Structureofthe Plasmodiumfalciparum Circumsporozoite ... · min; 85 °C for 5 s; hold at 4 °C. The resulting cDNA was used immediately or stored at 20 °C. The following probe and

Structure of the Plasmodium falciparum CircumsporozoiteProtein, a Leading Malaria Vaccine Candidate*□S

Received for publication, April 27, 2009, and in revised form, July 17, 2009 Published, JBC Papers in Press, July 24, 2009, DOI 10.1074/jbc.M109.013706

Matthew L. Plassmeyer‡, Karine Reiter‡, Richard L. Shimp, Jr.‡, Svetlana Kotova§, Paul D. Smith§, Darrell E. Hurt¶,Brent House�, Xiaoyan Zou�, Yanling Zhang‡, Merrit Hickman‡, Onyinyechukwu Uchime‡, Raul Herrera‡,Vu Nguyen‡, Jacqueline Glen‡, Jacob Lebowitz§, Albert J. Jin§, Louis H. Miller‡, Nicholas J. MacDonald‡, Yimin Wu‡,and David L. Narum‡1

From the ‡Malaria Vaccine Development Branch, NIAID, National Institutes of Health, Rockville, Maryland 20852, §Laboratory ofBioengineering and Physical Science, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health,Bethesda, Maryland 20892, ¶Bioinformatics and Computational Biosciences Branch, Office of Cyber Infrastructure andComputational Biology, NIAID, National Institutes of Health, Bethesda, Maryland 20892, and �United States Navy, Naval MedicalResearch Center, Silver Spring, Maryland 20910

ThePlasmodium falciparum circumsporozoite protein (CSP)is critical for sporozoite function and invasion of hepatocytes.Given its critical nature, a phase III human CSPmalaria vaccinetrial is ongoing.TheCSP is composedof three regions as follows:an N terminus that binds heparin sulfate proteoglycans, a fouramino acid repeat region (NANP), and a C terminus that con-tains a thrombospondin-like type I repeat (TSR) domain.Despite the importance of CSP, little is known about its struc-ture. Therefore, recombinant forms of CSP were produced byexpression in both Escherichia coli (Ec) and then refolded(EcCSP) or in the methylotrophic yeast Pichia pastoris (PpCSP)for structural analyses. To analyze the TSR domain of recombi-nant CSP, conformation-dependent monoclonal antibodiesthat recognized unfixedP. falciparum sporozoites and inhibitedsporozoite invasion of HepG2 cells in vitro were identified.Thesemonoclonal antibodies recognized all recombinant CSPs,indicating the recombinant CSPs contain a properly folded TSRdomain structure. Characterization of both EcCSP and PpCSPby dynamic light scattering and velocity sedimentation demon-strated that both forms of CSP appeared as highly extended pro-teins (Rh 4.2 and 4.58 nm, respectively). Furthermore, high res-olution atomic force microscopy revealed flexible, rod-likestructures with a ribbon-like appearance. Using this informa-tion, we modeled the NANP repeat and TSR domain of CSP.Consistent with the biochemical and biophysical results, therepeat region formed a rod-like structure about 21–25 nm inlength and 1.5 nm in width. Thus native CSP appears as a glyco-sylphosphatidylinositol-anchored, flexible rod-like protein onthe sporozoite surface.

Malaria caused by Plasmodium falciparum is a serious globalhealth issue, resulting in an estimated 1.5 million deaths annu-ally, primarily among infants and young children. Ongoing

multifaceted global intervention strategies to control malariainclude drug treatment, insecticide usage, bed-net use, and vac-cine development. However, parasite and mosquito controlmeasures have met with limited success resulting from anincreased drug and insecticide resistance within the Plasmodiaand mosquito populations, respectively. Vaccine developmentrepresents an encouraging approach given that previous animaland human studies using irradiated sporozoites demonstratedthe feasibility of producing an efficacious vaccine (1–3). Al-though the exact immunologic correlates of protection remainelusive, an abundance of evidence indicates that protectionagainst liver stage parasites is complex, involving multipleimmune mechanisms (4–11).To date, the majority of the pre-erythrocytic stage vaccine

development has focused on the circumsporozoite protein(CSP),2 the predominant surface antigen on sporozoites. CSPcan be segmented into three regions as follows: the N-terminalregion containing region I; the central repeat region; and theC-terminal region containing the thrombospondin-like type Irepeat (TSR). Initial CSP vaccine development focused on thecentral repeat region that contains the immunodominant B cellepitope (12). However, vaccine constructs quickly evolved toincorporate both the central repeat region containing the B cellepitopes and the C terminus containing the TSR domain, T cellepitopes, and B cell epitopes (13, 14). Currently, the mostadvanced and moderately effective malaria vaccine, RTS,S, iscomposed of a portion of the central repeat and the C-terminalregions linked to the hepatitis B surface antigen (15). However,recent studies have highlighted the physiological importance ofthe N-terminal region (16–19). Rathore et al. (19) not onlydemonstrated the role of the N-terminal region in liver cell

* This work was supported, in whole or in part, by National Institutes of Health(Intramural Research Program, including NIAID and National Institute ofBiomedical Imaging and Bioengineering).

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Fig. 1.

1 To whom correspondence should be addressed: Malaria Vaccine Develop-ment Branch, 5640 Fishers Ln., Twinbrook 1, Rockville, MD 20852. Tel.: 301-435-2185; Fax: 301480-1962; E-mail: [email protected].

2 The abbreviations used are: CSP, circumsporozoite protein; TSR, throm-bospondin-like type I repeat; mAb, monoclonal antibody; ISI, inhibition ofsporozoite invasion; ECM, Eagle’s essential minimum complete media;DTT, dithiothreitol; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymeth-yl)propane-1,3-diol; PBS, phosphate-buffered saline; SEC-MALS-QELS-HPLC, size exclusion chromatography-multiangle light scattering-quasi-elastic light scatter detection; high pressure liquid chromatography; qRT,quantitative real time; r, recombinant; AFM, atomic force microscopy;EcCSP, E. coli CSP; PpCSP, P. pastoris CSP; ESI-MS, electrospray ionization-mass spectrometry; -ML, mechanical microfluidization lysis; -CL, chemicaldetergent lysis.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 39, pp. 26951–26963, September 25, 2009Printed in the U.S.A.

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attachment but also identified alongwithAncsin andKisilevsky(16) an epitope within the N-terminal region that interactedwith liver cells through heparin sulfate (18). Moreover, thisepitope was not only found to be immunogenic but the result-ing antibodies were determined to be inhibitory in a sporozoiteinvasion assay (18). Peptides corresponding to the N-terminalregion (PpCS-(22–110) and PpCS-(65–110)) were also recog-nized by sera obtained from individuals living in malaria-en-demic regions (17).To better understand the structure of CSP and to produce

good quality recombinant protein for human vaccine-di-rected studies, we generated full-length and near full-lengthrecombinant CSP. We examined two expression systems,Escherichia coli and Pichia pastoris, to determine their fea-sibility to generate CSP. To assist the characterization of therCSPs, we generated a panel of monoclonal antibodies (mAbs)that were characterized biologically prior to being used toexamine the rCSPs. Additionally, each of the rCSP moleculeswas extensively biochemically and biophysically characterized.The results collated together have enabled the molecular mod-eling of CSP as a long flexible, rod-like protein.

