expression and purification of the recombinant protective antigen of bacillus anthracis

8
Expression and Purification of the Recombinant Protective Antigen of Bacillus anthracis Pankaj Gupta, 1 S. M. Waheed, 1 and R. Bhatnagar 2 Centre for Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India Received November 30, 1998, and in revised form February 19, 1999 Protective antigen (PA) is a major component of the vaccine against anthrax. The structural gene for the 83-kDa PA was expressed as fusion protein with 63 Histidine residues in Escherichia coli. Expression of PA in E. coli under the transcriptional regulation of the T5 promoter yielded an insoluble protein aggre- gating to form inclusion bodies. The inclusion bodies were solubilized in 6 M guanidine–HCl and the protein was purified under denaturing conditions using nickel nitrilotriacetic acid (Ni-NTA) affinity chromatogra- phy. The denatured protein was renatured by gradual removal of the denaturant while immobilized on the Ni-NTA column. The protein was then purified using Mono-Q column on FPLC. The yield of the purified recombinant PA (rPA) from this procedure was 2 mg/ liter of the culture. The rPA had an apparent molecu- lar mass of 83 kDa as determined by SDS–PAGE. Anti- sera to native PA recognized the fusion protein. The rPA was biologically as well as functionally active. Thus, the recombinant PA may be used to develop an effective recombinant vaccine against anthrax. © 1999 Academic Press Anthrax is a zoonotic disease whose etiologic agent is a gram-positive sporulating bacteria, Bacillus anthra- cis. Human beings acquire it via infected animals or contaminated animal products. Virulence of the bacte- ria is due to two major antigens viz., antiphagocytic capsular antigen, which is unique among bacterial cap- sules consisting of poly-D-glutamic acid and tripartite anthrax toxin (10,26,27). Since the former does not protect against anthrax infection, the latter is of main importance due to pathological and immunological rea- sons. Anthrax toxin has three components: protective antigen (PA; 83 kDa), lethal factor (LF; 90 kDa), and edema factor (EF; 89 kDa), of which no single compo- nent is toxic but combination of PA and either of LF or EF leads to pathogenesis in laboratory animals (39). The genes for these three protein components are present on a single 185-kb plasmid, pXO1, while an- other plasmid, pXO2, contains the genes responsible for poly-D-glutamic acid capsule synthesis (27). An- thrax toxin, like other bacterial toxins, fits the A-B model of classification of toxins, where B (PA in this case) is the binding moiety, which binds to the cell surface receptors, and LF/EF are alternate catalytic A moieties (38,40). Anthrax toxin is unusual in two re- spects. First, A and B domains are two distinct pro- teins. Second, the LF and EF have the identical PA binding domains. Thus each of the catalytic compo- nents exhibit competitive binding for PA forming two distinct toxins (22,23). EF and PA together form edema toxin, whereas LF and PA together form lethal toxin (20,22,38). PA binds to cell surface receptors, where it is cleaved by furin-like cellular proteases generating a cell-bound, 63-kDa protein (PA63) (19,37). This cleav- age of the 20-kDa amino terminal fragment exposes a high affinity binding site on PA63, to which EF or LF binds, which is subsequently internalized by receptor- mediated endocytosis into the lumen of acidic intracel- lular compartments, the endosomes (29,33). The chan- nel forming activity of PA63 in lipid bilayers probably brings about the translocation of EF and LF across the endosomal membrane into the cytosol (23). EF is a calcium/calmodulin-dependent adenylate cyclase, which increases the intracellular cAMP levels, thereby causing edema (20,40). Lethal toxin as the name sug- gests is lethal for several species (7,8,38). Mouse peri- toneal macrophages and macrophage-like cell lines such as J774A.1, RAW 264.7, etc., are sensitive to anthrax lethal toxin (7). It causes over-production of certain cytokines such as IL-1b and TNF-a in its target cells (13). Recent reports indicate that LF acts as an endopeptidase. It cleaves the amino terminus of mito- gen-activated protein kinase kinases 1 (MAPKK1), which contain the MAPK binding site. LF, thus pre- 1 Both the authors have contributed equally to the paper. 2 To whom correspondence and reprint requests may be addressed. Fax: (91) 11-6198234. E-mail: [email protected] and [email protected]. Protein Expression and Purification 16, 369 –376 (1999) Article ID prep.1999.1066, available online at http://www.idealibrary.com on 369 1046-5928/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.

