Scholarship for providing financial support to

A.J.C.

References

Au LC, Yang FY, Yang WJ, Lo SH, Kao CF. 1998. Gene synthesis by a LCR-

based approach: high-level production of leptin-L54 using synthetic

gene in Escherichia coli. Biochem Biophys Res Commun 248:200–

203.

Banki MR, Feng L, Wood DW. 2005. Simple bioseparations using self-

cleaving elastin-like polypeptide tags. Nat Methods 2:659–661.

Bokman SH,WardWW. 1981. Renaturation of Aequorea green-fluorescent

protein. Biochem Biophys Res Commun 101:1372–1380.

Bradford MM. 1976. A rapid and sensitive method for the quantitation of

microgram quantities of protein utilizing the principle of protein dye

binding. Anal Biochem 72:248–254.

Chiang CF, Okou DT, Griffin TB, Verret CR, Williams MNV. 2001. Green

fluorescent protein rendered susceptible to proteolysis: Positions for

protease-sensitive insertions. Arch Biochem Biophys 394:229–235.

Chong S, Montello GE, Zhang A, Cantor EJ, Liao W, Xu MQ, Benner J.

1998. Utilizing the C-terminal cleavage activity of a protein splicing

element to purify recombinant proteins in a single chromatographic

step. Nucleic Acids Res 26:5109–5115.

Chow DC, Dreher MR, Trabbic-Carlson K, Chilkoti A. 2006. Ultra-high

expression of a thermally responsive recombinant fusion protein in

E. coli. Biotechnol Prog 22:638–646.

Conley AC, Mohib K, Jevnikar AM, Brandle JE. 2009. Plant recombinant

erythropoietin attenuates inflammatory kidney cell injury. Plant

Biotechnol J 7:183–199.

Cutt JR, Dixon DC, Carr JP, Klessig DF. 1988. Isolation and nucleotide

sequence of cDNA clones for the pathogenesis-related proteins PR1a,

PR1b and PR1c of Nicotiana tabacum cv. Xanthi nc induced by TMV

infection Nucleic Acids Res 16:9861.

Doran PM. 2006. Foreign protein degradation and instability in plants and

plant tissue cultures. Trends Biotechnol 24:426–4232.

Fiedler U, Phillips J, Artsaenko O, Conrad U. 1997. Optimization of scFv

antibody production in transgenic plants. Immunotechnology 3:205–

216.

Floss DM, Sack M, Stadlmann J, Rademacher T, Scheller J, Stoger E, Fischer

R, Conrad U. 2008. Biochemical and functional characterization of

anti-HIV antibody-ELP fusion proteins from transgenic plants. Plant

Biotechnol J 6:379–391.

Ge X, Filipe CDM. 2006. Simultaneous phase transition of ELP tagged

molecules and free ELP: An efficient and reversible capture system.

Biomacromolecules 7:2475–2478.

Ge X, Trabbic-Carlson K, Chilkoti A, Filipe CD. 2006. Purification of an

elastin-like fusion protein by microfiltration. Biotechnol Bioeng

95:424–432.

Giddings G, Allison G, Brooks D, Carter A. 2000. Transgenic plants as

factories for biopharmaceuticals. Nat Biotechnol 18:1151–1155.

Gomord V, Denmat LA, Fitchette-Laine AC, Satiat-Jeunemaitre B, Hawes

C, Faye L. 1997. The C-terminal HDEL sequence is sufficient for

retention of secretory proteins in the endoplasmic reticulum (ER)

but promotes vacuolar targeting of proteins that escape the ER. Plant J

11:313–325.

Guda C, Lee SB, Daniell H. 2000. Stable expression of a biodegradable

protein-based polymer in tobacco chloroplasts. Plant Cell Rep 19:257–

262.

Hobbs SLA, Kpodar P, DeLong CMO. 1990. The effect of T-DNA copy

number, position and methylation on reporter gene expression in

tobacco transformants. Plant Mol Biol 15:851–864.

Hood EE, Gelvin SB, Melchers LS, Hoekema A. 1993. New Agrobacterium

helper plasmids for gene transfer to plants. Transgenic Res 2:208–218.

Huang Z, Mason HS. 2004. Conformational analysis of hepatitis B surface

antigen fusions in an Agrobacterium-mediated transient expression

system. Plant Biotechnol J 2:241–249.

Huang Z, Dry I, Webster D, Strugnell R, Wesselingh S. 2001. Plant-derived

measles virus hemagglutinin protein induces neutralizing antibodies in

mice. Vaccine 19:2163–2171.

Janssen BJ, Gardner RC. 1989. Localized transient expression of GUS in leaf

discs following cocultivation with Agrobacterium. Plant Mol Biol

14:61–72.