EXPERIMENTAL PROCEDURES

Antibody Production—To generate antibodies toward CSP,we utilized a Saccharomyces cerevisiae-expressing CSP (ScCSP)clone generated by David Kaslow.3 The 3D7 DNA sequence(GenBankTM accession number XM_001351086) correspond-ing to CSP amino acids Phe20 to Ser384 was cloned into theSaccharomyces expression vector YEpRPEU3 using the cloningsitesNheI andApaI. The resulting transcribed gene contained aHis6 tag. The sequence of CSP was verified prior to fermenta-tion. Fermentation material was produced as described previ-ously (20) and purified using a two-step purification scheme:nickel affinity chromatography followed by size exclusion chro-matography. Purified ScCSP was supplied to A&G Pharmaceu-tical, Inc. (Columbia, MD) to generate a panel of mAbs. Micewere boosted twice with a fragment ScCSP with an N-terminalsequence beginning at amino acid Ser301 (GenBankTM acces-sion number XM_001351086). Hybridoma supernatants werescreened byWestern blot against sporozoite lysates and confo-calmicroscopy of live sporozoites. Antibodies determined to bereduction-sensitive were isotyped and subclassed using theThermo Scientific Pierce rapid isotyping kit per the manufac-turer’s instructions (Thermo Fisher Scientific).Inhibition of Sporozoite Invasion (ISI) Assay—To ascertain

whether or not the mAbs generated toward the ScCSP had bio-logical significance, they were examined using an in vitro assayto determine their ability to inhibit sporozoite invasion ofHepG2 cells. In brief, analysis of the invasion of HepG2 cloneA16 cells (21) by P. falciparum sporozoites (strain NF54) in thepresence or absence of test anti-CSP IgG was performed usingqRT-PCR to determine the percent invasion of theHepG2 cells.The details for the ISI assay are provided below.ISI Assay, Preparation of Host Hepatocytes and P. falciparum

Sporozoites—Each well of a 48-well plate (Nalge Nunc Interna-tional, Rochester, NY) was coated with ECL cell attachment

matrix (catalog number 08-110, Millipore, Billerica, MA) inHanks’ balanced salt solution (Invitrogen) at 37 °C for 1 h. ThisECL solution was aspirated immediately prior to adding theHepG2 cells.HepG2 cells were maintained in Eagle’s essential minimum

complete media (ECM), Eagle’s essential minimum media(Invitrogen) containing 10% fetal bovine serum (Sigma), 1%100� L-glutamine, and 1% penicillin/streptomycin (10,000units/ml) (Invitrogen). In preparation for seeding, HepG2 cellswere trypsinized, washed, and resuspended in ECM to a finalconcentration of 300,000 cells/ml. After aspiration of ECL solu-tion, 300 �l of the HepG2 cell suspension was added to eachwell and incubated overnight at 37 °C.P. falciparum sporozoites (strain NF54) were grown in

Anopheles stephensi adult female mosquitoes until harvest.Mosquitoes were collected in a cylindrical chamber, placed at�20 °C for 5–10 min, and soaked in 70% ethanol. They werethen serially washed in Dulbecco’s modified Eagle’s medium(Invitrogen) containing penicillin/streptomycin plus anti-biotic-antimycotic (Invitrogen) and ECM. Each mosquito wasdissected by separating the head from the thorax and remov-ing the salivary glands from the head and/or thorax. Thesalivary glands were stored in ECM at room temperatureuntil use. After all mosquitoes were dissected, the salivaryglands were pooled and disrupted by passing through a 281⁄2-gauge needle. The sporozoites released from the salivaryglands were counted and diluted in ECM to a final concen-tration of 500,000 sporozoites/ml.ISI Assay, Sporozoite Invasion of HepG2 Cells, and Prepara-

tion of HepG2 Cells/Parasites for RNA Isolation—Each serumsample to be tested was diluted in ECM to 2� the desired finalconcentration. The anti-sporozoite (CSP) antibody, navy fal-ciparum sporozoite antibody 1 (NFS1) (developed by immuniz-ingmicewithwhole sporozoites), used as a positive control, wasdiluted to a concentration of 200 �g/ml. For each well, 55 �l ofthe test or control serumwasmixedwith 55�l of the sporozoitesuspension and incubated at room temperature for 20min. Oldmedium was aspirated, and then 100 �l of each sporozoite/serummixture was added to each well and incubated for 3 h at37 °C. After the 3-h invasion incubation, each well was washedtwice with fresh ECM and incubated overnight (15–21 h) at37 °C.To generate a standard curve for the qRT-PCR, sporozoites

were serially diluted in ECM (4,860 to 20 sporozoites), added toHepG2 monolayers. Total host-cell/parasite material was col-lected and stored at�80 °C until RNA extraction. Following anovernight incubation at 37 °C, HepG2 cells were washed atroom temperature, trypsinized at 37 °C, and transferred to anRNase-free microcentrifuge tube. Free sporozoites were re-moved by twice pelleting the host cells at 1500 rpm for 2 min.The pellet was stored at �80 °C or used directly for RNAextraction.ISI Assay, RNA Extraction, and qRT-PCR—RNA was ex-

tracted and isolated from host cell/parasite material using theRNeasy mini kit (Qiagen, Valencia, CA), and RNA quality wasassessed in a GeneQuant Pro DNA/RNA calculator (GEHealthcare) using quartz capillaries (GE Healthcare). OnlyRNA with an A260/280 ratio greater than 1.9 was used in further3 D. Kaslow, unpublished data.

Structure of Plasmodium falciparum CSP

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experiments. RNAwas uniformly diluted to�100 ng/ml, addedto PCR plates, and then transcribed to cDNA using the highcapacity cDNA reverse transcription kit (Applied Biosystems,Foster City, CA). Reaction volumes of 20 �l were used in aThermal Cycler (MJ Research Inc., Waltham, MA) set at thefollowing amplification cycle: 25 °C for 10 min; 37 °C for 120min; 85 °C for 5 s; hold at 4 °C. The resulting cDNA was usedimmediately or stored at �20 °C.The following probe and primers were used to detect parasite

18 S rRNA:Taqman�MGBprobe (Applied Biosystems, sequence5�-6FAM-CAGGTCTGTGATGTCC-MGBNFQ-3�); sequencedetection primer 1 (Applied Biosystems, sequence 5�-TAA CACAAG GAA GTT TAA GGC AAC A-3�); sequence detectionprimer 2 (AppliedBiosystems, sequence 5�-CGCGTGCAGCCTAGT TTA TCT-3�). To prepare the qRT-PCR plate, cDNA(�15–20 ng/ml), probe (250 nM), and primers (600 nM) weremixedwith TaqMan� Fast Universal PCRMasterMix (AppliedBiosystems) to a total volume of 20 �l per reaction in a Micro-Amp Fast Optical 96-well reaction plate (Applied Biosystems).The reaction plate was then placed in a 7500 Fast Real TimePCR system (Applied Biosystems) to quantify parasite 18 SrRNA using the default thermal cycling conditions as follows:stage 1 enzyme activation, 95 °C for 20 s; stage 2 (repeated 40times), melting at 95 °C for 3 s, annealing, and polymerizing at60 °C for 30 s. Results were analyzed automatically once aftereach runwas completed. Experimental sampleswere comparedwith the standard curve generated from host cell/sporozoitemixtures containing known numbers of sporozoites. The per-cent inhibition was determined by comparing the number ofsporozoites invaded after mAb treatment with the number ofsporozoites invaded without mAb treatment.SDS-PAGE and Western Blot—Sporozoite extract, equiva-

lent to�2� 106 sporozoites per lane, was loaded onto a 4–12%gradient BisTris gel under reducing and nonreducing condi-tions. Samples analyzed under reduced conditions were eithermixed with dithiothreitol (DTT) (Sigma) at a final concentra-tion of 50 mM (reduced) or mixed with DTT followed by treat-ment with iodoacetamide (Sigma) at a final concentration of200 mM (reduced and alkylated). Following the electrophoreticseparation, proteins were transferred onto a nitrocellulosemembrane for immunoblotting using standard methods (22,23). All washes were performed in 1� PBSwith 0.5%Tween 20.Development of the membrane was performed by incubatingthe nitrocellulose membrane with 0.5 �g/ml purified IgG fromtheCSP-specificmAbs diluted in blocking buffer (1�PBS, 0.5%Tween 20, and 5% skim milk). Primary antibody was detectedwith goat anti-mouse alkaline phosphatase-conjugated second-ary antibody. Detection was achieved using the 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium-componentphosphatase substrate (Kirkegaard & Perry Laboratories Inc.,Gaithersburg, MD), as per the manufacturer’s instructions.SDS-PAGE analysis of recombinant protein was performed on4–12% gradient BisTris polyacrylamide gels (Bio-Rad) undernonreducing and reduced/alkylated conditions. Immunoblot-ting was performed as described above using 4–12% gradientBisTris polyacrylamide gels (Bio-Rad) under nonreducing,reducing, and reduced/alkylated conditions.

