Molecular and Biochemical Parasitology 109 (2000) 179–184

Short communication

Expression, purification and characterization of a functionalregion of the Plasmodium 6i6ax Duffy binding protein�

Sheetij Dutta, Jon R. Daugherty 1, Lisa A. Ware, David E. Lanar,Christian F. Ockenhouse *

Department of Immunology, Walter Reed Army Institute of Research, 503 Robert Grant A6enue, Sil6er Spring,MD 20910, USA

Received 22 December 1999; received in revised form 30 March 2000; accepted 6 April 2000

Keywords: Malaria; Duffy binding protein; Baculovirus expression; Plasmodium 6i6ax

www.elsevier.com/locate/parasitology

Erythrocytes of Duffy-negative individuals areresistant to invasion by the parasite Plasmodium6i6ax [1]. A 140-kDa protein, the Duffy bindingprotein (PvDBP), has been identified as the para-site ligand for Duffy-glycoprotein receptor on hu-man erythrocytes [2,3]. PvDBP gene sequenceanalysis [4] reveals that it belongs to a family ofPlasmodium adhesion proteins found in severalPlasmodia including the P. falciparum erythro-

cyte-binding antigen (EBA-175) and the variantsurface antigen PfEMP-1 encoded by 6ar genes[5–7]. In the N-terminal region of PvDBP, a 324amino acid cysteine-rich stretch or ‘region II’(PvDBPrII), has been identified as the principalerythrocyte binding domain using transfectedCOS cells [8,9], making PvDBPrII an attractivevaccine candidate. Substantial quantities of cor-rectly folded PvDBPrII protein are needed toinitiate structural and immunological studies. Wehave expressed PvDBPrII gene in Escherichia colisystem; however, the protein is contained predom-inantly within the insoluble fraction (unpublishedobservation). The E. coli product did not showappreciable erythrocyte binding activity, similarto a previous report with E. coli-expressed regionII-containing construct [10]. In addition, there areno conformation-specific monoclonal antibodiesto analyze the structure of refolded PvDBPrIIprotein from E. coli. Hence, we have focused onthe eukaryotic baculovirus system for production

Abbre6iations: Bv-PvDBPrII, baculovirus recombinantPvDBPrII protein; IL-8, interleukin-8; PvDBPrII, region II ofP. 6i6ax Duffy binding protein.� Disclaimer: The opinions or assertions contained herein

are the private views of the authors and are not to beconstrued as official or as reflecting the views of the Depart-ment of the Army or the Department of Defense.

* Corresponding author. Tel.: +1-301-3199473; fax: +1-301-3199012.

E-mail address: chris.ockenhouse@na.amedd.army.mil (C.F.Ockenhouse).

1 Present address: Division of Vaccines and Related Prod-ucts Applications, OVRR¯CBER¯FDA, Rockville MD, USA.

0166-6851/00/$ - see front matter © Published by Elsevier Science B.V.

PII: S 0166 -6851 (00 )00244 -9

S. Dutta et al. / Molecular and Biochemical Parasitology 109 (2000) 179–184180

of PvDBPrII. Here we present the expression,purification and characterization of recombinantPvDBPrII protein produced in the insect cellsystem.

PCR primers, forward: 5%-atgcgcggccgctACG-ATCTCTAGTGCTATTATA-3% and reverse: 5%-atatgaattcTGTCACAACTTCCTGAGTATT-3%(restriction sites shown in lower case), were used toamplify PvDBPrII encoding gene (amino acidresidues 198–521; TISSAII…to…NTQEVVT)from genomic DNA of Salvador I strain of P.6i6ax. PCR product was cloned into the NotI–EcoR I sites of baculovirus transfer plasmidpBSV-8His [11] in-frame with a vector encoded

human complement regulatory factor H-like 1plasma protein (FHL-1) signal peptide on theamino-terminus (Fig. 1A). The construct also en-codes an enterokinase cleavage site followed by a8× histidine tag at the carboxy-terminus (Fig.1A). The vector pBSV-8His is designed to directexpressed proteins to a secretory pathway andallow easy purification of the product from culturesupernatant of insect cells [12]. The cloned insertwas evaluated by DNA sequencing, and recombi-nant PvDBPrII baculovirus clones were generatedessentially as described in the Clontech Laborato-ries BacPAK6 manual (Palo Alto, CA). Clonedvirus stock was amplified from a single plaque.

