Protein Expression and PuriWcation 41 (2005) 186–198

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Refolding, puriWcation, and crystallization of apical membrane antigen 1 from Plasmodium falciparum

Aditi Guptaa,1, Tao Baia,1, Vince Murphyb, Phillip Strikec, Robin F. Andersb, Adrian H. Batchelora,d,¤

a University of Maryland School of Pharmacy, 20 Penn Street, Baltimore, MD 21201, USAb Department of Biochemistry, Cooperative Research Center for Vaccine Technology, La Trobe University, Vic. 3086, Australia

c CSIRO Health Sciences and Nutrition, 343 Royal Parade, Parkville, Vic. 3052, Australiad The Walter and Eliza Hall Institute, 1G Royal Parade, Parkville, Vic. 3050, Australia

Received 1 December 2004, and in revised form 3 January 2005Available online 26 January 2005

Abstract

Extracellular domains of malaria antigens almost invariably contain disulphide linkages but lack N- and O-linked glycosylation.The best practical approach to generating recombinant extracellular Plasmodium proteins is not established and the problemsencountered when using a bacterial expression/refolding approach are discussed in detail. Limited proteolysis experiments were usedto identify a relatively non-Xexible core region of the Plasmodium falciparum protein apical membrane antigen 1 (AMA1), andrefolding/puriWcation was used to generate two fragments of AMA1. Several chromatographically distinct AMA1 variants wereidentiWed that are presumably diVerentially refolded proteins. One of these AMA1 preparations proved to be crystallizable and gen-erated two crystal forms that diVracted X-rays to 2 Å resolution. 2005 Elsevier Inc. All rights reserved.

Keywords: Malaria vaccine refolding puriWcation crystallization

Malaria parasites possess a multitude of proteinslocated on the surface of the parasite or in secretory ves-icles, that play a role in the process of host-cell invasion.The understanding of this complex process is only begin-ning to emerge but is best understood for the invasion ofPlasmodium falciparum into human red blood cells.There is great interest in synthesizing these extracellularPlasmodium proteins in order to elucidate their functionsin erythrocyte invasion and to test their potential asvaccines.

The antigen of interest in this paper is apical mem-brane antigen 1 (AMA1) from P. falciparum. AMA1,originally identiWed in Plasmodium knowlesi [1,2] and

¤ Corresponding author.E-mail address: abatchel@rx.umaryland.edu (A.H. Batchelor).

1 These authors contributed equally to this work.

1046-5928/$ - see front matter 2005 Elsevier Inc. All rights reserved.doi:10.1016/j.pep.2005.01.005

subsequently in P. falciparum [3], has been identiWed inall plasmodia and in all apicomplexan parasites exam-ined. However, the conservation in more evolutionarilydistant apicomplexa is restricted to the central region ofAMA1 [4,5]. Attempts to ‘knock out’ AMA1 have failedsuggesting that the AMA1 gene is essential [4,6]. Thefunction of AMA1 and its location in the micronemes[7,8] is consistent with a role in invasion, and antiseraraised to AMA1, or peptides that bind to AMA1, inhibitthe invasion of merozoites into erythrocytes [1,9–13]. Astriking feature of AMA1 is its polymorphisms. Exami-nation of all P. falciparum AMA1 sequences revealedmore than 50 polymorphic sites [14–16], whereas,because P. falciparum is a relatively recent human path-ogen only one or two neutral mutations would beexpected for a gene the size of AMA1 [17–19]. Thiswould suggest that AMA1 is a highly signiWcant target

A. Gupta et al. / Protein Expression and PuriWcation 41 (2005) 186–198 187

for naturally acquired immunity but would also lead tothe prediction that the acquired protective immunitywould be strain speciWc. Strain speciWcity of antibodyresponses to AMA1 has been observed for P. chabaudiin mice [20]. However, sera that recognize and inhibiterythrocyte invasion by P. falciparum merozoites areonly partially strain speciWc. Polymorphic rabbit anti-sera raised to strain 3D7 AMA1 completely inhibitederythrocyte invasion by strains 3D7 and D10, partiallyinhibited strain HB3 but were non-inhibitory for strainW2mef [11,21]. Similar results were observed by Ken-nedy et al. [22]. This would suggest that regions of theprotein that are critical to AMA1 function are evolu-tionarily constrained and are less polymorphic, althoughnot entirely non-polymorphic.

The expression of extracellular domains from malariaparasites presents us with an unusual practical problem.Several lines of evidence indicate that there is no N- orO-linked glycosylation of extracellular proteins [23–26]and yet proteins from plasmodia typically containnumerous potential N-linked glycosylation sites.Malaria antigens expressed using eukaryotic secretorysystems are invariably glycosylated, and glycosylationhas been shown to interfere with protein antigenicity[27,28]. Moreover, due to the Xexible nature of sugarchains, glycosylation would reduce the chances of crys-tallization. Various approaches have been taken to over-come the problem of aberrant glycosylation. Onesolution is to mutate potential N-linked glycosylationsites, and this has been used successfully in a number ofinstances [29,30]. However, this approach is risky if thestructure of the protein is not known. A diVerentapproach adopted by several groups is to express pro-teins of interest in bacteria and then to refold them invitro. Singh et al. [31] have been able to refold Plasmo-dium vivax DuVy binding antigen and have demon-strated that it is functionally active and able to bind toerythrocytes. Hodder et al. [11] and Dutta et al. [32] havebeen able to refold AMA1 from P. falciparum. Theydemonstrated cross antigenicity to native AMA1, but, aswith most malaria antigens, it is diYcult to conclusivelyprove that the preparation in question is correctly foldedbecause the function is not known. As an extension ofthis work, we have utilized recombinant full lengthextracellular AMA1 to identify proteolytically accessibleregions of the protein. Fragments were identiWed thatwere relatively resistant to proteases and these formedthe basis for a new round of expression constructs togenerate regions of AMA1 with potential for crystalliza-tion. We describe in detail the puriWcation protocols thatwere necessary to remove misfolded variants of AMA1that were mostly multimeric but also monomeric.

Thus far there have been no reports of the successfulcrystallization of extracellular proteins from plasmodiautilizing the refolding approach. However, one of ourAMA1 protein fragments turned out to be highly crys-

tallizable. This fragment yielded crystals in a variety ofconditions. Two of these conditions yielded crystals thatdiVracted X-rays to 2 Å resolution.