Immunofluorescence Assay and Confocal Microscopy—Im-munofluorescence assay analysis was performed as per stand-ard protocol (24). Sporozoites collected by the Ozaki methodwere washed with RPMI medium and incubated with blockingmedia (1� PBS, 3% bovine serum albumin) at 4 °C (25). Allprimary and secondary antibody incubations were carried outat 4 °C and separated by extensive washingwith PBS containing0.05% Tween 20 (Bio-Rad). The samples were mounted undercoverslips using Vectashield hard-set mounting medium andthen stored at 4 °C until images were acquired by confocalmicroscopy as described previously (24). A Leica SP2 confocalmicroscope using Leica confocal software was used for imageacquisition. All imageswere collected by using a PLAPO�100/1.4 oil immersion objective and a confocal zoom of �6. Alexa488 conjugate was excited by an argon laser (488 nm). Allimages were collected as three-dimensional data sets (z-stacks)with a step size of 0.1221 nm between the successive opticalsections. De-convolution of all image stacks was performedusing Huygens Essential (Scientific Volume Imaging) toimprove themaximum resolution of the data, as well as tomin-imize background noise. Deconvolved images were saved andanalyzed through Imaris image analysis software (version 5.7.2,Bitplane). Confocal images in this study are displayed as maxi-mum projection of the three-dimensional image stacks.Expression and Production of EcCSP—The amino acid

sequence ofCSP (Indian isolate, GenBankTM accession numberAAN87606) was used to generate a codon-optimized syntheticgene for expression in E. coli (GeneArt, Regensburg, Germany).The construct, corresponding to amino acids Leu19 to Ser411 ofthe full-length CSP, was subcloned into the E. coli pET-24a�

expression vector downstream of the T7 promoter using theNdeI and XhoI restriction sites (EMD Chemicals Inc., Gibbs-town, NJ). The resulting transcribed gene incorporates theadditional amino acid sequence LEHHHHHH. RecombinantEcCSP was expressed using E. coli BL21(DE3) cells. Fermenta-tion of BL21(DE3)-EcCSPwas performed in a 5-liter bioreactorusing defined medium at 37 °C, as described previously (26).When the absorbance at 550 nm reached 40–45, the tempera-ture was reduced to 25 °C and induced by adding isopropyl1-thio-�-galactopyranoside to a final concentration of 1mM for3 h. Cell culture was harvested by centrifugation and stored at�80 °C.The frozen cell pellet was disrupted using either mechanical

lysis or chemical lysis. For mechanical lysis, the cell pellet wasresuspended in 10 volumes of lysis buffer (20 mM Tris, pH 8.0,100 mM NaCl, 1 M urea, 5 mM DTT) and passed through amicrofluidizer (M110Y, Microfluidics Corp., Newton, MA)three times at 19,000 pounds/square inch. The lysate was cen-trifuged for 30 min at 10,000 � g to pellet and remove theinclusion bodies and cellular debris. EcCSP was then capturedfrom the soluble fraction using a nickel-Sepharose Fast Flow(GE Healthcare) column under reducing and denaturing con-ditions using a buffer containing 6 M guanidine-HCl and 5 mM

DTT.For chemical lysis, the cell pellet was resuspended in 10 vol-

umes of lysis buffer (20 mM citrate, 1% SB3-12 (Sigma), 0.1%ABS-14 (Sigma), 75 mM NaCl, pH 7.7) and mixed for 2 h atroom temperature. Following the addition of lysis buffer to the




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cell pellet, 25 units of benzonase (Novagen/EMD Chemicals,Inc., Gibbstown, NJ) perml of lysis buffer was added. The lysatewas centrifuged for 30 min at 10,000 � g to pellet inclusionbodies and cellular debris. Inclusion bodies were washed withwater and then solubilized in solubilization buffer (11 mM Tris,111 mM sodium phosphate, 6 M guanidine-HCl, pH 8.0) over-night at room temperature. Solubilized EcCSP was capturedunder reducing and denaturing conditions using a nickel-Sepharose 6 Fast Flow (GE Healthcare) column.Nickel-captured EcCSP was refolded on a Sephacryl S-300

HR column. A sample load of �2–3% of a column volume wasloaded on a pre-equilibrated column with refold buffer 1 (20mM sodium phosphate, 50 mM guanidine-HCl, 1 M urea, 500mM arginine, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, pH 7.2)and eluted from the column using refold buffer 2 (20 mM

sodium phosphate, 1 M urea, 500 mM arginine, 150 mM NaCl, 1mMEDTA, pH7.2).EcCSPwas subsequently dialyzed and puri-fied on a Q-Sepharose HP (GE Healthcare) column and pol-ished using a size exclusion Superdex 75 (GE Healthcare)column.Expression and Production of PpCSP—As with the E. coli

expression of CSP, the amino acid sequence of CSP (Gen-BankTM accession number AAN87606) was used to produce aPichia codon optimized synthetic gene for expression in themethylotrophic yeast P. pastoris (Invitrogen). A shortened CSPconstruct corresponding to amino acids Gly86 to Ser410 wascloned into the XhoI and XbaI sites of the Pichia expressionvector pPICZ�A as per the manufacturer’s instructions. Theresulting transcribed gene lacked a His6 tag. Prior to lineariza-tion with SacI and transformation into P. pastoris, the genesequence of CSP was verified. Recombinant protein was pro-duced in 5-liter bioreactors as described previously (22, 27). Ascalable purification process will be detailed elsewhere. How-ever, in brief, secreted PpCSP was subjected to depth filtration(Sartorius,Goettingen,Germany), followed by capture on aCMHyper Z column (Pall, East Hills, NY), purification byQ-Sepha-rose HP column (GE Healthcare), and polished with a sizeexclusion Superdex 75 column (GE Healthcare). The final bulkantigen was diluted to 1.0 mg/ml in saline, pH 7.4, sterile-fil-tered, and stored at �80 °C.BIAcore Chip Preparation and Interaction Studies—Surface

plasmon resonance measurements were performed using aBIAcore 3000 biosensor, operating with the BIAcore Controlsoftware (BIAcore 3000, BIAcoreAB, Sweden). All experimentswere performed at 25 °C. Three kinetic binding assays wereemployed to ascertain the binding properties of the recombi-nant CS proteins. Three mAbs, 1G12, 4B3, and 4C2, wereimmobilized to the CM5 sensor chip using an amine couplingkit according to the manufacturer’s instructions (GE Health-care). The CM5 chip surface was activated with an equal molarmix ofN-hydroxysuccinimide/N-ethyl-N-(dimethylaminopro-pyl)carbodiimide, followed by immobilization ofmAbs 1G12 in10 mM HEPES, pH 6.8, 4B3 in 10 mM acetate, pH 6.5, or 4C2 in10 mM acetate, pH 6.0. Unreacted sites were blocked with eth-anolamine (GEHealthcare). Each of themAbswas immobilizedin either flow cell 2 or 4 at 600 response units, whereas flow cells1 and 3 were activated and then de-activated to serve as refer-ence surfaces. For the interaction studies, recombinant CS pro-