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Expression and Purification of the Recombinant ProtectiveA

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Protein Expression and Purification 16, 369–376 (1999)Article ID prep.1999.1066, available online at http://www.idealibrary.com on

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ntigen of Bacillus anthracis

ankaj Gupta,1 S. M. Waheed,1 and R. Bhatnagar2

entre for Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India

eceived November 30, 1998, and in revised form February 19, 1999

edema factor (EF; 89 kDa), of which no single compo-nETpoftmcsmstbndt(icahbmlnbecwcgtsaccegw

Protective antigen (PA) is a major component of theaccine against anthrax. The structural gene for the3-kDa PA was expressed as fusion protein with 63istidine residues in Escherichia coli. Expression ofA in E. coli under the transcriptional regulation of

he T5 promoter yielded an insoluble protein aggre-ating to form inclusion bodies. The inclusion bodiesere solubilized in 6 M guanidine–HCl and the proteinas purified under denaturing conditions using nickelitrilotriacetic acid (Ni-NTA) affinity chromatogra-hy. The denatured protein was renatured by gradualemoval of the denaturant while immobilized on thei-NTA column. The protein was then purified usingono-Q column on FPLC. The yield of the purified

ecombinant PA (rPA) from this procedure was 2 mg/iter of the culture. The rPA had an apparent molecu-ar mass of 83 kDa as determined by SDS–PAGE. Anti-era to native PA recognized the fusion protein. ThePA was biologically as well as functionally active.hus, the recombinant PA may be used to develop anffective recombinant vaccine against anthrax. © 1999

cademic Press

Anthrax is a zoonotic disease whose etiologic agent isgram-positive sporulating bacteria, Bacillus anthra-

is. Human beings acquire it via infected animals orontaminated animal products. Virulence of the bacte-ia is due to two major antigens viz., antiphagocyticapsular antigen, which is unique among bacterial cap-ules consisting of poly-D-glutamic acid and tripartitenthrax toxin (10,26,27). Since the former does notrotect against anthrax infection, the latter is of mainmportance due to pathological and immunological rea-ons. Anthrax toxin has three components: protectiventigen (PA; 83 kDa), lethal factor (LF; 90 kDa), and

1 Both the authors have contributed equally to the paper.2 To whom correspondence and reprint requests may be addressed.

ax: (91) 11-6198234. E-mail: [email protected] [email protected].

046-5928/99 $30.00opyright © 1999 by Academic Pressll rights of reproduction in any form reserved.

ent is toxic but combination of PA and either of LF orF leads to pathogenesis in laboratory animals (39).he genes for these three protein components areresent on a single 185-kb plasmid, pXO1, while an-ther plasmid, pXO2, contains the genes responsibleor poly-D-glutamic acid capsule synthesis (27). An-hrax toxin, like other bacterial toxins, fits the A-Bodel of classification of toxins, where B (PA in this

ase) is the binding moiety, which binds to the cellurface receptors, and LF/EF are alternate catalytic Aoieties (38,40). Anthrax toxin is unusual in two re-

pects. First, A and B domains are two distinct pro-eins. Second, the LF and EF have the identical PAinding domains. Thus each of the catalytic compo-ents exhibit competitive binding for PA forming twoistinct toxins (22,23). EF and PA together form edemaoxin, whereas LF and PA together form lethal toxin20,22,38). PA binds to cell surface receptors, where its cleaved by furin-like cellular proteases generating aell-bound, 63-kDa protein (PA63) (19,37). This cleav-ge of the 20-kDa amino terminal fragment exposes aigh affinity binding site on PA63, to which EF or LFinds, which is subsequently internalized by receptor-ediated endocytosis into the lumen of acidic intracel-

ular compartments, the endosomes (29,33). The chan-el forming activity of PA63 in lipid bilayers probablyrings about the translocation of EF and LF across thendosomal membrane into the cytosol (23). EF is aalcium/calmodulin-dependent adenylate cyclase,hich increases the intracellular cAMP levels, thereby

ausing edema (20,40). Lethal toxin as the name sug-ests is lethal for several species (7,8,38). Mouse peri-oneal macrophages and macrophage-like cell linesuch as J774A.1, RAW 264.7, etc., are sensitive tonthrax lethal toxin (7). It causes over-production ofertain cytokines such as IL-1b and TNF-a in its targetells (13). Recent reports indicate that LF acts as anndopeptidase. It cleaves the amino terminus of mito-en-activated protein kinase kinases 1 (MAPKK1),hich contain the MAPK binding site. LF, thus pre-