Kapila J, De-Rycke R, Angenon G. 1997. An Agrobacterium-mediated

transient gene expression system for intact leaves. Plant Sci 122:101–

108.

Kay R, Chan A, Daly M, McPherson J. 1987. Duplication of CaMV 35S

promoter sequences creates a strong enhancer for plant genes. Science

236:1299–1302.

Kusnadi AR, Nikolov ZL, Howard JA. 1997. Production of recombinant

proteins in transgenic plants: Practical considerations. Biotechnol

Bioeng 56:473–484.

Lichty JJ, Malecki JL, Agnew HD, Michelson-Horowitz DJ, Tan S. 2005.

Comparison of affinity tags for protein purification. Protein Expr Purif

41:98–105.

Lim DW, Trabbic-Carlson K, Mackay JA, Chilkoti A. 2007. Improved non-

chromatographic purification of a recombinant protein by cationic

elastin-like polypeptides. Biomacromolecules 8:1417–1424.

Lin M, Rose-John S, Grotzinger J, Conrad U, Scheller J. 2006. Functional

expression of a biologically active fragment of soluble gp130 as an ELP-

fusion protein in transgenic plants: purification via inverse transition

cycling. Biochem J 398:577–583.

Ma JK, Barros E, Bock R, Christou P, Dale PJ, Dix PJ, Fischer R, Irwin J,

Mahoney R, Pezzotti M, Schillberg S, Sparrow P, Stoger E, Twyman

RM. 2005. Molecular farming for new drugs and vaccines. Current

perspectives on the production of pharmaceuticals in transgenic plants.

EMBO Rep 6:593–599.

Menassa R, Nguyen V, Jevnikar A, Brandle J. 2001. A self-contained system

for the field production of plant recombinant interleukin-10. Mol

Breed 8:177–185.

Menassa R, Du C, Yin ZQ, Ma S, Poussier P, Brandle J, Jevnikar AM. 2007.

Therapeutic effectiveness of orally administered transgenic low-alka-

loid tobacco expressing human interleukin-10 in a mouse model of

colitis. Plant Biotechnol J 5:50–59.

Menkhaus TJ, Bai Y, Zhang C, Nikolov ZL, Glatz CE. 2004. Considerations

for the recovery of recombinant proteins from plants. Biotechnol Prog

20:1001–1014.

Meyer DE, Chilkoti A. 1999. Purification of recombinant proteins by fusion

with thermally-responsive polypeptides. Nat Biotechnol 17:1112–

1115.

Meyer DE, Chilkoti A. 2002. Genetically encoded synthesis of protein-based

polymers with precisely specified molecular weight and sequence by

recursive directional ligation: Examples from the elastin-like polypep-

tide system. Biomacromolecules 3:357–367.

Co

Meyer DE, Chilkoti A. 2004. Quantification of the effects of chain length

and concentration on the thermal behavior of elastin-like polypeptides.

Biomacromolecules 5:846–851.

Meyer DE, Trabbic-Carlson K, Chilkoti A. 2001. Protein purification by

fusion with an environmentally responsive elastin-like polypeptide:

effect of polypeptide length on the purification of thioredoxin. Bio-

technol Prog 17:720–728.

Palmer E, Freeman T. 2004. Investigation into the use of C- and N-terminal

GFP fusion proteins for subcellular localization studies using reverse

transfection microarrays. Comp Funct Genomics 5:342–353.

Patel J, Zhu H, Menassa R, Gyenis L, Richman A, Brandle J. 2007. Elastin-

like polypeptide fusions enhance the accumulation of recombinant

proteins in tobacco leaves. Transgenic Res 16:239–249.

Puig O, Caspary F, Rigaut G, Rutz B, Bouveret E, Bragado-Nilsson E, Wilm

M, Seraphin B. 2001. The tandem affinity purification (TAP)method: a

general procedure of protein complex purification. Methods 24:218–

229.

Ramirez N, Ayala M, Lorenzo D, Palenzuela D, Herrera L, Doreste V, Perez

M, Gavilondo JV, Oramas P. 2002. Expression of a single-chain Fv

antibody fragment specific for the hepatitis B surface antigen in

transgenic tobacco plants. Transgenic Res 11:61–64.

Raucher D, Chilkoti A. 2001. Enhanced uptake of a thermally responsive

polypeptide by tumor cells in response to its hyperthermia-mediated

phase transition. Cancer Res 61:7163–7170.

Rouillard JM, LeeW, Truan G, Gao X, Zhou X, Gulari E. 2004. Gene2Oligo:

oligonucleotide design for in vitro gene synthesis. Nucleic Acids Res

32:W176–W180.