teins were serially diluted in Hanks’ balanced solution buffer(GEHealthcare) at concentrations from10 nM to 1�M. Sampleswere injected at a flow rate of 30 �l/min for 2 min. The sensorchip was regenerated using two injections at 30 �l/min of 10mM glycine, pH 3.0, for 30 and 10 s with a 2-min stabilizationperiod. Data were analyzed using BIAevaluation (GE Health-care) and fit a 1:1 model.For the indirect kinetic analysis, mAb 4B3 was immobilized

on a CM5 chip as stated previously. CS protein was diluted inHanks’ balanced solution buffer to a concentration of 100 nM.CS protein was injected as stated above. 4C2 and 1G12 werediluted in Hanks’ balanced solution buffer at two concentra-tions, 50 and 500 nM, and injected similarly to the CS protein.The sensor chip was regenerated using two injections at 30�l/min of 10 mM glycine, pH 3.0, for 30 s and 10 s with a 2-minstabilization period. As a negative control, 4C2 was diluted to50 nM, 10 times the KD, and injected as stated above.Circular Dichroism—To examine secondary structure,

samples in 1� PBS were diluted 1:10 in deionized (MilliQ)water. CD spectra were recorded over the wavelength range185–260 nm in a 1-mm path length quartz cuvette using astep size of 0.2 nm, a slit bandwidth of 1.0 nm, and a signalaveraging time of 1.0 s. Analysis of temperature on secondarystructure was performed in 5 °C temperature increments from5 to 80 °C. Secondary structure content was calculated usingthe DICHROWEB web server (28, 29).SEC-MALS-QELS-HPLC—To assess the identity, purity,

and solution aggregation state of purified EcCSP and PpCSP,analytical size exclusion chromatography with in-line mul-tiangle light scattering (MALS) and quasi-elastic light scat-ter detection (QELS) was performed. Chromatography wasdone on an Alliance HPLC system (Waters, Milford MA) con-nected to a multiangle DAWN EOS light scattering detectorand a quasi-electric light scattering detector (Wyatt Technol-ogy, Santa Barbara, CA). Samples were prepared and analyzedas described by Tsai et al. (30).Analytical Ultracentrifugation Characterization of EcCSP

and PpCSP—Boundary sedimentation velocity measurementswere made in Optima XLA analytical ultracentrifuge (Beck-man-Coulter Instruments). Sedimentation velocity analysiswas performed at 20 °C at a rotor speed of 58,000 rpm. Thecentrifuge cell was filled with 400 �l of protein solution at aconcentration of 1.0 mg/ml PpCSP or EcCSP.Absorbance scans were obtained at 235 nm. Sedimentation

coefficient distribution analysis to deconvolute the boundaryvelocity data into sedimenting species was performed asdescribed previously (31), using the public domain softwareSedfit developed by Peter Schuck. The result of this computa-tional analysis is a presentation of the distribution of sediment-ing species in the form of a c(s) versus s plot. In this computa-tional treatment, the sedimentation boundary velocity datawere subjected to maximum entropy regularization statisticalanalysis for the most parsimonious distribution of sedimentingspecies (31) that best fits the data. Aweight average shape factor(f/f0) is extracted from the boundary spreading, and this allowsfor the conversion of the c(s) versus s distribution to a c(M)versus M graphical presentation, thereby providing the molarmass of each sedimenting peak. The software program




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Sednterp developed by Hayes, Laue, and Philo was also used todetermine the partial specific volume of the PpCSP and theviscosity (�) and density (�) for the PBS solution used in thesedimentation analyses and for the hydrodynamic analysis to bereported below.Atomic Force Microscopy (AFM)—Samples for AFM were

prepared and analyzed basically as described by Tsai et al. (30)with modifications as noted. Biological AFM imaging of theprotein products was carried out under a range of conditions,both in fluid and air, using gentle tapping-mode AFM, mostlywith a PicoForce Multimode AFM (Veeco, CA) consisting of aNanoscope� V controller, a type E scanner head, and a sharp-ened TESP-SS (Veeco, CA) or similar AFM cantilever (30, 32).For rCSP visualization, suitable protein attachment was readilyachieved by 1min of incubation of 7 �l of�1 �g/ml solution ofrCSP in PBS, pH 7.4, buffer on freshly peeled mica substrates,followed by rinsing with �1 ml of deionized water and com-plete drying under an inert gas flow. The sample was thensealed into the instrument compartment dehumidified byDrierite� particles (30). AFM images were evaluated withinthe Nanoscope software (version 7.2, Veeco, CA), andexported to Image J (version 1.41o, National Institutes ofHealth, Bethesda) andMathcad (version 14, Mathsoft, MA) forfurther analyses and display.Modeling—Tertiary structure modeling of the truncated

N-terminal domain was not attempted. Secondary structurewas predicted using PSIPRED (33) and JUFO (34, 35).The repeat region was modeled by building up experimen-

tally determined structures of the repeating unit (NPNA). Thecrystal structure of Ac-ANPNA-NH2 (36) was aligned to theaverage NMR structure of (NPNA)3 at each of the NPNArepeats (37) to verify the agreement between them (0.47, 0.46,and 0.39 Å C� root mean square deviation). The NMR struc-ture of (NPNA)3 was extended by aligning residues 1–8 of asecond (NPNA)3 to residues 5–12 of the first. Repeating thisprocedure (NPNA)43 was built up, corresponding to residues103–274 of the native CSP sequence. Mutations were intro-duced into the N terminus of the repeat region to correctlymodel the DPNA and NPNV repeats found there. Side chainsrotamers were optimized using SCWRL3 (38).The TSR domain was modeled using the automatic thread-

ing and homology modeling service LOMETS (39). High con-fidence models were selected and evaluated for the presence ofthe characteristic disulfide formation of the TSR. Alignmentsby SAM-T02 (40), FUGUE (41), and PPA-II (39) to TSR tem-plate 1lsl (42) generated acceptable models using MODELLER(43).Secondary structure composition for the repeat region and

the TSR domain was measured by DSSP (44), and structureanalysis and visualization were accomplished using Chimera(45) and PyMOL (46).

RESULTS

Generation and Characterization of CSP-specific mAbs—Toevaluate the folding of recombinant CSP expressed in E. coli(EcCSP) andP. pastoris (PpCSP) (Fig. 1), a panel of 18mAbswasfirst screened by Western blot analysis against sporozoitelysates to identify reduction-sensitive mAbs. Representative

Western blots for three of the mAbs are shown in Fig. 2A. Both1G2 and 1B3 displayed the characteristic CSP doublet bandingpattern at �60 kDa (47, 48) on both nonreduced and reducedplus alkylated sporozoite lysates, as represented by 1G2 (Fig.2A). These mAbs also recognize the NANP repeats (data notshown). In contrast, three other mAbs, 1G12, 4B3, and 4C2,maintained reactivity with the nonreduced sporozoite lysatebut displayed a markedly reduced interaction with the reducedand alkylated sporozoite lysate, depicted by 1G12 and 4C2 (Fig.2A). To further characterize the mAbs for biological signifi-cance, the panel of mAbs was examined for reactivity to livesporozoites by confocal microscopy. All three mAbs reactedwith viable sporozoites, demonstrating a characteristic surfacestaining. Shown in Fig. 2B are maximum projection images

FIGURE 1. Schematic of the E. coli and P. pastoris CSP constructs. The dia-gram represents the full-length CSP gene (top) and the shortened CSP aminoacid sequences Leu19–Ser411 and Gly86–Ser410 corresponding to the EcCSP(middle) and PpCSP (bottom) constructs, respectively. The signal sequence,region 1, repeat region, TSR domain, and glycosylphosphatidylinositol (GPI)signal are shown. The start site for the ScCSP degradative product used toimmunize mice, Ser301, as indicated under “Experimental Procedures,” is indi-cated by an asterisk on the diagram of the full-length CSP gene.