Fig. 1. Expression and purification of the recombinant Bv-PvDBPrII construct. (A) Schematic view of the PvDBP gene (top)showing the conserved amino acid blocks with other EBPs; shaded areas are N- and C-terminal cysteine-rich regions (N-, C-cys);transmembrane (TM). Expressed Bv-PvDBPrII construct (bottom) shows the extra residues encoded by vector pBSV-8His; (*)indicates translation stop. (B) Coomassie-stained SDS-PAGE analysis of Bv-PvDBPrII during various stages of purification. Lane1, 6.5× concentrated baculovirus culture supernatant; 2, flow-through of Ni2+ column; 3, pooled fractions containing Bv-PvDBPrII eluted off Ni2+ column and loaded on SP Sepharose; 4, flow-through of the SP Sepharose column; 5, 1 M NaCl eluateof SP Sepharose containing pure Bv-PvDBPrII protein indicated by the arrow. Molecular weight in kDa indicated on left.

S. Dutta et al. / Molecular and Biochemical Parasitology 109 (2000) 179–184 181

The 367 amino acid long Bv-PvDBPrIIpolypeptide has a predicted molecular weight of41 kDa after FHL-1 signal peptide cleavage. Ex-pressed Bv-PvDBPrII secreted into the culturesupernatant migrated close to 40 kDa on SDS-PAGE as detected by western blot with anti-6×histidine monoclonal antibody (data not shown).The Bv-PvDBPrII appeared as a compact bandon non-reducing gels confirming that the majorityis present as a single conformer. Its mobility onSDS-PAGE was decreased upon reduction withDTT confirming its disulphide bonded nature,and the protein did not seem to undergo extensiveglycosylation in the baculovirus system as as-sessed by staining the SDS-PAGE with the Gel-Code® Glycoprotein Staining Kit (Pierce,Rockford, IL) (data not shown). N-terminusamino acid sequencing of purified Bv-PvDBPrIIprotein revealed the first six amino acids as Thr–Ile–Ser–Ser–Ala–Ile which corresponds to thefirst six residues of native PvDBPrII, confirmingthat the signal peptide was efficiently cleaved dur-ing secretion. We found it unnecessary to cleavethe 8-histidine tag at the C-terminus, as it had noeffect on erythrocyte binding ability of Bv-PvDBPrII.

Recombinant Bv-PvDBPrII was produced inlarge scale using a Bellco Bioreactor with a 10-lworking volume. Sf21 insect cells were grown, inserum-free medium (Cyto-SF9, Kemp Biotech-nologies, Frederick, MD), culture was maintainedat 27°C and the dissolved oxygen level was at 60%of air saturation. At a density of 1.5×106 cellsml−1 the culture was infected with recombinantPvDBPrII baculovirus. Seventy-two hours afterinfection, supernatant was harvested and concen-trated at 4°C using a 10 000 MWCO hollow-fiberultrafilter (A/G Technology, Woburn, MA) to 1.5l (a 6.5-fold reduction in volume). The concen-trated supernatant was aliquoted and stored at−80°C. Bv-PvDBPrII constituted about 8% oftotal protein in the 6.5-fold concentrated bac-uloviral culture supernatant (Fig. 1B, lane 1) asestimated by densitometric analysis of Coomassiestained SDS-PAGE gels.