Materials and methods

IdentiWcation of proteolytically accessible regions in AMA1 ectodomain

Refolded full-length AMA1 was produced asdescribed previously [11]. The proteases elastase andsubtilisin (obtained from Sigma) were dissolved at a con-centration of 1 mg/ml in 10 mM acetic acid–NaOH, pH5, 50% glycerol and stored at ¡20 °C. AMA1 was treatedwith serial dilutions of protease for 10 min at 30 °C inbuVer containing 20 mM Tris–HCl, pH 8, 100 mM NaCl(and 2 mM CaCl2 for subtilisin). Typically 2 �g AMA1was treated with 3-fold serial dilutions of protease rang-ing from 3 to 0.01�g. Reactions were terminated by theaddition of SDS-containing electrophoresis samplebuVer combined with heating at 90 °C for 2 min. Proteol-ysis reactions were then analysed by polyacrylamide gelelectrophoresis. To purify the resulting fragment, theappropriate reaction with elastase was scaled up 20-fold,terminated by addition of 0.05% triXuoroacetic acid(TFA), and puriWed by reverse phase chromatographyon a 2.1 £ 100 mm C8 column (Brownlee) using 0.05%TFA and an acetonitrile gradient. The resulting non-reduced fragment was analysed by N-terminal sequenc-ing and by MALDI-TOF mass spectrometry. Disulphidebonds were then reduced by dialysis of the sample into20 mM Tris–HCl, pH 8, 100 mm NaCl and subsequentaddition of 6 M guanidine hydrochloride (GdnHCl) and20 mM dithiothrietol followed by incubation at roomtemperature for 30 min. The resulting reduced fragmentswere further puriWed by reverse phase chromatographyand analysed by further rounds of N-terminal sequenc-ing and mass spectrometry.

Plasmid constructs

AMA1 coding sequences were ampliWed from geno-mic DNA prepared from P. falciparum strain 3D7, gen-erously provided by Pauline Crewther. Upstreamprimers 5�-aattatatgggtaatccatgg and 5�-atagtaga acatat-gaattatatgggtaatccatgg were used together with twodownstream primers (5�-gatccgagctca tgttacttctgccct-tctttct or 5�-gatccgagctcattcaacttcgatgggatggga) to gen-erate four DNA fragments. Two of the fragmentsencoded AMA1 domains I + II + III whilst two shorterfragments encoded AMA1 domains I + II. PCR mixturescontaining 2 U of Vent polymerase (New England Bio-labs) and 0.2 mM deoxynucleotides in the recommendedbuVer were incubated at 94, 45, and 68 °C each for 1 minfor 30 cycles. PCR products were isolated by phenol/

188 A. Gupta et al. / Protein Expression and PuriWcation 41 (2005) 186–198

chloroform extraction and ethanol precipitation, cleavedwith NdeI and SacI (New England Biolabs) and puriWedby electrophoresis in 1% agarose gels in 40 mM Tris–ace-tic acid, pH 8, 1 mM EDTA. P. falciparum DNA ishighly susceptible to thymidine dimer formation due tothe high AT content in the genome. DNA fragmentswere therefore not exposed to UV. Instead, edge slices ofthe gel were stained with ethidium bromide in order tolocate the positions of the DNA fragments. DNA wasisolated from the agarose slice using the Geneclean IIprotocol, followed by a further round of phenol/chloro-form extraction and ethanol precipitation. AMA1cDNA fragments were ligated into one of two expressionvectors: a non tag vector p�8a used in a previous crystal-lographic study [33] cleaved with NdeI and SacI orpPROEXHTb (Invitrogen) cleaved with SfoI and SacI.pPROEXHTb codes for a hexa-histidine N-terminal tagseparated from the AMA1 coding sequence with a linkercontaining a TEV protease cleavage site. The resultingplasmids were sequenced using pPROEXHTb vectorprimers (5�-cgacatcataacggttctggca and 5�-tatcagac cgct-tctgcgttc) and internal AMA1 sequence primers (5�-aggaccattttcttgagctgc and 5�-gcagctcaagaaaataatggtcc).In spite of the precautions taken several clones withmutations were generated, as is often the case with P.falciparum DNA. However, we were able to obtain thefollowing clones that were mutation free: pBB117 (histagged AMA1 domains I + II + III), pBB119 (his taggedAMA1 domains I + II), and pBB120 (non-tagged AMA1domains I + II).

Protein expression

Expression plasmids pBB117 and pBB119 were trans-formed into Escherichia coli BL21(DE3) cells containingthe supplementary rare tRNA expression plasmid pRIL(Stratagene) and selected on Luria broth-agar platescontaining 100 �g/ml ampicillin (to select for pBB117and pBB119) and 30 �g/ml tetracycline to select forpRIL. Log phase cells were frozen in 10% glycerol to actas starter cultures. Ten millilitres of Luria broth (LB, 5 gyeast extract, 10 g tryptone, 10 g NaCl/L) supplementedwith ampicillin and tetracycline was innoculated with5 �l of frozen stock culture and grown to conXuenceovernight. Ten millilitre overnight cultures were addedto 1 L of LB containing ampicillin and tetracycline andincubated with shaking at 37 °C for 3 h. Cultures werethen induced with 1 mM IPTG for a further 3 h. Some ofthe protein preparations were made utilizing richermedia (35 g tryptone, 20 g yeast extract, 5 g NaCl/L) ormedia supplemented with 2% glucose. However, use ofricher media made little diVerence to protein yields (Fig.3). Moreover, the pRIL plasmid was not necessary forAMA1 expression but was used for most protein prepa-rations because it improved protein yields by about 25%(data not shown).

Protein solubilization

As expected proteins resulting from pBB117, pBB119,and pBB120, expressed to high yields (approximately0.2 mg/ml LB), but were insoluble. One litre LB cell pel-lets were stored frozen and resuspended in 200 ml of30 mM Tris–HCl, pH 8, 200 mM NaCl, 2 mM EDTA,0.2 mM PMSF, and 0.1% Triton X-100, by pipetting,treatment with 0.1 mg lysozyme and sonication. Mostprotein impurities were solubilized by this wash buVer.AMA1 proteins remained insoluble and were repelletedby centrifugation at 8000g for 30 min. The AMA1 pro-tein pellets were then washed a second time by sonin-ation into 20 mM Tris–HCl, pH 8, 0.5 mM EDTA, 2 Murea and repelleted. The importance of this second washstep is uncertain. We included it because we were con-cerned about carry over of Triton X-100 into the refold-ing process. Examination of the AMA1 proteins byreduced SDS–PAGE indicated bands of approximately80% purity (Fig. 3). However, if samples were notreduced the band was smeared and largely consisted ofhigher molecular weight disulphide-linked multimers ofmisfolded AMA1 (Fig. 4). The proteins wereresolubilized in 200 ml of 20 mM Tris–HCl, pH 8, 2 mMof 2-mercaptoethanol (2ME), and 6 M guanidine hydro-chloride (GdnHCl) by rotating the centrifugation bottlesat room temperature overnight. In some preparations2ME was not used at this stage. In the absence of 2MEapproximately 50% of the AMA1 pellet was not solubi-lized in GdnHCl. The use of reduced or non-reducedprotein dissolved in GdnHCl made no diVerence to sub-sequent refolding.