FIGURE 2. Characterization of CSP-specific mAbs. A, representative immu-noblot of a sporozoite lysate reacted with three mAbs, 1G12, 4C2, and 1G2,under nonreducing (NR) and reducing � alkylated (R/A) conditions. B, confo-cal microscopy analysis of live sporozoites. Sporozoites were stained with theindicated monoclonal antibody at 4 °C and detected using species-specificsecondary antibodies coupled with Alexa 488.




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from confocal microscopy using mAbs 1G12, 4C2, and 1G2.Because the reduction-sensitive mAbs recognized CSP on thelive parasites, we examined their ability to inhibit invasion ofliver cells by liveP. falciparum sporozoites (strainNF54), wherethe invasion rate ranges between 0.3 and 6%. 1G12, 4B3, and4C2 exhibited inhibitory activity of sporozoite invasion ofHepG2 cells (Table 1).Expression, Purification, and Biological Characterization of

EcCSP and PpCSP—Two different synthetic CSP genes wereused to express CSP in E. coli and P. pastoris (Fig. 1). Followingexpression and purification as described under “ExperimentalProcedures,” two forms of refolded and purified EcCSP weregenerated and identified based on the lysis method, EcCSP-ML(mechanical microfluidization lysis) and EcCSP-CL (chemicaldetergent lysis). The integrity and purity of EcCSP-ML andEcCSP-CL, as well as the PpCSP, are shown byCoomassie Blue-stained SDS-polyacrylamide gel (Fig. 3A) and immunoblot (Fig.3B). A reduction-sensitive highmolecularmass bandmigratingat 120 kDa is only observed in EcCSP-CL (Fig. 3A) likelybecause of the presence of an N-terminal free cysteine (seebelow). As shown in Fig. 3B, EcCSP-ML/CL and PpCSP reactwith the mAb, 1G12, in a reduction-sensitive manner byWest-ern blot. Similar reactivity byWestern blot usingmAbs 4C2 and4B3 was observed with all three recombinant CS proteins,EcCSP-ML/CL and PpCSP (data not shown). mAbs 1G12, 4C2,and 4B3 were also shown to react by Western blot with a pro-tein product corresponding to the TSR domain in a reduction-sensitive manner (data not shown). These three reduction-sen-sitivemAbswere isotyped and subclassed and determined to beIgG1.

The interaction of EcCSP-ML and PpCSP with the threereduction-sensitive mAbs, 1G12, 4B3, and 4C2, was furthercharacterized by surface plasmon resonance. To examine theaffinity or KD value of the three mAbs, 1G12, 4B3, and 4C2, toEcCSP-MLandPpCSP, each of themAbswas amine-coupled tothe surface of a CM5 chip (see supplemental Fig. 1A for exam-ple 4C2). The KD value of the mAbs toward the two recombi-nantCSproteinswas comparable. The affinity of the reduction-sensitive mAbs 4B3, 4C2, and 1G12 for EcCSP-ML was 8, 40,and 100 nM, respectively. Similarly for PpCSP, the affinity of thethree mAbs 4B3, 4C2, and 1G12 was 4, 40, and 60 nM. Analysisby surface plasmon resonance demonstrated the mAb combi-nations 4B3/4C2 and 4B3/1G12 shared an overlapping epitope(see supplemental Fig. 1B).Additional biological and biophysical characterization of the

three recombinant CS proteins using electrospray ionization-

mass spectrometry (ESI-MS), N-terminal sequencing, andendotoxin concentration was performed to ascertain the iden-tity and purity of the purified proteins. Using reversed phase-HPLC, the primary elution peak from each of the samples wasanalyzed by ESI-MS (Table 2). The observed mass for the non-reduced form of PpCSP was 33,078.9 Da (Table 2), which iswithin 1 Da of the nonreduced theoretical mass 33,079.9 Da,indicating the biochemical integrity of the protein is good.Results obtained with EcCSP-CL showed that the observedmass is similar to the theoreticalmass (Table 2). Identity of bothproteins was confirmed by N-terminal sequence analysis,which yielded the expectedN-terminal sequence for EcCSP-CL

TABLE 1ISI assay using P. falciparum (strain NF54) sporozoites and HepG2liver cellsHepG2 liver cells were invaded by P. falciparum sporozoites in the presence ofanti-CSP mAbs at the indicated concentrations. Quantification of percent inhibi-tion was performed as described under “Experimental Procedures.”

Antibody Isotype Regionrecognized Concentration Percent inhibition

� S.D.

�g/ml1G2 IgG1 Repeat 190.0 72.6 � 16.21G12 IgG1 TSR 196.0 54.7 � 29.64B3 IgG1 TSR 155.0 62.9 � 24.84C2 IgG1 TSR 178.5 32.4 � 20.2

FIGURE 3. Biophysical and biochemical characterization of EcCSP andPpCSP by SDS-PAGE and immunoblot. A, Coomassie Blue-stained SDS-PAGE of 1 �g of purified recombinant EcCSP and PpCSP under nonreduced(NR) and reducing � alkylated (R/A) conditions. B, representative immunoblotof 0.5 �g of purified recombinant EcCSP-ML (lanes 1, 4, and 7), EcCSP-CL (lanes2, 5, and 8), and PpCSP (lanes 3, 6, and 9) under nonreducing (lanes 1–3), reduc-ing (lanes 4 – 6), and reducing � alkylated (lanes 7–9) conditions, and shownare results obtained using mAb 1G12.




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and PpCSP (Table 2). Contrary to EcCSP-CL and PpCSP, themajor observed mass for EcCSP-ML did not correspond to theexpected theoretical mass for the designed product. Furtherexamination based on mass spectrometry and N-terminalsequencing analyses identified a mixture of several differentCSP species withN-terminal truncations (Table 2). Analysis forendotoxin concentration of our purified proteins showed endo-toxin levels ranged between 5 and 68 endotoxin units/mg(Table 2).Biophysical and Biochemical Characterization of EcCSP and

PpCSP, CD Spectroscopy, and Thermal Stability—CD spectraof purified PpCSP and EcCSP were generated to examine theirsecondary structures. Analysis of the far-UV spectra for sec-ondary structure outputs for PpCSP using the Dichrowebserver (28, 29) indicated PpCSP was composed of 10 � 4%�-helix, 23 � 7% �-sheet, 17 � 5% �-turn, and 49 � 13% ran-dom structure. Similar spectral results were obtained for bothof the EcCSPs. All three recombinant proteins were evaluatedfor changes in secondary structure because of heating by a stepprocedure that generated a temperature ramp in 5 °C incre-ments, beginning at 5 °C and ending at 80 °C.As shown in Fig. 4,a change in the secondary structure of PpCSP was observedwith increased temperature as indicated by increased intensityin negative ellipticity between 215 and 235 nm and a decreasefrom 190 to 215 nm (Fig. 4). The reversibility of the tempera-ture-induced secondary structure transitions was shown by thecooling back to either 5 or 20 °C from 80 °C, where secondarystructure was reformed, as indicated by a shift in the ellipticityspectra of the heated samples back toward the original 5 °Cspectra. Similar results were also obtained for EcCSP-ML andEcCSP-CL (data not shown). The spectra forPpCSP in the near-UV, 250–320 nm, showed very little signal, which is indicativeof a low amount of tertiary structure (data not shown). Theseresults were consistent with results obtained from EcCSP-MLand EcCPS-CL (data not shown).Biophysical and Biochemical Characterization of EcCSP and