Highly purified fractions of the PvDBPrII wereobtained using a two-step purification protocolwhich was carried out at room temperature. A

typical purification run was started with 50 ml ofconcentrated supernatant, it was cleared by cen-trifugation at 10 000×g for 15 min and 5 ml of10× PBS (1.5 M NaCl, 17 mM KH2PO4, 50 mMNa2HPO4, pH 7.4) was added to it. The superna-tant was passed over a 5-ml Ni-NTA SUPER-FLOW column (Qiagen; Valencia, CA), on aFPLC system (Waters, MILLIPORE; Milford,MA). The column was washed extensively with1× PBS followed by PBS containing 500 mMNaCl and 40 mM imidazole. Bv-PvDBPrII waseluted with a 40 to 200 mM imidazole lineargradient in 50 mM Tris, 0.05% Triton X-100 (pH7.0). Triton X-100 enhanced the recovery andpurity during the second step of purification. Thefirst step of purification on Ni2+ column resultedin up to 63–73% pure protein fractions (Fig. 1B,Lane 3). Elution fractions containing Bv-PvDBPrII were pooled, diluted fourfold in thesame buffer without imidazole, and loaded on a1-ml SP Sepharose Fast Flow cation exchangecolumn (Amersham–Pharmacia Biotech; Piscat-away, NJ). The column was washed with 50 mMTris buffer containing 100 mM NaCl (pH 7.0)and purified Bv-PvDBPrII was eluted in 50 mMTris (pH 7.0) containing 1 M NaCl. Elutedprotein was dialyzed against 1× PBS overnightand stored at −20°C. Estimated purity of thefinal product was 92–96% (Fig. 1B, Lane 5). Anaverage of 2 mg Bv-PvDBPrII protein waspurified per liter of the baculovirus supernatant.

Purified Bv-PvDBPrII protein was analyzedfurther for its biological activity using an erythro-cyte binding assay (see Fig. 2A legend). Bv-PvDBPrII protein bound Duffy-positive humanerythrocytes (Fig. 2A) while no binding was ob-served with Duffy-negative erythrocytes. Positivebinding was also shown with Aotus monkey ery-throcytes, but not with Rhesus monkey erythro-cytes. This data corroborates the observedbinding properties for native PvDBP from P.6i6ax culture supernatant [2]. In order to evaluatethe specificity of this interaction we pretreatedhuman Duffy-positive erythrocytes with eithertrypsin or chymotrypsin, and assayed thesetreated erythrocytes for Bv-PvDBPrII-binding ac-tivity. Chymotrypsin which is known to removethe Duffy antigen from erythrocytes abolished the

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binding of Bv-PvDBPrII to Duffy-positive ery-throcytes (Fig. 2B). Trypsin treatment which hasno effect on the Duffy antigen did not cause anyreduction in Bv-PvDBPrII binding toerythrocytes.

Recombinant IL-8 has been shown to competewith PvDBPrII for binding to the Duffy antigenon erythrocytes [8,13]. Inhibition of Bv-PvDBPrIIbinding to human Duffy-positive erythrocytes wasdone by pre-incubating erythrocytes with twoconcentrations of recombinant human IL-8 (R&Dsystems; Minneapolis, MN) for 30 min at 37°Cfollowed by erythrocyte binding assay. IL-8 at0.25 mM concentration inhibited approximately75% Bv-PvDBPrII binding (Fig. 2C) and close to90% inhibition was observed at 2.5 mM estimatedby densitometer.

In order to assess whether the protein wasantigenic, human immune sera collected fromadults attending a malaria clinic on the Thai–Burmese border as part of a larger study examin-ing immune responses to P. falciparum and P.6i6ax antigens (data to be presented elsewhere)were tested by immunoblot for reactivity to re-combinant Bv-PvDBPrII. A preliminary examina-tion indicated that PvDBPrII was indeedantigenic, in that the protein reacted to antibodiespresent in some serum samples (Fig. 2D, lanes1,2,4,6 top panel). Furthermore, these same serumsamples were also analyzed for their ability toblock Bv-PvDBPrII binding to human Duffy-pos-itive erythrocytes. Binding inhibition was done byincubating 1 mg Bv-PvDBPrII with 10 ml of hu-man serum (final dilution 1:5) for 1 h at roomtemperature prior to its addition to the erythro-cyte suspension (Fig. 2D, bottom panel). Al-though some of the sera showed the presence oferythrocyte binding-inhibitory antibodies (lanes 2and 6, bottom panel), other samples (lanes 1 and4, bottom panel) had no detectable anti-PvDBPblocking activity despite being highly reactive toPvDBPrII on immunoblot.