Protein refolding

AMA1 proteins were diluted into 20 mM Tris–HCl,pH 8, 6 M GdnHCl such that the Wnal protein concentra-tion was 20 �g/ml. Fifty millilitres of dilute protein wasdialysed against 3.5 L of 20 mM Tris–HCl, pH 8, 0.5 mMEDTA, 1 M urea, 200 mM NaCl, 2 mM 2ME, and0.2 mM cystamine–HCl overnight at 4 °C. The dialysisbag was then transferred into 3.5 L of 20 mM aceticacid–NaOH, pH 5, 0.5 mM EDTA for a further over-night dialysis. At this stage a signiWcant proportion ofthe AMA1 (approximately 20%) had precipitated out ofsolution, but this consisted of multimeric misfoldedAMA1. Sodium azide (0.02%) was added to dialysedsamples that were stored at 4 °C for up to 2 months.

Protein puriWcation prior to tag cleavage

Refolded protein contained a signiWcant amount ofmisfolded protein precipitate which was removed byplacing a coarse Wlter over a Wne 0.2 �m PES Wlter (Corn-ing). The starting volume for a typical protein prepara-tion was approximately 2 L (40 mg protein). This was

A. Gupta et al. / Protein Expression and PuriWcation 41 (2005) 186–198 189

concentrated 20-fold by ion exchange using a HiTrap SPSepharose column (Amersham–Pharmacia), eluting with20 mM Tris–HCl, pH 8, 0.5 M NaCl. Protein was thenloaded onto a 5 ml HiTrap chelating column (Amer-sham–Pharmacia) that had been charged with 10 ml of0.1 mM CoCl2 and eluted in 20 mM Tris–HCl, pH 8,0.5 M NaCl with a 0–400 mM imidazole gradient. AMA1eluted in 140 mM imidazole. Approximately 10% ofAMA1, presumably misfolded protein in which thehexahistidine tag is no longer exposed, eluted in 20 mMimidazole (Fig. 6A).

Tag cleavage using TEV protease

Cleavage with TEV presented a practical diYcultybecause TEV protease is an intracellular protease and itis active under reducing conditions. Treatment ofrefolded AMA1 with low concentrations of reducingagent (1 mM 2ME) resulted in the non-reduced SDS–PAGE band being detectably more diVuse (data notshown). However, TEV protease retained reasonableactivity in low concentrations of reducing agents thatdid not appear to aVect AMA1. TEV protease (contain-ing 5 mM dithiothrietol, DTT) was diluted 10-fold into20 mM Tris–HCl, pH 8 and further diluted 20-fold intorefolded AMA1 (in 20 mM Tris–HCl, pH 8, 0.5 M NaCl,and 140 mM imidazole) such that the Wnal DTT concen-tration was 0.025 mM. TEV proteolysis was allowed toproceed for 2 days at room temperature. One hundredmicrolitres of TEV protease (1000 U) was capable of cut-ting »8 mg of refolded AMA1.

Protein puriWcation following tag cleavage

Following TEV cleavage AMA1 proteins were dia-lysed into 20 mM Tris–HCl, pH 8 and the total proteinpreparation (approximately 40 mg) loaded onto an 8 mlmono Q column (Amersham–Pharmacia, HR 10/10).Protein was eluted using a NaCl gradient, with the prin-cipal component eluting in 70 mM NaCl (Fig. 6B). Size-exclusion chromatography was carried out using a Seph-acryl S200 26/60 column (Amersham–Pharmacia) in20 mM acetic acid–NaOH, pH 5.5, 0.5 mM EDTA. Voidvolume for the 26/60 column was 70 ml with a totalvolume of 320 ml. AMA1 I + II + III protein (predictedmolecular weight 47.6 kDa) eluted at a volume of 155 ml,and co-eluted with a 44 kDa size exclusion marker (Bio-Rad), indicating that it was monomeric. The principalimpurity eluted at a volume of 130 ml, with a predictedmolecular weight of 100 kDa consistent with it beingmostly dimeric protein (Fig. 6C). AMA1 I + II (predictedmolecular weight 38.4 kDa) eluted slightly anomalouslyat a volume of 175 ml, corresponding to a size of 25 kDa.Presumably, this was due to AMA1 I + II sticking to thecolumn slightly. Protein puriWed by size-exclusion chro-matography was loaded onto an 8 ml mono S column

(Amersham–Pharmacia, HR 10/10) and eluted in 20 mMacetic acid–NaOH, pH 5.5, 0.5 mM EDTA with a NaClgradient. For both AMA1 I + II + III and AMA1 I + IItwo principal peaks were observed. A smaller peak elutedin 150–160 mM NaCl, whereas the major peak eluted in180–190 mM NaCl (Fig. 7). Each fraction was dialysedinto deionized water, treated with 0.02% sodium azide asa preservative and concentrated by centrifugation over5 kDa molecular weight Wlters (VivaScience). Proteinsthat eluted in 150–160 mM NaCl from the mono S col-umn did not concentrate eYciently and associated withthe 5 kDa membrane such that signiWcant losses wereobserved. Proteins that eluted in 180–190 mM NaCl fromthe mono S column concentrated eYciently. AMA1 I + IIwas not particularly soluble and could be concentratedto 3 mg/ml. AMA1 I + II + III was highly soluble andcould be concentrated to at least 20 mg/ml.

Binding of AMA1 to monoclonal antibody 1F9

Binding of mAb 1F9 to the chromatographically dis-tinct refolded AMA1 species was assessed by native gelelectrophoresis. mAb 1F9 was puriWed from hybridomasupernatants using protein G Sepharose, eluted using100 mM glycine–HCl, pH 2.7 and dialysed into phos-phate buVered saline. Binding reactions contained 2 �gof mAb 1F9 and 2�g of AMA1 and were carried out in50 mM Tris–HCl, pH 7.5 + 15% glycerol. Proteins wereseparated in 50 mM Tris–HCl, pH 7.5, 7.5% polyacryl-amide (37.5:1 mono:bis acrylamide, Bio-Rad) at 100 Vfor 1.5 h in a running buVer of 50 mM Tris–acetic acid,pH 8. Gels were stained in 0.05% Coomassie blue in 10%acetic acid, 25% isopropanol for 30 min before destain-ing in 10% acetic acid.