PpCSP, SEC-MALS-QELS-HPLC—Each of the three recombi-nant CS proteins was examined by analytical SEC-MALS-QELS-HPLC to determine solution state, molar mass, andhydrodynamic radius. Analysis of PpCSP by SEC-HPLC indi-cated 93.6%of the proteinwasmonomeric in solutionwith 6.4%product-related impurities. Examination of the EcCSPs bySEC-HPLC indicated EcCSP-ML was composed of two popu-lations, amonomer population at 79.6% and a dimer population

at 20.4%, whereas EcCSP-CL was composed of two additionalspecies, yielding a composition of 24% aggregates, 33% dimer,17%monomer, and 26% product-related degradants. Examina-tion of the monomer peaks from SEC-HPLC reveals PpCSP,EcCSP-ML, and EcCSP-CL have similar retention times (Table2), similar to the retention time of immunoglobulinG (158 kDa)at �17 min (data not shown). Further analysis of the three CSproteins by in-line MALS indicated the weight average molarmass of the monomeric peaks corresponded to the theoreticalmass of each protein (Table 2 and Fig. 5A). To further charac-terize the recombinant CS proteins, the hydrodynamic radii(Rh) of the monomeric recombinant CS proteins were exam-ined byQELS. The Rh of PpCSPwas 4.2 nm (Fig. 5B), indicatingthe monomeric form of PpCSP is highly extended comparedwith a typical globular protein like bovine serum albumin (66.4kDa) for whichRh �3.4 (30). Similarly, theRh ofEcCSP-MLwas4.7 nm. For EcCSP-CL, there was insufficient signal for themonomer to obtain a valid Rh value.Biophysical and Biochemical Characterization of EcCSP and

PpCSP, Boundary Sedimentation Velocity Characterization ofPpCSP—To better characterize the recombinant CS proteins,an orthogonal approach was used to determine the mass andhydrodynamic radius. Boundary sedimentation velocity wasused to obtain the sedimentation coefficient distribution of thePpCSP sample. The absorbance scans of the sedimentingboundaries versus time (data not shown) were analyzed by thecomputational program Sedfit to give the sedimentation distri-bution of protein components shown in Fig. 5C. The weightaverage frictional ratio (f/f0) of 2.10 was extracted in the fittingprocess to account for boundary diffusion. Integration of theentire c(s) peak gives a weight average uncorrected s value of1.90 S. Correcting for the effects of the density and viscosity ofPBS, we obtained an s20,w value of 1.96 S. The weight averagesedimentation coefficient coupled with the weight average f/f0provides for the conversion of the c(s) distribution into a c(M)versus M distribution as shown in the inset of Fig. 5C. Theweight average molar mass of the major peak is 30,980 (Table2). This is in excellent agreement with the sequencemolarmassof 33,076. Using the latter and the s20,w value of 1.96 S, weobtain the following hydrodynamic results: a calculated f/f0 of2.19 and a Stokes radius of 4.58 nm. Based on the large f/f0 forCSP, it is reasonable to model the shape of this protein as acylinder. Using this hydrodynamic model with an estimatedhydration of 0.3g of water per g of protein, the CSP cylinder or

TABLE 2Biochemical and biophysical characterization of EcCSP-ML, EcCSP-CL, and PpCSP

EcCSP-ML EcCSP-CL PpCSP

N-terminal sequencea Mixture MLFQEYQX GREGKDEDMLFQ26YGS65LKK

Theoretical MS 42,126.5 Da 42,125.5 Da 33,079.9 DaObserved ESI-MS Mass ranging from 36,403 to 41,158 Da 42,154.0 Da 33,078.9 DaSEC-HPLC monomer peak retention time 16.83 min 16.08 min 16.60 minSEC-MALS-HPLC (weight average molar mass) 40,900 Da 52,000 Da 30,700 DaSedimentation velocity (weight average molar mass) 39,139 Da 42,409.5 Da 30,980 DaEndotoxin 5.3 EUb/mg 67.7 EU/mg 5.0 EU/mg

a TheN-terminal sequence forEcCSP-CL andPpCSPmatches the expected sequence, corresponding to amino acids Leu19 andGly86, respectively.EcCSP-MLcontains amixtureof N-terminal sequences. The indicated amino acid start sites correspond to the amino acid positions within the Indian isolate (amino acid GenBankTM accession numberXP_001351122.1).

b EU indicates endotoxin units.




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rod would have length and diameter dimensions of 20.9 and1.52 nm, respectively.A sedimentation analysis of EcCSP-ML was also performed

under identical conditions used for PpCSP (results not shown).It is of interest to compare the sedimentation and hydrody-namic properties of EcCSP-MLwith PpCSP. For the former weobtained an uncorrected s value of 2.16 S, which corrects to ans20,w value of 2.26 S. The increase in s would be anticipatedgiven the molar mass of 41,743 for EcCSP. The weight averagef/f0 from the c(s) versus s analysis was 2.16. The weight averagemolar mass from the c(M) versusM analysis was 39,139 in goodagreement with the sequence molar mass (Table 2). An f/f0 of2.26 was obtained from the sequence molar mass and s20,wvalue. The Stokes radius for EcCSP is 5.09 nm. Again modelingthis CSP construct as a cylinder or rod, we obtain length anddiameter dimensions of 34 and 1.6 nm, respectively. It is evidentthat the sedimentation and hydrodynamic behavior of PpCSPand EcCSP-ML are quite comparable. A similar analysis wasperformed with EcCSP-CL in which comparable results were

obtained with EcCSP-ML and PpCSP. The weight averagemolar mass determined from the sedimentation analysis ofEcCSP-CL is presented in Table 2. The AFM results that will bepresented below support the rod/ribbon shape modeling forCSP.Biophysical and Biochemical Characterization of EcCSP and

PpCSP, AFM—AFM images directly reveal that PpCSP proteinis an extended linear and flexible molecule with a variabletwisted ribbon-like appearance (Fig. 6A). In agreement with thesolution studies, AFM images suggest about 92% PpCSP pro-tein mass is in a monomeric state with the measured proteinvolume around the value of 33 nm3 (Fig. 6B). The balance of theprotein mass suggests a small amount of dimers and very spo-radic occurrence of small oligomers. The protein particles arevaried in shape, but all are consistent with the extended twistedribbon forms, when quantified by the histogram of the circular-ity, defined as 4��area/(perimeter)2 (Fig. 6C). Assuming astraight ribbon shape for a fully extendedmolecule, thesemeas-ured circularity values suggest the typical length to width ratio

FIGURE 4. CD analysis of PpCSP in aqueous solution. The ellipticity (degrees cm2/dmol) was plotted as a function of wavelength (nm). Raw data measuredin millidegrees was converted into ellipticity (degrees cm2/dmol). Spectra were obtained at 5, 20, and 80 °C and at 5 and 20 °C following heating at 80 °C. Inset,ellipticity (degrees cm2/dmol) was plotted as a function of wavelength (nm) during heating. Spectra were obtained in 5 °C increments beginning at 5 °C;however, data are presented in 10 °C increments beginning at 5 °C.