Further immunologic characterization indicatedthat Bv-PvDBPrII is immunogenic in addition tobeing antigenic. Anti-PvDBPrII antibodiesaffinity-purified from rabbit serum immunizedwith Bv-PvDBPrII (in Freund’s adjuvant), reactedwith merozoites within late stage schizonts on

Fig. 2. Biological and immunological characterization of Bv-PvDBPrII protein. (A) Erythrocyte binding assays were per-formed using Aotus, Rhesus, and human erythrocytes(Duffy-positive (pos), Duffy-negative (neg)). Erythrocytes (12ml of 50% hematocrit) in PBS were added to 500 ml PBScontaining 0.1% BSA along with 1 mg Bv-PvDBPrII proteinand incubated at 37°C for 30 min. Erythrocytes werewashed twice with 0.5 ml PBS and bound protein eluted in30 ml 1 M NaCl containing 1 mM PMSF. Eluted proteinwas detected on a western blot immunostained with affinitypurified polyclonal anti-Bv-PvDBPrII as the primary anti-body. The blot was developed with SuperSignal Chemi-lu-minescent substrate reagent (Pierce) and exposed on KodakX-Omat Blue XB-1 film (Eastman Kodak, Rochester, NY).(B) Effect of trypsin or chymotrypsin enzyme treatment onBv-PvDBPrII binding to human Duffy-positive erythrocytes.(C) Competitive inhibition of Bv-PvDBPrII binding to hu-man Duffy-positive erythrocytes with 0, 0.25 and 2.5 mMIL-8. (D) Recognition of Bv-PvDBPrII by human immunesera in immunoblot (serum dilution 1:1000; top panel). In-hibitory activity of Bv-PvDBPrII binding to Duffy-positivered cells using the same set of sera (bottom panel). (E)Indirect immunofluorescence assay, on methanol fixed Aotuserythrocytes infected with P. 6i6ax Sal I strain, with affinitypurified 20 mg ml−1 rabbit anti-Bv-PvDBPrII antibodies.Fluorescence image of a mature schizont (arrow) containingindividual merozoites appearing as dots (magnification ×1000).

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indirect immunofluorescence assay (Fig. 2E), simi-lar to the pattern observed previously in P.knowlesi schizonts [14], further establishing thenear native structure of the Bv-PvDBPrII.

Plasmodium antigens, such as the PvDBPrII,containing a large number of cysteine and aro-matic amino acids pose a problem of insolubilityduring expression in the E. coli system. Althoughthe total yield of PvDBPrII produced in E. coli isconsiderably larger than baculovirus, the fractionof correctly folded and functional molecules islow as indicated by poor erythrocyte bindingcharacteristics (Dutta et al., unpublished data).The baculovirus system offers several advantagesover prokaryotic systems including secretion ofthe expressed protein into the culture supernatant,correct folding, signal peptide cleavage and post-translational modifications. Bv-PvDBPrII ex-pressed in baculovirus system showed biologicalcharacteristics similar to those reported for nativeP. 6i6ax PvDBP protein from culture supernatantor COS cell expressed region II. Immunologicalcharacteristics of Bv-PvDBPrII confirms the nearnative structure of recombinant protein. Humanimmune sera from individuals residing in an en-demic area not only recognized Bv-PvDBPrII onwestern blot and ELISA (data not shown), but insome cases were able to block Bv-PvDBPrII bind-ing to erythrocytes. Interestingly, the presence ofhigh titer antibodies to recombinant PvDBPrIIdid not necessarily correlate with its ability toblock erythrocyte binding in a preliminary analy-sis of persons exposed to and infected with P.6i6ax. Further investigation is required to examinethe nature of epitopes which elicit inhibitory anti-bodies and strategies are needed to optimize theproduction of inhibitory antibodies. Although it ispossible that the activity observed in these in vitroerythrocyte binding assays may not reflect all ofthe possible anti-parasite effects present in vivo,and because of the limitations of in vitro cultureof P. 6i6ax, the significance of these results awaitconfirmation in the in vivo primate models of P.6i6ax. Nevertheless, the baculovirus-expressedDuffy binding protein is an attractive vaccinecandidate and plans are underway to immunize

non-human primates with Bv-PvDBPrII and chal-lenge with P. 6i6ax in the near future.