369

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370 GUPTA, WAHEED, AND BHATNAGAR

nd inhibits the MAPK signal transduction pathway.owever, the exact mechanism of cell death is not yet

stablished (5).In the former USSR live spore vaccine of Sterne is

eing used while in UK currently available vaccineonsists of alum-precipitated cell-free filtrate of Sternetrain (12,28,36,41). In the US, the vaccine is alumi-um hydroxide-adsorbed cell-free filtrate of cultures ofnoncapsulating nonproteolytic strain of B. anthracis

15,16,41). In all these currently available vaccines, PAs the major component of vaccine against anthrax9,18). Although these vaccines have proven effica-ious, however, they have certain limitations. Vaccinesary from lot to lot, depending on levels of PA produc-ion and presence of impurities such as traces of activeoxin components LF and EF (3,12,17,28). Apart fromhis, immunization requires six booster doses followedy annual boosters. Therefore, for developing a betteruman anthrax vaccine homogenous preparations ofA are essential (4,6,25).The genes for all three protein exotoxins have been

loned and sequenced (20,23,42,44). Culture superna-ants of B. anthracis have been the major source forurifying PA (21,31). However, working with B. an-hracis cultures requires P-3 containment facilities.part from this, PA preparation from B. anthracis isften contaminated with LF or EF. Earlier workersave tried to express and purify the PA from otherxpression hosts such as B. subtilis, baculovirus, etc.urification of PA from B. subtilis required aerobicrowth in rich media and enormous amount of PA wasegraded due to a number of proteases secreted by B.ubtilis (18,23). Baculovirus vectors expressed PA innsect cells; however, purification could not be possibleue to low yields (14). Although PA has been expressedn Escherichia coli, attempts were not very successfuln purifying the protein as it involved conventionalurification strategies. The protein undergoes exten-ive degradation during the purification process30,32,35). Our recent efforts are focused on developingsystem for rapid and efficient purification of PA. Weave expressed the full-length PA gene as a fusionrotein with 63 Histidine residues in E. coli. Recom-inant protein was purified using metal chelate affinityhromatography and anion exchange chromatography.he studies reported here are a part of continuingesearch that will lead to the development of moreotent minimally reactogenic vaccine against anthrax.

ATERIALS AND METHODS

eagents and Supplies

The enzymes and chemicals used for DNA manipu-ation were purchased from Boehringer-MannheimGermany); Life Technologies (U.S.A.); Amersham Inc.UK); and New England Biolabs (U.S.A.). The oligonu-

echnologies for Molecular Medicine, Yale Universityedical School, U.S.A.). The PCR was performed onerkin–Elmer thermal cycler using DNA amplificationit from Perkin–Elmer (U.S.A.). DNA purification kit,el extraction kit, expression vector pQE30, E. coliG13009 cells, and Ni-NTA agarose were obtained

rom Qiagen (Germany). Agarose (Sea Kem GTG) wasrom FMC Corp. (U.S.A.). Mono-Q column was pur-hased from Pharmacia Biotech (Sweden). Cell culturelasticwares were obtained from Corning (U.S.A.).PMI 1640, Dulbecco’s modified Eagle’s medium