Crystallization of AMA1

Crystallizations were attempted using the hangingdrop method using 1.5�l protein + 1.5�l well solution in21.5 °C constant temperature cabinets (Torrey-Pines Sci-entiWc). Well solutions included crystal screens I and II(Hampton Research), a variety of polyethylene glycolsincluding PEG 4000 + buVer, PEG 4000 + 100 mM lith-ium sulphate + buVer, lithium sulphate + buVer, andammonium sulphate + buVer. No crystals were observedfor AMA1 I + II + III. In contrast, AMA1 I + II crystal-lized in many conditions including virtually all PEGconditions tested. Two conditions yielded crystals thatdiVracted X-rays to 2 Å resolution: 20 mM MES, pH 6,10 mM MnCl2, 8–10% PEG 3350, or 100 mM Tris–HCl,pH 8.5, 100 mM Li2SO4, and 30% PEG 3350.

Crystal freezing and diVraction

Crystals grown in 100 mM Tris–HCl, pH 8.5, 100 mMLi2SO4, and 30% PEG 3350 were already suspended in a

190 A. Gupta et al. / Protein Expression and PuriWcation 41 (2005) 186–198

solution appropriate for freezing. Crystals grown in20 mM Tris–HCl, pH 8, 8% PEG 3350, and 10 mMMnCl2 were placed over a well solution containing88 mM MES, pH 6, 35% PEG 3350, and 44 mM MnCl2and allowed to ‘dehydrate’ at 21.5 °C for at least 1 week.Crystals were transferred to nylon loops (Hampton) anddipped into liquid nitrogen before being mounted adja-cent to an Oxford Cryosystems Cryostream and main-tained at 100 K. DiVraction studies were carried out inhouse using a rotating anode Rigaku micromax 7 gener-ator coupled to an Raxis IV++ image plate detector.Data processing was carried out using the CrystalClear/D*TREK software package [34].

Results

IdentiWcation of proteolytically accessible regions in AMA1 ectodomain

Full-length AMA1 ectodomain, extending from thesignal cleavage site to the predicted transmembranedomain, was prepared as described previously [11].BrieXy, this protein was expressed in E. coli, puriWedunder denaturing conditions by Ni aYnity chromatogra-phy, refolded by rapid dilution, and puriWed by ionexchange and reverse phase chromatography. AMA1ectodomain was treated with serial dilutions of elastasein order to identify proteolytically accessible regions onthe protein. Elastase was used because it is relativelynon-speciWc, having a preference for cutting immediatelyafter amino acids with small non-polar side chains. Thebest results were obtained if AMA1 was treated with alarge amount of protease for a short period, typically 10minutes. Results of a representative experiment are showin Fig. 1. Elastase cleaved full-length AMA1 ectodomainwith an apparent molecular weight of 70 kDa to yield asingle sub-fragment with an apparent weight of approxi-mately 50 kDa. Similar results were obtained with adiVerent protease, subtilisin. However, subtilisin wasmore eYcient at proteolyzing AMA1 and increasingamounts of subtilisin resulted in the complete destruc-

Fig. 1. Treatment of full-length AMA1 ectodomain with elastase.Threefold serial dilutions of elastase were added to 2 �g of AMA1 andnon-reduced samples separated by SDS–PAGE.

tion of AMA1 ectodomain (data not shown). Theseresults indicated that AMA1 mostly consisted of a rela-tively non-Xexible region that was protease resistant butalso contained more exposed proteolytically accessibleregions.

To identify the elastase susceptible sites within theAMA1 ectodomain, the proteolysis reaction was scaledup, and the resulting sub-fragment puriWed by reversephase chromatography. N-terminal sequencing yieldedthe sequence at position 104 of the P. falciparumsequence, 104-NYMGNPYT. [In hindsight the N-termi-nal sequencing reaction should have yielded threesequences, but one sequence was dominant.] Mass spec-trometry indicated that the size of the fragment was44.225 kDa. The elastase treated AMA1 was examinedby reduced SDS–PAGE and shown to consist of threefragments of apparent molecular weights of approxi-mately 30, 10, and 7 kDa. Reduction and puriWcation ofAMA1 by reverse phase chromatography yielded thetwo small fragments. For reasons that are not clear wewere unable to isolate the 30 kDa fragment. N-terminalsequencing and mass spectrometry were used to identifythe smaller fragments: an N-terminal 385-FKADRYK-SHG, 9.013 kDa fragment, and an N-terminal 461-KLNDNDDEGN 6.287 kDa fragment. These resultswere consistent with the smaller fragments consisting ofthe peptides extending from 385-FKADR to ESKRI-460 (9.008 kDa) and from 461-KLNDN to RRAEV-516(6.294 kDa) within the AMA1 sequence. Fortuitously,the N-terminal sequence that dominated for the non-reduced fragment corresponded to the N-terminalsequence of the missing 30 kDa reduced fragment, andfrom the size of the non-reduced fragment (44.225 kDa)it was possible to deduce the composition of the 30 kDaelastase-resistant portion of AMA1: 104-NYMGN toKQYEQ-355 which has a molecular weight of28.926 kDa. The positions of the elastase resistant frag-ments in relation to the disulphide positions are shownin Fig. 2.

Four regions of AMA1 ectodomain were identiWed asbeing susceptible to AMA1 proteolysis. The cleavage siteat the N-terminus (before NYMGN) removed a longprosequence that is unique to P. falciparum and P. rei-chenowi AMA1 [35]. The cleavage site at the C-terminus(RRAEV-516) is seven amino acids downstream of thelast disulphide bonded cysteine in domain III. There wasa single clip after position 460 (SKRI-KLND) withindomain III, and a 32 amino acid fragment extendingfrom 356-HLTD to PTGA-384 was cut from domain II.Since performing this analysis Howell et al. [26,36] haveidentiWed the cleavage sites of AMA1 that occur duringerythrocyte invasion. The natural cleavage sites were insimilar but not identical locations to the elastase sites.The natural prosequence cleavage site was seven aminoacids N-terminal to the elastase site. The C-terminal sitewas one amino acid C-terminal to the elastase site. The

A. Gupta et al. / Protein Expression and PuriWcation 41 (2005) 186–198 191

elastase clip in domain III was three amino acids N-ter-minal to a clip in domain III that occurs in 30% ofAMA1 released into culture supernatant. We do notascribe any physiological relevance to these coinci-dences, but these results were useful to us in that theyconWrmed our Wndings and indicated that that thesewere proteolytically susceptible regions of AMA1.