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to be between two and six. Restricting to the more typicalPpCSPmonomers, having ameasured protein volume betweenabout 25 to 41 nm3, the histogram of the molecular area istightly distributed and fallswithin the range of�120 to 220nm2

(Fig. 6D). Taken together, theAFM images suggest an “average”ribbon shape of the dimensions around 25 � 7 � 0.2 nm forPpCSPmonomers as appeared onmica surfaces. AFM imagingforEcCSP also reveals twisted ribbon-like appearancewith rod-like dimensions, but with more complex monomer-dimer oli-gomerization hierarchies (data not shown). Therefore, our highresolution AFM images fully support the interpretations of thestructures deduced from the solution measurements.Modeling of CSP—Molecular modeling as detailed under

“Experimental Procedures” was successful for the repeat regionand for part of the TSR domain. The overall secondary struc-ture composition for the combination of the repeat region, theTSR domain, and the truncated N-terminal domain corre-sponding to the EcCSP construct was about 5% �-helix, 27%�-sheet, 28% �-turn, and 36% random structure, in good agree-ment with the circular dichroism measurements. Stretches ofabout 45 residues before and after the repeat region were pre-dicted to have little secondary structure. The TSR domain itselfcontains characteristic disulfide bonds between Cys334 andCys369 and between Cys338 and Cys374 and forms an extendedstructure about 4.9 nm in length and 1.3 nm in width. It may befollowed by a membrane-associated helix from about Val377 tothe C terminus (Fig. 7A). The repeat region forms a stem-likesuperhelix about 18 nm in length and 1.5 nm in width com-posed of regular �-turns with a pitch of about 28 residues or 7repeat units (NPNA). Substitution of DPNA in the first turn ofthe superhelix adds a �5 formal charge to the otherwise elec-trically neutral structure and imparts a significant negativeelectrostatic potential to the N terminus of the repeat region(Fig. 7, B–D).

DISCUSSION

Recently, multiple vaccine trials examining the effectivenessof RTS,S, a CSP vaccine, have reported efficacy rates againstinfection ranging from 30 to 66% and against clinical malaria inchildren 1–4 years at 30% (49–51). However, the RTS,S con-struct lacks the N-terminal region of CSP, which has beenshown to be a critical functional domain (16–19).Our objectivewas to generate and subsequently characterize recombinantCSP incorporating the N terminus, repeat region, and C termi-nus, with the intention of gaining a better understanding of thestructure of CSP. Here we describe the bench scale productionand the extensive biological, biochemical, and biophysical char-acterization of CSP derived from two expression systems,E. coli and P. pastoris. The recombinant products from eachexpression system were similar in all of the biochemical andbiophysical characteristics reported here providing strong sup-port to the conclusion that CSP is a highly extended, rod-likeprotein on the surface of sporozoites.

Procedures.” The molar mass distribution c(M) versus M shown in the inset iscalculated from the c(s) versus s distribution using the fitted weight averagef/f0. The differential c(s) scale is in units of absorbance per Svedberg unit, anddifferential c(M) scale is in units of absorbance per molar mass multiplied by100,000.

FIGURE 5. Biochemical characterization of PpCSP by SEC-MALS-QELSHPLC and analytical ultracentrifugation. A, analysis of PpCSP by SEC-MALS-HPLC provided the molar mass distribution of the main peak (molarmass line indicated by arrow) compared with the absorbance at 280 nm.B, QELS goodness of fit of the autocorrelation function plot at the apex of thepeak. C, sedimentation coefficient distributions and molar mass obtainedfrom the boundary sedimentation velocity data (data not shown) of thePpCSP sample by the computational analysis described under “Experimental




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To biologically characterize the rCSP and to demonstratethat both EcCSP and PpCSP have structural compatibility tonative CSP, we generated a panel of mAbs to purified ScCSP.The S. cerevisiae expression system was not selected for pro-duction of recombinant CSP because of low levels of expres-sion (data not shown). To facilitate production of mAbs spe-cific to the TSR domain of CSP, mice were boosted with apurified degraded product corresponding to the TSR domainas determined byN-terminal sequencing (data not shown). Thesubsequent CSP-specific mAbs were screened byWestern blotagainst sporozoite lysates, confocalmicroscopy of live sporozo-ites, and a sporozoite invasion inhibition assay to determine

their biological activity as well asto determine their applicability forexamination of recombinant CSPconformation. The results obtainedby Western blot of the sporozoitelysate demonstrated mAbs 1G12,4B3, and 4C2 were reduction-sen-sitive, indicating they recognize aconformation-dependent epitope(Fig. 2A), whereas confocal mi-croscopy results showed the char-acteristic surface reactivity to livesporozoites for the whole mAbpanel (Fig. 2B). Finally, mAbs 1G2,1G12, 4B3, and 4C2 were examinedfor their ability to inhibit sporozoiteinvasion of liver cells (Table 1). Allthree conformation-dependent mAbs,1G12, 4B3, and 4C2, were able todemonstrate inhibition of liver cellinvasion. These data are consistentwith results published by Roggeroet al. (52) demonstrating antibod-ies generated to a 102-mer peptidecorresponding to the C-terminalregion of CSP are able to inhibitsporozoite invasion of hepatomacells.To assess folding of the TSR do-

main in the purified recombinantproteins, Western blot analysis ofEcCSP-ML, -CL, and PpCSP wasperformed using 1G12, 4B3, and4C2 (Fig. 3). Reactivity was dimin-ished following treatment withDTT and further reduced follow-ing subsequent treatment withiodoacetamide, which preventedreformation of the disulfide bondsin the TSR domain (Fig. 3). Giventhat 1G12, 4B3, and 4C2 reactedsimilarly with native CSP (Fig. 2A),the results indicate EcCSP andPpCSP share a common conforma-tional epitope in the TSR domainwith native CSP. Further examina-

tion of the interaction between the recombinant proteins andmAbs 1G12, 4B3, and 4C2 via BIAcore indicated only slightaffinity differences between EcCSP-ML and PpCSP with therespective mAbs, indicating both EcCSP-ML and PpCSP likelyshare a common structural epitope. Interaction betweenEcCSP-CL and the conformationally sensitive mAbs was notexamined using BIAcore because of the propensity ofEcCSP-CL to form aggregates in solution.Biochemical characterization of purified EcCSP-ML showed

that neither the N-terminal sequence nor the mass spectrumdirectly corresponded to the expected results. The N-terminalsequence contained a mixture of sequences (Table 2), which is

FIGURE 6. AFM characterization of PpCSP deposited from PBS, pH 7.4, on Mica. A, panel of representativeimages, in three-dimensional plots (70 nm square viewed from the scan direction vertically, colored scale barfor height up to 0.4 nm bottom left, and 30 nm bars in the x-y representing scale), showing PpCSP proteinmonomer shapes as seen in AFM topographies of uniformly dispersed PpCSP particles on a mica surface.B, mass distribution histogram from �2600 computed particles reveals that about 92% PpCSP is seen in amonomeric state (under the blue curve centered at the monomer volume of 33 nm3). C, histogram of thecircularity, defined as 4��area/(perimeter)2, of these particles suggests flexible rod-like molecules, showingtwisted ribbon-like morphologies with a typical length to width ratio between 2 and 6. D, histogram of themolecular area for 711 PpCSP monomers, having a more typical measured protein volume between 0.75 and1.25 of the monomer value in (B) reveals a distribution range of �120 to 200 nm2.