DMEM), Hank’s balanced salt solution (HBSS), fetalalf serum (FCS), trypsin, 3-(4,5-dimethylthiazol-2-yl),--diphenyltetrazolium bromide (MTT), bovine serumlbumin (BSA) 3-{(3-cholamidopropyl) dimethyl ammo-io}-1- propanesulfonic acid (Chaps), isopropyl-thio-b--galactopyranoside (IPTG), and other chemicals wereurchased from Sigma Chemical Co. (U.S.A.). J774A.1,macrophage-like cell line was obtained from Ameri-

an Type Culture Collection (ATCC) (U.S.A.). Mediaomponents for bacterial growth were purchased fromi-Media Laboratories (India). PA purified from B.nthracis was obtained as a generous gift from Dr.tephen H. Leppla (NIDR, NIH, U.S.A.).

lasmid Construction

PA was expressed as fusion protein with 63 Histi-ine affinity tag using the vector pQE30 (QiaexpressIAGEN). This vector contains T5 promoter for high

evel expression, ribosome binding site, 63 Histidineoding sequences, followed by a multiple cloning site.he vector also contains two lac operator sequences.or high level expression, SG13009 (pREP4) cells con-aining multiple copies of plasmid pREP4, which car-ies lacIq gene encoding the lac repressor, were used.he plasmid pREP4 contains a kanamycin resistanceene as a selection marker.Plasmids pXO1 and pQE30 were purified using DNA

urification kit as described in the manual. The PAene was amplified by polymerase chain reactionPCR), using pXO1 (26) as a template and primers thatdded BamHI and KpnI sites to the 59 and 39 ends ofhe PCR product respectively (Fig. 1). The amplifiedCR product and plasmid pQE30 were digested withestriction enzymes BamHI and KpnI. The digestedroducts were separated on 1% agarose gel. The bandsere excised and the DNA was eluted using the gelxtraction kit. The digested PCR product and the vec-or were ligated overnight at 14°C and transformednto E. coli SG13009 (pREP4) competent cells. Prepa-ation and transformation of competent E. coliG13009 bacteria were performed according to proce-ures described by Maniatis et al. (34). The transfor-ation mixture was plated on Luria agar plates con-

aining 100 mg of ampicillin per milliliter and 25 mg of

kanamycin per milliliter. The plates were incubated for1spbd

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371PURIFICATION OF THE RECOMBINANT PROTECTIVE ANTIGEN

6 h at 37°C. Colonies appearing on the plate werecreened for the recombinant plasmid pMW1 by mini-reparations of plasmid DNA (34). The desired recom-inant plasmid was confirmed by restriction enzymeigestion with BamHI and KpnI.

xpression of Protective Antigen

For localizing the expressed recombinant protein inhe cell, the E. coli strain SG13009 (pREP4) carryinghe recombinant plasmid pMW1 was grown at 37°C inuria broth with 100 mg of ampicillin per milliliter and5 mg of kanamycin per milliliter at 250 rpm in 500-mlasks. When A600 reached 1.0, IPTG was added to anal concentration of 1 mM. After 5 h of induction, cellsere harvested by centrifugation at 4000g for 20 min.eriplasm, cytosol, and inclusion bodies were checked

or the presence of PA. To check for periplasmic local-zation the pellet from 100 ml culture was resuspendedn 10 ml 30 mM Tris–Cl, EDTA 1 mM, 20 % sucrose, pH.0, and incubated in ice for 10–15 min in 50-ml tubes.ample was centrifuged at 8000g at 4°C for 10 min.upernatant was removed and the pellet was resus-ended gently in 10 ml ice-cold 5 mM MgSO4 andncubated in ice for 10 min. Tubes were centrifuged at000g at 4°C for 10 min. The supernatant (periplasmicxtract) was collected. To check for cytosolic localiza-ion of the recombinant protein, the pellet from 100 mlulture was resuspended in 5 ml of sonication buffer50 mM Na-phosphate, pH 7.8, 300 mM NaCl). Cellsere sonicated at 4°C (1-min bursts/1-min cooling/00–300 W) for 5 cycles. The lysate was centrifuged at0,000g for 30 min. Supernatant (cytosolic extract) wasollected while the pellet (insoluble matter) was solu-ilized in 5 ml of 8 M urea, 0.1 M Na-phosphate, 0.01 Mris–Cl, pH 8.0, by stirring for 1 h at room tempera-ure. Tubes were centrifuged at 10,000g for 30 min,nd the supernatant (inclusion bodies) was collected.xpression of PA was established by SDS–PAGE andestern blot analyses of the samples collected.