Rymerson R, Menassa R, Brandle JE. 2002. Tobacco, a platform for the

production of recombinant proteins. In: Erickson L, Brandle J,

Rymerson RT, editors. Molecular farming of plants and animals for

human and veterinary medicine. Amsterdam. Kluwer: p 1–32.

Scheller J, Henggeler D, Viviani A, Conrad U. 2004. Purification of spider

silk-elastin from transgenic plants and application for human chon-

drocyte proliferation. Transgenic Res 13:51–57.

Scheller J, Leps M, Conrad U. 2006. Forcing single-chain variable fragment

production in tobacco seeds by fusion to elastin-like polypeptides.

Plant Biotechnol J 4:243–249.

Shamji MF, Betre H, Kraus VB, Chen J, Chilkoti A, Pichika R, Masuda K,

Setton LA. 2007. Development and characterization of a fusion protein

between thermally responsive elastin-like polypeptide and interleukin-

1 receptor antagonist: Sustained release of a local antiinflammatory

therapeutic. Arthritis Rheum 56:3650–3661.

Shimazu M, Mulchandani A, Chen W. 2003. Thermally triggered purifica-

tion and immobilization of elastin-OPH fusions. Biotechnol Bioeng

81:74–79.

Silhavy D,Molnar A, Lucioli A, Szittya G, Hornyik C, TavazzaM, Burgyan J.

2002. A viral protein suppresses RNA silencing and binds silencing-

generated, 21- to 25-nucleotide double-stranded RNAs. EMBO J 21:

3070–3080.

Stiborova H, Kostal J, Mulchandani A, Chen W. 2003. One-step metal-

affinity purification of histidine-tagged proteins by temperature-trig-

gered precipitation. Biotechnol Bioeng 82:605–611.

Sudarshana MR, Plesha MA, Uratsu SL, Falk BW, Dandekar AM, Huang

TK, McDonald KA. 2006. A chemically inducible cucumber mosaic

virus amplicon system for expression of heterologous proteins in plant

tissues. Plant Biotechnol J 4:551–559.

Terpe K. 2003. Overview of tag protein fusions: from molecular and

biochemical fundamentals to commercial systems. Appl Microbiol

Biotechnol 60:523–533.

Trabbic-Carlson K, Liu L, Kim B, Chilkoti A. 2004a. Expression and

purification of recombinant proteins from Escherichia coli: Comparison

of an elastin-like polypeptide fusion with an oligohistidine fusion.

Protein Sci 13:3274–3284.

Trabbic-Carlson K, Meyer DE, Liu L, Piervincenzi R, Nath N, LaBean

T, Chilkoti A. 2004b. Effect of protein fusion on the transition

temperature of an environmentally responsive elastin-like poly-

peptide: A role for surface hydrophobicity? Protein Eng Des Sel

17:57–66.

nley et al.: ELP Fusion Optimization for Expression and Purification 11

Biotechnology and Bioengineering

Tsien RY. 1998. The green fluorescent protein. Annu Rev Biochem 67:509–

544.

Twyman RM, Stoger E, Schillberg S, Christou P, Fischer R. 2003. Molecular

farming in plants: host systems and expression technology. Trends

Biotechnol 21:570–578.

Urry DW. 1988. Entropic elastic processes in protein mechanisms. I. Elastic

structure due to an inverse temperature transition and elasticity due to

internal chain dynamics. J Protein Chem 7:1–34.

Urry DW. 1997. Physical chemistry of biological free energy transduction as

demonstrated by elastic protein-based polymers. J Phys Chem B

101:11007–11028.

Urry DW, Luan CH, Parker TM, Gowda DC, Prasad KU, Reid MC, Safavy

A. 1991. Temperature of polypeptide inverse temperature transition

depends on mean residue hydrophobicity. J Am Chem Soc 113:4346–

4348.

12 Biotechnology and Bioengineering, Vol. xxx, No. xxx, 2009

Voinnet O, Rivas S, Mestre P, Baulcombe D. 2003. An enhanced transient

expression system in plants based on suppression of gene silencing by

the p19 protein of tomato bushy stunt virus. Plant J 33:949–956.

Waugh DS. 2005. Making the most of affinity tags. Trends Biotechnol

23:316–320.

Wu K, Malik K, Tian L, Hu M, Martin T, Foster E, Brown D, Miki B. 2001.

Enhancers and core promoter elements are essential for the activity of a

cryptic gene activation sequence from tobacco, tCUP. Mol Gen Geno-

mics 265:763–770.

Yang Y, Li R, Qi M. 2000. In vivo analysis of plant promoters and

transcription factors by agroinfiltration of tobacco leaves. Plant J

22:543–551.

Zhang X, Urry DW, Daniell H. 1996. Expression of an environmentally

friendly synthetic protein-based polymer gene in transgenic tobacco

plants. Plant Cell Rep 16:174–179.