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consistent with the disparity observed between the theoreticalESI-MS and the observed ESI-MS. Given that EcCSP-ML wascaptured from the soluble fraction following mechanical dis-

ruption of the E. coli cellular membrane, it is feasible that CSPwas exposed to active E. coli proteases, which consequently ledto a mixed N-terminal population of EcCSP. Subsequent mod-ification of the lytic process frommechanical to detergent lysisleads to the production of purified EcCSP-CL for which theN-terminal sequence and ESI-MS results were comparablewith the expected results. The instability of the designed Nterminus of EcCSP-ML may be due to the presence of thePEXEL domain that accounts for an additional proteolytic site(53–55). This is further supported by the absence of any detect-able nativeCSPdimers thatwould be expected if a free thiol waspresent as detected by Western blot (Fig. 2). Similar to EcCSP-CL, N-terminal and ESI-MS results for PpCSP corresponded tothe designed product. Evaluation of each of the three recombi-nant proteins for impurities indicated the endotoxin levelswerewithin acceptable levels for human studies (Table 2).Both EcCSP and PpCSP were examined by CD to first exam-

ine secondary and tertiary structure. Analysis of the resultsfrom the far-UV examination of both proteins using theDichroweb server indicates that both EcCSP and PpCSP con-tain very little �-helical structure, whereas�40% of the proteinis composed of �-sheets and �-turns and 49% random struc-ture. Inspection of both proteins in the near-UV indicated verylittle tertiary structure. This would fit, because the only struc-tural domain based on sequence parsimony is the TSR domain,which has an elongated anti-parallel two �-sheet fold (42, 56).However, more important for vaccine development is knowl-edge of the thermal stability or temperature effects on the struc-ture. Examination of both proteins via a temperature rampindicated the recombinant proteins underwent an alteration ofsecondary structure upon heating. Furthermore, we sought tounderstand the following: should a temperature excursion from4 °C or room temperature occur during storage or formulationif the secondary structure would reform upon cooling? Theresults for both EcCSP and PpCSP indicated secondary struc-ture is regained following cooling from 80 °C back to either 4 or20 °C. Consequently, if there is a short temperature deviation,the resulting change in temperature is unlikely to have a detri-mental effect on secondary structure for either recombinantprotein.Further biophysical studies of the EcCSP and PpCSP by SEC-

MALS andQELS indicated a predominantmonomeric solutionstate for both EcCSP-ML and PpCSP, whereas EcCSP-CLaggregated in solution as mentioned previously. The weightaverage molar mass of 52 kDa for EcCSP-CL is 10 kDa largerthan the theoretical mass of our designed product, which islikely because of an incomplete separation between the mono-mer and dimer peak. Furthermore, QELS data indicate EcCSPand PpCSP are extended molecules based on the observedhydrodynamic radii, which was corroborated by sedimentationvelocity. Both QELS and sedimentation velocity data supportthe concept that CSP acquires a nonglobular highly extendedstructure. To further investigate and begin to resolve the struc-tural features of CSP, we examined all three recombinant pro-teins by high resolution AFM that permitted visualization ofindividual rCSP molecules. Such studies allowed the examina-tion ofmonomeric rCSP using an orthogonal technique to bothSEC-QELS and sedimentation velocity in the solution state of

FIGURE 7. Molecular models. A, TSR domain of CSP (Tyr319 to Ser375) was mod-eled by homology. Protein Data Bank entry 1lsl is shown in gray, and the modelfor the CSP TSR domain in green. The important disulfide bonding pattern isretained in the model (arrows). Red indicates portions of the TSR region withpoorly predicted structure. B, NMR (green) and crystal (pink) structures for theNPNA repeat agree to less than 0.5 Å root mean square deviation. C, NMR struc-ture was extended by sequential superposition of the NPNA repeats. This view isdown the long axis. D, repeat region forms a long stem-like superhelix composedof regular �-turns. The electrostatic potential mapped to the solvent-accessiblesurface of the repeat region indicates an area of significant negative charge in thefirst turn of the superhelix. E, scaled graphic depiction of CSP extending from thelipid membrane relative to PfAMA1. The graphical image of PfAMA1 was gener-ated from a composite of the structural data for PfAMAI and PvAMAI (Protein DataBank codes 2Q8A and 2J4W, respectively). The TSR domain is represented in blue,repeat region in red, and the N terminus in purple. The N-terminal depiction hasbeen added solely to symbolize the N-terminal space. The shape of the N termi-nus is a graphical illustration and is not based on structural data.




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CSP. The AFM images and the subsequent measurements viadirect visualizations on a substrate indicate that CSP is anextended linear, twisted ribbon-like, and flexible molecule withmeasurements centered around 25 � 7 � 0.2 nm.

These data are the first direct evidence demonstrating CSPhas an elongated structure and are consistent with previousstudies that examined the structure of the NANP repeat regionof CSP. Several studies examined conformationally constrainedNANP mimetics by NMR and CD (37), cyclic ANPNAA pep-tides by NMR (57), and Ac-ANPNA-NH2 peptides by crystal-lography (36), all of which demonstrated the NPNA motifadopts a �-turn (58). Speculation by Nanzer et al. (59) suggeststhat by affixingmultipleNPNArepeats together, a stable “stem-like” structure may be formed. Our SEC-MALS-QELS, sedi-mentation velocity, and AFMdata support the concept that theNANP repeat region of CSP does form a stem-like structure,creating an elongated and flexible molecule. Molecular model-ing also supports the stem-like nature of the repeat region (Fig.7,A–E). In addition to the repeat region, the TSR domain is alsoan elongated structure. A long linker exists between the N-ter-minal domain and the repeat region. Another linker existsbetween the repeat region and the TSR domain. These ele-ments combine to support the experimental observations ofnonglobularity and multiple morphology with a three-dimen-sional shape of �21–25 � 1.5 � 1.5 nm. The differencebetween the AFMmeasurements versus the solution and com-putational measurements is partly due to compression by theAFM tip. Although gentle AFM can quantitatively characterizedelicate biological samples (30, 32), it is logical for the CSPprotein with its particular elongation, flexibility, and high con-tent of random coiled structure to be visualized in the twistedribbon-like forms between the substrate and the AFM tip.Overall, there is very good agreement for the relative shape inboth the length and the cross-sectional area of CSP between thedifferent analytical methods.Sporozoite entry ofmammalian cells has beenwell studied in

vitro (60, 61) and characterized to include both traversal of cellsin which no parasitophorous vacuole is formed and infection ofcells accompanied by formation of a parasitophorous vacuole(62–65). However, the definitive role for the CS protein withinthis traversal and infection process is unclear. An abundance ofevidence suggests both the N-terminal domain and C-terminaldomain are involved in the invasion process of host organisms,ranging from motility to liver cell attachment via heparin sul-fate proteoglycans displayed on the surface of receptors (16–19, 66, 67). How the unique structure of the CS protein as anextended, flexible rod-like structure impacts on the process ofgliding, traversal, or infection is worthy of further evaluation.Here we have generated extensively characterized rCSP

expressed from two expression systems. PpCSP correspondedto our designed construct and was purified to greater than99% purity, as determined by SEC-HPLC, without the use ofan affinity tag and remained monomeric in solution followingpurification. These data not only present evidence of a highlypurified rCSP, but they provide the first analytical evidencedescribing CSP as an elongated, flexible rod-like protein on thesurface of sporozoites.

Acknowledgments—We are thankful to Dominique Jones for collect-ing the CD data; Drs. Owen Schwartz and Juraj Kabat (BiologicalImaging, Research Technologies Branch, National Institutes ofHealth) for advice and help on confocal microscopy; Dr. Michael B.Murphy (GE Healthcare, BIAcore) for assistance with the BIAcore;Olga Muratova for providing sporozoites for Western blot analysis;Drs. Carl Hammer and Mark Garfield for mass spectrometry andprotein sequencing, respectively.

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Albert J. Jin, Louis H. Miller, Nicholas J. MacDonald, Yimin Wu and David L. NarumOnyinyechukwu Uchime, Raul Herrera, Vu Nguyen, Jacqueline Glen, Jacob Lebowitz,Smith, Darrell E. Hurt, Brent House, Xiaoyan Zou, Yanling Zhang, Merrit Hickman,

Matthew L. Plassmeyer, Karine Reiter, Richard L. Shimp, Jr., Svetlana Kotova, Paul D.Malaria Vaccine Candidate

Circumsporozoite Protein, a LeadingPlasmodium falciparumStructure of the

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Structureofthe Plasmodiumfalciparum Circumsporozoite ... · min; 85 °C for 5 s; hold at 4 °C. The resulting cDNA was used immediately or stored at 20 °C. The following probe and