Design of AMA1 expression constructs for protein crystallography

The use of limited proteolysis to identify regions of aprotein that are relatively protease resistant and byinference more likely to crystallize is a relatively wellestablished technique and is documented in a number ofpapers [37,38]. The limited proteolysis results suggestedthat the most promising fragment extended from 104-NYMGN to RRAEV-516, encompassing AMA1domain I + II + III (i.e., full-length ectodomain but with-out the N-and C-terminal sequences removed by elas-tase) (Fig. 2). However, we considered the chances of thisfragment crystallizing were not high because the elastaseresults predicted that there were at least two Xexible cen-tral regions remaining in this construct: the YEQH toPTGA fragment in domain II and the SKRI-KLND clipin domain III. The amino acid sequence adjacent to thedomain III clip is extremely hydrophilic which suggestedthat this region formed a long Xexible loop, subsequentlyconWrmed by the NMR structure of domain III [39].

The AMA1 sequence from Toxoplasma gondii is simi-lar in domains I + II, but divergent in domain III [4,5].Similarly, the AMA1-related gene, MAEBL, is homolo-gous in domains I + II but not domain III [40]. Thisobservation suggested that domain I + II formed anindependently folded unit. In addition, Hodder and oth-ers were able to express and refold isolated domain IIIindicating that domain III was an independently foldingunit [39]. This suggested to us that ‘domains I + II + III’in fact consisted of two independently folding domains:I + II and III. We therefore designed a second construct

consisting of domains I + II. The designed C-terminus ofthe domain I + II was an educated guess based on analignment of AMA1 sequences in the domain II to IIIlinker. We decided on a C-terminus (terminating withPIEVE-438) Wve amino acids N-terminal to the Wrstdomain III cysteine. The domain I + II fragment ulti-mately turned out to be crystallizable but its solubilitywas relatively low (3 mg/ml in water at 4 °C), whereas thesolubility of domain I + II + III was high (>20 mg/ml inwater at 4 °C). This suggests that although domain I + IIfolds independently of domain III there are contactsbetween the two folding units.

Protein expression and refolding of AMA1 fragments

AMA1 domains I + II and I + II + III fragments werecloned into the bacterial expression vector,pPROEXHTb, containing an N-terminal hexa-histidinetag followed by a TEV protease cleavable linker. Expres-sion from a non-tagged vector was also attempted butthis resulted in translation initiation at internal methio-nines, 214-MIPDN and 273-MFCFR, and refolding ofthe non-tagged protein, although possible, requiredmore stringent conditions and had to be carried out over3 days (Fig. 3 and data not shown).

The internal translation initiation and refolding prob-lems were not encountered with the his-tagged proteins.Both his-tagged domains I+II and I+II+II expressed tohigh levels to yield a single product (Fig. 3). Once a refold-ing protocol had been determined, refolding was reproduc-ible and in fact relatively robust. Virtually all of thecomponents in the refolding buVer used (20mM Tris–HCl,pH 8, 0.5mM EDTA, 1M urea, 200mM NaCl, 2mM2ME, and 0.2mM cystamine–hydrochloride) could bechanged. The pH could be altered from pH 8.5 to pH 7.5.EDTA (0.5mM) could be dispensed with. 2ME could bevaried from 10 to 0.5mM. Refold conditions were similarfor AMA1 domains I+II although because AMA1domains I+II+III was more soluble it could be refolded ina lower salt concentration (50mM NaCl) (data not shown).

Fig. 2. Diagram of AMA1 showing the disulphide connectivities within each ‘domain’ and the signal and transmembrane (TM) sequences. The threefragments that were resistant to elastase (limited proteolysis) are shown along with the positions of the two open reading frames used in attempts tocrystallize AMA1.

192 A. Gupta et al. / Protein Expression and PuriWcation 41 (2005) 186–198

Refolding was principally monitored by examiningthe refolded proteins by non-reduced SDS–PAGE.Unsuccessful refolds result in an increased fraction ofhigh molecular weight disulphide-linked multimericAMA1 aggregates. However, as is discussed below, wealso detected misfolded AMA1 variants that weremomomeric by gel Wltration chromatography and co-migrated with refolded AMA1 on non-reduced SDS–PAGE gels. Monomeric misfolded AMA could bedetected to a limited extent by SDS–PAGE by examin-ing band diVuseness. For example, increasing the con-centration of urea to 2 M or greater increased the yieldof refolded protein but resulted in a more ‘fuzzy’ bandthat we assumed resulted from diVerentially foldedAMA1 variants (data not shown). Similarly, reducingthe concentration of cystamine resulted in a more diVuseband, and, attempts to refold the AMA1 fragments byrapid dilution resulted in more diVuse bands. A suY-ciently low concentration of AMA1 was necessary foreYcient folding. Increasing protein concentrations togreater than 20 �g/ml increased the fraction of multi-meric disulphide-linked protein. This severely restrictedthe scaling up of this procedure. It was only possible torefold 1 mg of protein per dialysis bag. A typical protein

Fig. 3. Protein expression from AMA1 ‘crystallography constructs’.His6-AMA1 I + II consists of domains I + II cloned into the His tagvector pPROEXHTb. Similarly, His6-AMA1 I + II + III consists ofdomains I + II + III cloned into pPROEXHTb. AMA1 I + II wasexpressed from vector, p�8a, that results in non-tagged protein. Alter-nate lanes result from bacteria that were grown in enriched media orLuria broth. Inclusion bodies were washed with buVer containing 0.1%Triton X-100 and reduced samples were separated by SDS–PAGE.

preparation therefore required that at least 40 50 mlrefolds were carried out to yield approximately 2 L ofdilute refolded protein. An example of a successful pro-tein refolding experiment is shown in Fig. 4. In this case4 £ 50 ml refolds are analysed by non-reduced andreduced SDS–PAGE. The resulting non-reduced bandsare not diVuse and are virtually identical to the reducedbands indicating that most material is refolding success-fully. In addition there was little batch variability indi-cating that the refolding was reproducible.

Protein puriWcation

Details of the protein puriWcation protocols used forAMA1 domains I + II and I + II + II are described inMaterials and methods. A single gel showing the puriW-cation of one batch of AMA1 I + II + III is shown inFig. 5. PuriWcation protocols for the AMA1 domainI + II and I + II + III were essentially identical. In a typ-ical protein puriWcation the starting point was 40 mg ofrefolded protein in 2 L of 20 mM acetic acid–NaOH,pH 5, 0.5 mM EDTA, and 0.02% sodium azide. Therefolded protein was puriWed over SP-Sepharose

Fig. 4. Example of four successful refold experiments monitored bynon-reduced and reduced SDS–PAGE sample separations. Fivemicrolitres of refolded His6-AMA1 I + II + III is added per lane. Pre-refolded His6-AMA1 I + II + III separated non-reduced by SDS–PAGE in a diVerent gel is shown to highlight the disappearance ofmost disulphide-bonded aggregates.

Fig. 5. PuriWcation of AMA1 I + II + II. Approximately 1/50,000 of thetotal protein preparation at each stage is added per lane and non-reduced samples separated by SDS–PAGE. The last 3 lanes contain1 �l samples of the three peaks separated over mono S beads.