urification of Protective Antigen

As PA was mainly localized in the inclusion bodies,he protein was purified under denaturing conditions.he pellet from 2 liters of culture was resuspended in0 ml of buffer containing 6 M GuHCl, 0.1 M Na-hosphate, pH 7.8, and 300 mM NaCl. Cells weretirred at room temperature for 1 h. Lysate was cen-rifuged at 10,000g for 30 min at 4°C. The supernatantas mixed with 8 ml of 50% slurry of Ni-NTA resin andllowed to stir at room temperature for 45 min, andhen the resin was loaded carefully into a 1.6-cm diam-ter column. The column was washed with 10 columnol of buffer containing 8 M urea, 0.1 M Na-phosphate,H 7.8, and 300 mM NaCl. The resin was then washedith a gradient of 8 to 0 M urea in buffer containing 0.1

ate the slow removal of the urea. The resin was thenashed with a buffer containing 0.1 M Na-phosphate,H 6.0, and 500 mM NaCl. The recombinant proteinas eluted with a gradient of 0–500 mM Imidazole in.1 mM Na-phosphate, pH 7.0, and 10% glycerol. Theractions were analyzed on SDS–PAGE and those con-aining the protein were pooled and dialyzed against

10E5 (Tris 10 mM and EDTA 5 mM, pH 8.0) buffer.he dialyzed sample was loaded onto the Mono-Q col-mn. The protein was eluted with a gradient of 0 to 1

NaCl in T10E5. The rPA was dialyzed against 10 mMepes overnight and stored in aliquots at 270°C.

uantitation of PA

The fold purification of PA at different column stagesas determined by calculating the amount of protein

equired to kill 50% of J774A.1 cells (EC50) when incu-ated with LF (1 mg/ml) at 37°C. The protein waseasured by the method of Lowry et al. (24).

eceptor Binding Assay

Receptor binding assay was performed with J774A.1ells plated in 12-well plates using the radio-iodinatedA as described earlier (1). In brief, cells were cooled

or 15 min and the medium was replaced with coldinding medium (DMEM containing 1% BSA and 25M Hepes, pH 7.4). Native PA (125I-PA, 0.95 3 107

pm/mg)/recombinant PA (125I-PA, 1.12 3 107 cpm/mg) 1g/ml was added in the binding medium and cells were

ncubated for 12 h at 4°C. The cells were washed fourimes with cold HBSS, solubilized in 0.5 ml of 100 mMaOH, and counts were taken in a gamma counter.onspecific binding of 125I-PA to cells was determinedy incubating the cells with 100-fold excess of PA.rotein content of the cells/well was 1.12 6 0.05 mg.rom the amount of radioactivity bound to cells,mount of PA bound per milligram of cell protein wasetermined.

n Vitro Binding of Recombinant PA to LF

To study the binding of PA to LF, PA molecule wasleaved with trypsin. PA (native as well as recombi-ant) at 1.0 mg/ml was incubated with trypsin (1 ng/mgrotein) for 30 min at 25°C in 25 mM Hepes, 1 mMaCl2, and 0.5 mM EDTA (11). Trypsin was inacti-ated by adding PMSF (1 mM). Trypsin nicked PA (1g/ml) was incubated with LF (1 mg/ml) in Tris, pH 9.0,ontaining 2 mg/ml Chaps for 15 min. Samples werepplied to 4–15% polyacrylamide gradient Phast gelsPharmacia LKB Biotechnology Ltd., native buffertrips). Gels were stained in Coomassie brilliant blue-250, destained, and dried (11).