Acknowledgements

We thank the National Research Council(Washington, DC) for funding S. Dutta; Peter F.Zipfel at Bernhard Nocht Institute for TropicalMedicine, Hamburg, Germany for the plasmid;Gregory E. Garcia and Deborah R. Moorad atWRAIR for N-terminal sequencing; Connie L.Howard at the Walter Reed blood bank; Christo-pher W. Kemp at Kemp Biotechnologies for fer-mentation; Patrick E. Duffy and V. Ann Stewartat WRAIR for P. 6i6ax parasites; David C. Milesat WRAIR for photography.

References

[1] Miller LH, Mason SJ, Clyde DF, McGinniss MH. Theresistance factor to Plasmodium 6i6ax in blacks. TheDuffy-blood-group genotype, FyFy. N Engl J Med1976;295:302–4.

[2] Wertheimer SP, Barnwell JW. Plasmodium 6i6ax interac-tion with the human Duffy blood group glycoprotein:identification of a parasite receptor-like protein. Exp Par-asitol 1989;69:340–50.

[3] Barnwell JW, Nichols ME, Rubinstein PJ. In vitro evalu-ation of the role of the Duffy blood group in erythrocyteinvasion by Plasmodium 6i6ax. J Exp Med1989;169:1795–802.

[4] Fang XD, Kaslow DC, Adams JH, Miller LH. Cloning ofthe Plasmodium 6i6ax Duffy receptor. Mol Biochem Para-sitol 1991;44:125–32.

[5] Adams JH, Sim BK, Dolan SA, Fang X, Kaslow DC,Miller LH. A family of erythrocyte binding proteins ofmalaria parasites. Proc Natl Acad Sci USA1992;89:7085–9.

[6] Okenu DM, Malhotra P, Lalitha PV, Chitnis CE,Chauhan VS. Cloning and sequence analysis of a geneencoding an erythrocyte binding protein from Plasmod-ium cynomolgi. Mol Biochem Parasitol 1997;89:301–6.

[7] Peterson DS, Miller LH, Wellems TE. Isolation of multi-ple sequences from Plasmodium falciparum genome thatencode conserved domains homologous to those in ery-throcyte-binding protein. Proc Natl Acad Sci USA1995;92:7100–4.

[8] Chitnis CE, Miller LH. Identification of the erythrocytebinding domains of Plasmodium 6i6ax and Plasmodiumknowlesi proteins involved in erythrocyte invasion. J ExpMed 1994;180:497–506.

S. Dutta et al. / Molecular and Biochemical Parasitology 109 (2000) 179–184184

[9] Ranjan A, Chitnis CE. Mapping regions containing bind-ing residues within functional domains of Plasmodium6i6ax and Plasmodium knowlesi erythrocyte-bindingproteins. Proc Natl Acad Sci USA 1999;96:14067–72.

[10] Fraser T, Michon P, Barnwell JW, Noe AR, Al-Yaman F,Kaslow DC, Adams JH. Expression and serologic activityof a soluble recombinant Plasmodium 6i6ax Duffy bindingprotein. Infect Immun 1997;65:2772–7.

[11] Kuhn S, Zipfel PF. The baculovirus expression vectorpBSV-8His directs secretion of histidine-tagged proteins.Gene 1995;162:225–9.

[12] Prodinger WM, Schoch J, Schwendinger MG, Hellwage J,

Parson W, Zipfel PF, Dierich MP. Expression in insectcells of the functional domain of CD21 (complementreceptor type two) as a truncated soluble molecule using abaculovirus vector. Immunopharmacology 1997;38:141–8.

[13] Horuk R, Chitnis CE, Darbonne WC, Colby TJ, RybickiA, Hadley TJ, Miller LH. A receptor for the malarialparasite Plasmodium 6i6ax : the erythrocyte chemokinereceptor. Science 1993;261:1182–4.

[14] Adams JH, Hudson DE, Torii M, Ward GE, Wellems TE,Aikawa M, Miller LH. The Duffy receptor family ofPlasmodium knowlesi is located within the micronemes ofinvasive malaria merozoites. Cell 1990;63:141–53.

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