A. Gupta et al. / Protein Expression and PuriWcation 41 (2005) 186–198 193

(essentially a concentration step) and eluted into20 mM Tris–HCl, 0.5 M NaCl. The protein was thenpuriWed over a Co2+ nitrilotriacetic acid aYnity col-umn (Amersham–Pharmacia). The principal refoldfraction eluted in 120 mM imidazole, whereas approxi-mately 10% of the protein eluted in 20 mM imidazole(Fig. 6A). Analysis of this fraction by non-reducedSDS–PAGE suggested that it contained a mixture ofimpurities, but mostly consisted of AMA1 of the cor-rect size but was presumably misfolded such that thehexahistidine tag was no longer exposed (data notshown).

Following Co2+ aYnity chromatography the hexa-histidine tag was removed by TEV protease (Fig. 5).We have not attempted to purify and crystallize thetagged protein. The tag in the expression vector used,pPROEXHTb, is relatively long (23 amino acids) andwe anticipated that this would inhibit cystallization.TEV-cleaved proteins were then puriWed over the highresolution ion exchange columns, mono Q and mono S,with a buVer exchange/gel Wltration step in between.For the mono Q puriWcation proteins were dialysedinto 20 mM Tris–HCl, pH 8 and eluted in a NaCl gradi-ent. The principal folded component consisted ofapproximately 80% of loaded protein and eluted in80 mM NaCl (Fig. 6B). The remainder of the protein

Fig. 6. 280 nm absorbance proWles resulting from AMA1 eluting froma variety of columns. (A) Elution from a Co2+ aYnity column in20 mM Tris, pH 8, 0.5 M NaCl with a 0–400 mM imidazole gradient.The 140 and 20 mM peaks refer to the imidazole concentration inwhich the protein is eluting. (B) Elution from a mono Q column in20 mM Tris, pH 8 with a 0–1 M NaCl gradient. The 80 and 150 mMpeaks refer to the concentration of eluting NaCl. (C) Chromatogramshowing the elution of AMA1 I + II + III from an S200 size exclusioncolumn. The position of elution of 158 and 44 kDa markers is shown.

eluted in a broad 100–300 mM NaCl peak, with a smallprotein spike eluting in 150 mM NaCl (Fig. 6B).Reloading of fractions of this broad peak yielded thesame broad peak, conWrming that it was chromato-graphically distinct from the principal 80 mM NaClpeak (data not shown). Examination of the 100–300 mM NaCl peak by non-reducing SDS–PAGE indi-cated that it consisted of multimeric disulphide-linkedmisfolded impurities (data not shown). The sharp150 mM NaCl peak contained AMA1 that was appar-ently monomeric by non-reduced SDS–PAGE. Thiswas conWrmed by size-exclusion chromatography (datanot shown). As expected the refolding reaction gener-ated misfolded AMA1 variants that formed aggregates.However, the monomeric nature of the protein in the150 mM NaCl peak indicated that it was possible for asingle AMA1 molecule to refold to generate diVerentsoluble chromatographic forms.

Following mono Q puriWcation proteins were puri-Wed and buVer exchanged into 20 mM acetic acid–NaOH, pH 5.5 using a Sephacryl S200 size-exclusioncolumn. AMA1 I + II + III comigrated with a 45 kDasize-exclusion marker indicating that the protein wasmostly monomeric (Fig. 6C). AMA1 I + II elutedslightly anomalously with an apparent molecularweight of 25 kDa, and is presumably binding slightly tothe column (data not shown). For both I + II + II andI + II dimeric impurity was detected, approximately 3%of the protein (Fig. 6C).

PuriWcation of the monomeric AMA1 was then car-ried out over mono S beads in pH 5.5 acetic acid–NaOHwith a NaCl gradient. AMA1 I + II + III eluted in twomajor peaks: at 150 mM NaCl and in 180 mM NaCl

Fig. 7. Comparison of the puriWcation of AMA1 domains I + II + IIIand I + II over mono S beads. 280 nm absorbance resulting fromAMA1 eluting from the mono S column in a 0–1 M NaCl gradient.Twenty millimolar acetic acid–NaOH, pH 5.5 was used as a buVer. Theconcentration of eluting NaCl is shown for each peak.

194 A. Gupta et al. / Protein Expression and PuriWcation 41 (2005) 186–198

(Fig. 7). However, a detailed examination of the chro-matographic proWle indicated that there were otherminor peaks. There was a small peak just before the150 mM peak, a peak between the 150 and 180 mMpeaks, and the 180 mM peak was asymmetric suggestingthat it contained more than one component. Remark-ably, reloading of each of the individual peaks onto themono S column resulted in the same peak, conWrmingthat the each peak contained a chromatographically dis-tinct protein (data not shown). Each peak containedrefolded AMA1 that was indistinguishable by non-reduced SDS–PAGE (Fig. 5).

A similar proWle was observed when AMA1 I + II waspuriWed over mono S beads (Fig. 7). There were twomain peaks eluting in 160 and 190 mM NaCl, and minorpeaks in between. The 190 mM peak was narrower andmore symmetrical than the AMA1 I + II + III 180 mMpeak suggesting that it contained one component. Anal-ysis of these peaks indicated that they containedrefolded AMA1 that was indistinguishable by non-reduced SDS–PAGE (data not shown). These resultsconWrmed the observation that AMA1 fragments couldrefold to generate distinct forms that werechromatographically distinct on ion-exchange columnsbut identical when examined by SDS–PAGE.

Analysis of chromatographically distinct AMA1 species by analytical reverse phase HPLC

Ion exchange puriWcation protocols identiWed thepresence of chromatographically distinct forms ofrefolded AMA1 that must presumably have a diVerentdistribution of charges on the protein surface. Exami-nation of these distinct forms by denaturing techniquessuch as SDS–PAGE (Fig. 9) suggested that the distinctforms were identical in size, and this was conWrmed bymass spectrometry (data not shown). Reduced disul-phide-linked proteins typically bind more tightly tohydrophobic columns than non-reduced proteins dueto the diVerential exposure of hydrophobic residues.We therefore hoped to probe the internal disulphidebond structure of the diVerent AMA1 forms by analyz-ing their chromatographic behaviour by reverse phaseHPLC. As shown in Fig. 8, AMA1 I + II + II elutedfrom a C4 column in 38% acetonitrile, whereas reducedAMA1 I + II + III eluted in 41% acetonitrile. Thesewere distinct peaks because a mixture of reduced andnon-reduced protein eluted as two peaks. All of themonomeric AMA1 I + II + III species eluted from a C4column at an identical position, in 38% acetonitrile(data not shown). This suggested that the burial ofhydrophobic groups by the disulphide-bond structurein the diVerent AMA1 I + II + III fractions was similar.Virtually identical results were observed for AMA1I + II except that AMA1 I + II eluted in 39% acetoni-trile (data not shown).