Cell Culture and Cytotoxicity Assay

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372 GUPTA, WAHEED, AND BHATNAGAR

The biological activity of the rPA was determined byhe cytotoxicity assay (2). Cytotoxicity was determinedy percentage viability of J774A.1 cells after incuba-ion with anthrax toxin using MTT dye. Macrophage-ike cell line J774A.1 was maintained in RPMI 1640

edium containing 10% heat-inactivated FCS. The celluspension was plated at 150 ml/well in 96-well flat-ottom plates, and cells were allowed to adhere byncubation at 37°C for 16 h (95% humidity and 5%O2). The next day medium and detached cells wereemoved by gentle aspiration and replaced (100 ml/ell) with RPMI containing 1.0 mg/ml LF and varying

oncentrations of PA purified from either B. anthracisr E. coli and incubated for 3 h at 37°C in a humidifiedO2 incubator. All experiments were done in tripli-

ates. After 3 h MTT dye dissolved in RPMI was addedo the cells to a final concentration of 0.5 mg/ml and theells were incubated for 30 min at 37°C to allow uptakend oxidation of the dye by viable cells. The mediumas replaced by 100 ml of 0.5% (w/v) sodium dodecyl

ulfate, 25 mM HCl in 90% isopropyl alcohol, and vor-exed to dissolve the precipitate. The absorptionas read at 540 nm using a microplate reader

Nunc.GMBH) and percentage cytotoxicity was calcu-ated (2).

ESULTS AND DISCUSSION

The main objective of this study was to express andurify functionally as well as biologically active PArom E. coli. To date culture supernatants of B. anthra-is have been the major source for purification of an-hrax toxin proteins (21,22,23). Although PA has beenurified from other sources such as B. subtilis andaculovirus etc. (17,23); however, purification of PA

rom E. coli has not been very successful due to exten-ive degradation of PA and the presence of largemount of E. coli proteins in comparison to PA. Singht al. tried purification of PA by guiding it to theeriplasm (35). Their purification procedure involvedime-consuming multistep conventional chromato-raphic techniques and their yield was 0.5 mg/liter. Inhis study we have described two-step purification in-olving metal chelate affinity chromatography and an-on exchange Mono-Q column on FPLC for rapid andfficient purification of PA and the yield from thisrocedure is 2 mg/liter of the culture.

xpression of Recombinant PA

The structural gene for the protective antigen wasloned in the BamHI and KpnI sites of the vectorQE30 to generate the construct pMW1 (Fig. 1). Thexpression vector pQE30 has a six-histidine codingequence added upstream of the multiple cloning sites.. coli SG13009 (pREP4) cells were transformed with

he recombinant plasmid pMW1. To establish the ex-ression of the recombinant protein, cells were grownnd induced by 1 mM IPTG. The presence of PA wasetermined by SDS–PAGE and Western blotting anal-ses of periplasm, cytosol, and inclusion bodies. Therotein was getting hyperexpressed and aggregating toorm inclusion bodies (Fig. 2).

urification of rPA

The recombinant PA was purified from inclusionodies under denaturing conditions. Cells containinghe recombinant construct were grown to an OD600 0.7o 1.0 and induced with 1 mM IPTG. After 5 h ofnduction, cells were harvested and the cell pellet wasysed by 6 M Gu.HCl. The inclusion bodies are solublen 6 M Gu.HCl or 8 M urea. Cell lysate was removed byentrifugation and the supernatant was stirred withi-NTA slurry for 45 min at room temperature. The

lurry was loaded onto a column and the denaturantas changed to 8 M urea by washing the slurry with 10

IG. 1. Construction of plasmid used for the expression of PA. Leftanel: plasmid pQE30, an expression vector containing P, T5 poly-erase promoter; R, synthetic ribosome binding site; ■, 63 Histidine

ffinity tag sequence; MCS, multiple cloning site; T, transcriptionalerminator; O, origin of replication and ampr, ampicillin resistanceene. BKXSPH in MCS are sites for restriction endonucleasesamHI, KpnI, XmnI, SalI, PstI, and HindIII, respectively. Rightanel: plasmid pXO1 containing the entire native PA gene (pag). TheA gene was amplified by PCR with new restriction sites added athe ends by primers P1 and P2 and cloned into the BamHI and KpnIites of pQE30 to generate construct pMW1. Sequence of the primers1 and P2 are 59 GCG CAG GCC GGA TCC GAA GTT AAA CAG 39nd 59 CCT AGA GGT ACC TTA TCC 39, respectively.

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373PURIFICATION OF THE RECOMBINANT PROTECTIVE ANTIGEN

olumn vol of 8 M urea. The protein was then rena-ured by the slow and gradient removal of the ureahile immobilized on the Ni-NTA column. The slurryas washed with a gradient of 8–0 M urea in sodiumhosphate buffer, pH 7.8. The advantage of using suchrefolding approach where the proteins are immobi-

ized on a column is that aggregation is prevented.roteins are immobilized so that the hydrophobic facesxposed in partially folded and folding proteins arehen not free to associate with one another. The rena-ured protein was then washed with sodium-phosphateuffer of pH 6.0. At pH 6.0 most of the contaminatingroteins were removed without affecting the binding ofhe recombinant protein to the Ni-NTA. The proteinas then eluted with a gradient of imidazole. Therotein eluted at 100–200 mM Imidazole concentra-ion. The protein after affinity chromatography wasbout 70% pure with small amount of degraded prod-

IG. 2. Electrophoretic analysis of E. coli-expressed PA. Proteinsere separated by 12% SDS–PAGE and stained with Coomassie blue

a) and Western blot of the E. coli proteins containing PA, developedith a rabbit polyclonal PA antibody (b). Lane A, E. coli SG13009

ells without the vector; Lane B, cells containing the vector pQE30ithout the PA gene; Lane C, cells containing the construct pMW1

uninduced); Lane D, cells expressing PA; Lane E, periplasmic pro-eins of cells expressing PA; Lane F, cytosolic proteins of cells ex-ressing PA; Lane G, inclusion bodies of cells expressing PA; Lane H,A purified from B. anthracis; and Lane M, molecular weight stan-ards.

cts and other impurities. The pooled fractions con-aining affinity purified PA were dialyzed against

10E5 buffer and subjected to anion exchange chroma-ography using Mono-Q column on FPLC. The proteinas eluted with a gradient of NaCl and was 95% purefter anion exchange chromatography (Fig. 3). The rPAas purified 3023-fold compared to the protein in the

ell lysate (Table 1). Meanwhile we also tried varioustrategies to avoid the formation of inclusion bodiesuch as growing the cultures at 28°C, reducing thencubation period after induction, and induction by lowPTG concentrations (0.1–0.5 mM). To purify the pro-ein under native conditions, the cell pellet was resus-ended in sonication buffer and sonicated at 4°C (1-in bursts/1-min cooling/200 –300 Watts) for five

ycles. The lysate was centrifuged at 10,000g andixed with 5 ml of Ni-NTA slurry. The slurry was

acked into a column (5.0 3 1.6 cm) and allowed toettle. The matrix was washed with a Na-phosphate,

TABLE 1

Purification of PA from Escherichia coli

FractionsVolume

(ml)Protein(mg/ml)

Activity(EC50)a

Purification(fold)b

ell lysatec 50 115.84 75.580 1ffinity purification 10 0.65 0.040 1890PLC 2 2.0 0.025 3023

a EC50 is defined as the concentration of PA (mg/ml) along with LF1 mg/ml) required to kill 50% of the J774A.1 cells. After 3 h ofncubation, viability was determined by MTT dye. The results rep-esent the mean of three experiments.

b Purification fold was determined by dividing EC50 for cell lysateith EC50 for fractions obtained from different columns.c Cell lysate prepared from 2 liters of culture.

IG. 3. Purification of E. coli-expressed PA. The proteins werenalyzed on 10 % SDS–PAGE and stained with Coomassie blue.ane A, Uninduced E. coli SG13009 cells; Lane B, cell lysate of cellsxpressing PA; Lane C, proteins after Ni-NTA affinity purification;ane D, PA after passing through Mono-Q column on FPLC; Lane E,A purified from B. anthracis; and Lane M, molecular weight stan-ards.