Binding of refolded and puriWed AMA1 fragments to monoclonal antibody 1F9

A major diYculty in working with malaria antigenswith roles in erythrocyte invasion is that the role of mostproteins is unknown and a functional assay is currentlynot possible. However, it is possible to demonstratecross-antigenicity between refolded AMA1 and parasitederived AMA1 [11,32], indicating that at least the struc-ture of certain epitopes is preserved. Of particular inter-est are the epitopes that are critical to AMA1 function,and are recognized by growth- or invasion-inhibitory

Fig. 8. Analysis of puriWed AMA1 I + II + III 165 mM mono S fractionby reverse phase HPLC. 280 nm absorbance resulting from AMA1elution from a C4 column in 0.05% TFA with a 0–100% acetonitrilegradient. The percentage of eluting acetonitrile is indicated. Proteinwas reduced by the addition of Tris, pH 8 buVer, 6 M GndHCl, and30 mM 2ME for 30 min at 37 °C. A mixture of reduced and non-reduced protein is shown in comparison to demonstrate that there aretwo peaks. Addition of 0.05% TFA prevents reduction after mixing.

Fig. 9. Native gel electrophoresis demonstrating the binding of inhibi-tory monoclonal antibody 1F9 to all puriWed AMA1 fractions. Twomicrograms of 1F9 was added to lanes 1, 2, 4, 6, 8, and 10. A molarexcess of AMA 1 domains I + II + III 150 mM mono S fraction wasadded to lanes 7 and 8 with the 180 mM fraction added to lanes 9 and10. Note that even though these proteins have the same mass and iden-tical chromatographic behaviour apart from on a mono S column theyhave distinct mobilities. A molar excess of AMA1 I + II 190 mM monoS fraction was added to lanes 2 and 3. As a negative control a refoldedfragment from the P. falciparum antigen EBA175 was added to lanes 4and 5.

A. Gupta et al. / Protein Expression and PuriWcation 41 (2005) 186–198 195

antibodies. One such antibody, 1F9 [41], was tested forits ability to bind to the various AMA1 preparationsdescribed above (Fig. 9). In this assay 1F9, the AMA1fragments and the 1F9–AMA1 complexes were sepa-rated by native gel electrophoresis. 1F9 migrated a shortdistance in the native gel, whereas AMA1 fragments,because they were smaller and with a larger negativecharge moved faster. Complex formation resulted in alower abundance of AMA1 fragment and the AMA1–1F9 complex migrated very slightly faster than uncom-plexed 1F9 in a band which was clearly more intense andless diVuse than 1F9 alone (Fig. 9). As a control, 1F9 isshown not to interact with a refolded subfragment fromthe P. falciparum EBA175 protein. Interestingly, theAMA1 I + II + III 150 and 180 mM fractions migrateddiVerently by native gel electrophoresis (Fig. 9). The150 mM band migrated faster in the gel buVered at pH7.5, indicating that it had a more negative charge. At pH5.5 the 150 mM fraction bound less well to mono S beadsconsistent with the protein in the150 mM fraction havinga more negative charge. These results conWrmed thatAMA1 was refolding to form chromatographicallydistinct heterogeneous forms.

Crystallization of AMA1

Crystallizations were mostly attempted with themajor fractions of AMA1 I + II and I + II + III, the 180–190 mM mono S peaks. We were unable to obtain crys-tals of the I + II + III protein, presumably because thisprotein contains too many Xexible regions. In contrast,AMA1 I + II was highly crystallizable. Small twinned orclumped crystals were obtained using many PEG orPEG + salt conditions. Obtaining individual crystalsproved to be considerably more diYcult and we havehad most success with two conditions: 20 mM Mes, pH6, 8% PEG 3350, 10 mM MnCl2 or 100 mM Tris–HCl,pH 8.5, 100 mM Li2SO4, 30% PEG 3350. It was diYcultto reproducibly grow large untwinned crystals in100 mM Tris–HCl, pH 8.5, 100 mM Li2SO4, and 30%

PEG 3350, and we have only obtained one crystal thatdiVracted X-rays to high resolution. This crystaldiVracted X-rays in spacegroup C2, unit cell 109.1, 38.4,and 71.7 Å, 90°, 93.7°, and 90°, and a mosaicity of 0.9°.Data collected using in house equipment extended to2.0 Å with a 6.5% overall R merge (33% outer shell),99.5% completeness with a 3.6-fold redundancy.

Large crystals (at least 0.05 mm in the smallest dimen-sion) could be reproducibly grown in 20 mM MES, pH 6,10 mM MnCl2, and 8% PEG 3350. However, if thesecrystals were placed directly into cryosolution (20 mMMes, pH 6, 10 mM MnCl2, and 35% PEG 3350) theydiVracted X-rays very poorly to 5 Å resolution (see Fig.10). A slow dehydration procedure, placing the crystalsover 88 mM Mes, pH 6, 44 mM MnCl2, and 35% PEG3350 for 1 week improved diVraction to 2.5–2 Å resolu-tion. Our best crystal diVracted X-rays to 1.9 Å with amosaicity of 0.3°. We have collected a dataset that is 99%complete with a 10-fold redundancy, spacegroup p31,unit cell 54.1, 54.1, and 214.0 Å, 90°, 90°, and 120°, over-all Rsym 5.0%, outer shell Rsym 30% at beamline X25 atthe National Synchrotron Light Source at Brookhaven(in collaboration with Dr. Michael Becker).

Discussion

The only malaria antigen thus far expressed and puri-Wed successfully for crystallographic studies is the C-ter-minal 11 kDa fragment of MSP1 with an apparentmolecular weight of 19 kDa, MSP119 [42]. Using the bac-ulovirus secretory system, Chitarra et al. [42] expressedthe Plasmodium cynomolgi MSP119 in a folded solubleform that was not glycosylated in spite of the presence ofone potential Asn-Met-Thr glycosylation site. In thecrystal structure of MSP19, the Asn and Thr within theAsn-Met-Thr sequence are solvent accessible andthe reason for this site not being glycosylated is notclear. Similarly, Garman et al. [43] successfully expressedand crystallized MSP119 from P. knowlesi using a

Fig. 10. Crystallization of AMA1 domains I + II and resulting diVraction pattern. (A) Example of a crystal grown in 20 mM MES, pH 6, 10 mMMnCl2, and 8% PEG 3350. (B) The resulting poor diVraction if the crystal is transferred directly into 25% PEG 3350. (C) Crystals slowly dehydratedinto 35% PEG consistently diVracted X-rays to high resolution with low mosaicity.

196 A. Gupta et al. / Protein Expression and PuriWcation 41 (2005) 186–198

Saccharomyces cerevisiae secretory expression system.This protein contained one glycosylation site close to theN-terminus of the protein. This region was disordered inthe structure and it is uncertain whether the site was infact glycosylated. Clearly, the use of a secretory expres-sion system does not always result in aberrant glycosyla-tion or glycosylation that is problematic forcrystallization.

There are several reported instances of aberrant gly-cosylation of malaria antigens expressed using secretoryexpression systems. Kocken et al. [29] expressed P. vivaxAMA1 in Pichia pastoris and detected heterogeneousglycosylation. Hodder et al. [28] expressed P. falciparumAMA1 using the baculovirus system and detected glyco-sylation. One possible solution to the glycosylationproblem is to mutate potential N-linked glycosylationsites. Mutation of two potential glycosylation sites (glu-tamine in place of asparagine) converted non-antigenicMSP142 into an antigenic form of the protein expressedin mouse milk [30]. Similarly, N-linked glycosylationsites were mutated in P. falciparum AMA1 to allowexpression of antigenic protein in P. pastoris [22,29].However, this did not fully prevent glycosylation asAMA1 expressed in P. pastoris was observed to includeantigen thought to be modiWed by glycation of lysineresidues [22].

In this paper, we have described a diVerent approachto the glycosylation problem, expression of protein inbacteria followed by refolding. In this procedure, AMA1expressed in bacteria is dissolved in GdnHCl andrefolded by dialysis into buVer containing 2 mM of 2-mercaptoethanol and 0.2 mM cystamine–HCl andpuriWed extensively using a 5 column protocol. Similarprocedures have been used previously [32,44] wheredenatured AMA1 was puriWed by Ni2+ chromatographyand refolded by dilution into a buVer containing reducedand oxidized glutathione. The protocol used here diVersfrom these methods in two ways. We adopted a dialysisrather than a rapid dilution refolding approach, andelected to purify by metal aYnity chromatography fol-lowing rather than preceding refolding (discussedbelow).

A refolding approach has been used successfully togenerate other malaria antigens apart from AMA1.Singh et al expressed P. vivax DuVy binding protein inbacteria, puriWed by Ni2+ chromatography in GdnHCl,and refolded by rapid dilution into 50 mM phosphate,pH 7.2, 1 mM reduced glutathione, 0.1 mM oxidized glu-tathione, 1 M urea, 0.5 M arginine. Pan et al. adopted anovel approach to generate soluble MSP-1. MSP-1expressed in bacteria was dissolved in GdnHCl andloaded onto a Ni2+ column but was then subjected to areducing urea gradient before being eluted in 50 mMTris–HCl, pH 7, 10% glycerol with an imidazole gradi-ent. Remarkably this protein formed a single discreteband by non-reduced SDS–PAGE.

In the refolding/puriWcation protocol developed herewe paid close attention to procedures that removed mis-folded or alternatively folded variants of AMA1 thatwere likely to inhibit crystallization. We found the mostuseful assay for monitoring alternative folding was non-reduced SDS–PAGE. Misfolded variants could be iden-tiWed on the basis of formation of higher molecularweight disulphide linked aggregates but also from thediVuseness of the monomeric band. We chose to refoldby dialysis because rapid dilution protocols resulted inslightly more diVuse bands by non-reduced SDS–PAGE.In addition, following refolding the protein was dialysedinto salt free buVer, which was useful in that a signiWcantfraction of the misfolded AMA1 aggregates precipitatedout of solution. We also considered it useful to performthe metal aYnity chromatography step following refold-ing because the principal impurity that eluted in 20 mMimidazole was misfolded AMA1 as judged by non-reduced SDS–PAGE. The most critical step in removinghigh molecular weight disulphide-linked aggregates wasthe puriWcation over mono Q beads (Figs. 5, 6B). Thebulk of monomeric refolded AMA1 eluted in 80 mMNaCl whereas multimeric AMA1 eluted in a broad 100–300 mM NaCl peak.

Interestingly, we identiWed several AMA1 variantsthat appeared to be correctly folded, as judged by non-reduced SDS–PAGE. The 150 mM NaCl mono Q frac-tion consisted of monomeric, correctly folded AMA1 asjudged by size exclusion chromatography, non-reducedSDS–PAGE and analytical reverse phase HPLC. Simi-larly, we identiWed AMA1 variants that were chromato-graphically distinct over mono S beads in a pH 5.5 saltgradient. These variants were identical as judged by non-reduced SDS–PAGE, size exclusion chromatography,reverse phase HPLC, and mass spectrometry but dis-played distinct electrophoretic mobility in non-denatur-ing polyacrylamide gels (Fig. 9). This diVerence inbehaviour may be due to a charge diVerence. Fractionsthat migrated faster by native gel electrophoresis (morenegatively charged) eluted, as would be expected, earlierfrom mono S beads in a salt gradient. One possibility isthat the less negatively charged species has been deami-nated at a lysine residue, and a terminal amine has beenconverted to an alcohol group. Another possibility isthat the 150 mM mono S species have an apparentlylower charge because they are partially misfolded andmigrate slower in native gels because they are less com-pact. This is considered unlikely because the overallcharge diVerences were also observed by isoelectricfocussing, a technique that is not dependent on the over-all shape of the protein (data not shown). Yet anotherpossibility is that the proteins are diVerentially foldedsuch that the local environment of a histidine residue isaltered such that it is diVerentially charged. Consistentwith this suggestion is the Wnding that the 150 mM monoS species do not concentrate eYciently and appeared to

A. Gupta et al. / Protein Expression and PuriWcation 41 (2005) 186–198 197

stick to the 5 kDa membrane during concentration. Thismay be because this protein fraction is partiallymisfolded and possesses more exposed hydrophobicresidues.

The bacterial expression/refolding approach oVersmany advantages for the generation of non-glycosylatedextracellular proteins. Most notably, ease of expression.However, our experience has shown that there is consid-erable microheterogeneity in the refolded protein prepa-rations. To generate homogeneous protein preparationsusing this approach a refolding procedure that mini-mizes variation in addition to an extensive puriWcationprotocol is required. By paying attention to these detailsit is possible to produce high quality protein prepara-tions that are suYciently homogeneous to generate crys-tals that diVract X-rays to 2 Å resolution.

Acknowledgments

We acknowledge the assistance of Pauline Crewther,Tony Hodder, Andrew Pearce, and Patrick Dueppen.The initial limited proteolysis experiment was carriedout at the Walter and Eliza Hall Institute when A.H.Bwas supported by a Colin Syme Fellowship.

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