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374 GUPTA, WAHEED, AND BHATNAGAR

H 6.0, buffer. Protein was eluted with gradients of 0nd 500 mM imidazole chloride. Fractions containinghe protein were further purified using anion exchangeono-Q column on FPLC. However, the yields after

urification were considerably reduced as the proteinnderwent extensive degradation and we could onlyurify 0.7–0.8 mg of rPA per liter of the bacterialulture. Thus we had to revert purifying the proteinnder denaturing conditions.

omparison of rPA with nPA

The rPA was compared to PA from B. anthracis byeceptor binding assay, by determining in vitro bindingf trypsin-nicked PA to LF, and finally by macrophageysis assay. To determine whether the rPA binds to cellurface receptors, J774A.1 cells were incubated with

IG. 4. Binding of PA to LF protein in solution. LF (1 mg) wasncubated with trypsin-nicked PA (1 mg) for 15 min and the samplesere analyzed on a nondenaturing 4–15% Phast gradient gel. Theel was stained with Coomassie blue. Lane A, PA purified from B.nthracis; Lane B, PA from E. coli; Lane C, LF from B. anthracis;ane D, PA from B. anthracis nicked with trypsin and incubatedith LF; and Lane E, PA from E. coli nicked with trypsin incubatedith LF.

Binding of PA to Cell Surface Receptorsa

Protein CPM PA (ng)PA/Cell Proteinb

(ng/mg)

nPA 76608 6 1254 8.06 6 0.12 7.20 6 0.12c

rPA 86464 6 1616 7.72 6 0.15 6.89 6 0.15

a J774A.1 cells were incubated with 1 mg of 125I-PA (nPA, nativeA, or rPA recombinant PA) for 12 h at 4°C. Cells were washed withBSS and solubilized in 100 mM NaOH. Radioactivity was counted

n a gamma counter.b Protein content of the cells per well was 1.12 6 0.05 mg as

etermined by the method of Lowry et al. (24).c The values are means 6 SD and are representative of three

ndividual experiments done in triplicate.

BSS, solubilized in 100 mM NaOH, and the radioac-ivity was measured. The protein content of the cellser well was 1.12 6 0.05 mg. From the amount ofadioactivity bound per milligram cell protein, PAound per milligram was also calculated. About 7.2 ngf the native PA bound per milligram cell protein,hile the binding of recombinant PA was 6.9 ng/mg cellrotein (Table 2). PA cleaved by trypsin has the abilityo bind to LF in vitro. Recombinant as well as nativeA was digested with trypsin and incubated with LF inbuffer containing Chaps. Samples were analyzed on 4

o 15% polyacrylamide gradient phast gels. It was ob-erved that like nPA, rPA could bind to LF and theobility of PA63 LF complex was retarded on nonde-

aturing phast gels (Fig. 4). To determine whether thePA is biologically active or not we performed the cy-otoxicity assay. Macrophage-like cell line J774A.1,hich is sensitive to anthrax lethal toxin, was used (5).arious concentrations of rPA or nPA were added to

IG. 5. Biological activity of PA purified from B. anthracis and E.oli. J774A.1 cells were incubated with varying concentrations of PAlone or in combination of LF (1 mg/ml) for 3 h at 37°C. Cell viabilityas determined by MTT assay as described under Materials andethods. E, PA from B. anthracis, Œ, PA from E. coli alone, and F,A from B. anthracis in combination of LF; �, PA purified from E.oli in combination of LF.

the cells in combination with LF (1 mg/ml) and incu-bazt5mwia

intstrtthpf

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Ivins, B. E. (1985) Demonstration of a capsule plasmid in B.

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375PURIFICATION OF THE RECOMBINANT PROTECTIVE ANTIGEN

ated for 3 h. After 3 h, viability was determined bydding MTT dye. Live cells oxidized the dye to forma-on crystals while the dead cells did not. The precipi-ate was solubilized and optical density was read at40 nm from which percentage cytotoxicity was deter-ined (Fig. 5). It was observed that rPA along with LFere able to lyse the macrophages and showed biolog-

cal activity comparable to that of PA obtained from B.nthracis.The currently available vaccine is unsatisfactory as

ts composition is not very well defined and the immu-ity is not long lasting and it has reactogenicity due tohe presence of contaminants such as LF or EF. Re-earch shows that these vaccines may not give protec-ion against all natural strains of anthrax. For thiseason there is a need to focus attention to addresshese problems and the present work is an attempt inhis direction. The work has been focused to obtainighly purified, minimally reactogenic recombinantrotective antigen for an attractive alternative in theuture anthrax vaccine.

CKNOWLEDGMENT

This work was supported by Department of Biotechnology, Gov-rnment of India, New Delhi.

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