Expression and Purification of the Recombinant Protective Antigen of Bacillus anthracis

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

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 aggregating 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 chromatography. 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 molecular mass of 83 kDa as determined by SDS–PAGE. Antisera 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 anthracis. Human beings acquire it via infected animals or contaminated animal products. Virulence of the bacteria is due to two major antigens viz., antiphagocytic capsular antigen, which is unique among bacterial capsules 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 reasons. Anthrax toxin has three components: protective antigen (PA; 83 kDa), lethal factor (LF; 90 kDa), and 1

Both the authors have contributed equally to the paper. To whom correspondence and reprint requests may be addressed. Fax: (91) 11-6198234. E-mail: [email protected] and [email protected]. 2

1046-5928/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.

edema factor (EF; 89 kDa), of which no single component 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 another plasmid, pXO2, contains the genes responsible for poly-D-glutamic acid capsule synthesis (27). Anthrax 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 respects. First, A and B domains are two distinct proteins. Second, the LF and EF have the identical PA binding domains. Thus each of the catalytic components 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 cleavage 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 receptormediated endocytosis into the lumen of acidic intracellular compartments, the endosomes (29,33). The channel 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 suggests is lethal for several species (7,8,38). Mouse peritoneal 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 mitogen-activated protein kinase kinases 1 (MAPKK1), which contain the MAPK binding site. LF, thus pre369

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vents the association of MAPKK1 with its substrate and inhibits the MAPK signal transduction pathway. However, the exact mechanism of cell death is not yet established (5). In the former USSR live spore vaccine of Sterne is being used while in UK currently available vaccine consists of alum-precipitated cell-free filtrate of Sterne strain (12,28,36,41). In the US, the vaccine is aluminum hydroxide-adsorbed cell-free filtrate of cultures of a noncapsulating nonproteolytic strain of B. anthracis (15,16,41). In all these currently available vaccines, PA is the major component of vaccine against anthrax (9,18). Although these vaccines have proven efficacious, however, they have certain limitations. Vaccines vary from lot to lot, depending on levels of PA production and presence of impurities such as traces of active toxin components LF and EF (3,12,17,28). Apart from this, immunization requires six booster doses followed by annual boosters. Therefore, for developing a better human anthrax vaccine homogenous preparations of PA are essential (4,6,25). The genes for all three protein exotoxins have been cloned and sequenced (20,23,42,44). Culture supernatants of B. anthracis have been the major source for purifying PA (21,31). However, working with B. anthracis cultures requires P-3 containment facilities. Apart from this, PA preparation from B. anthracis is often contaminated with LF or EF. Earlier workers have tried to express and purify the PA from other expression hosts such as B. subtilis, baculovirus, etc. Purification of PA from B. subtilis required aerobic growth in rich media and enormous amount of PA was degraded due to a number of proteases secreted by B. subtilis (18,23). Baculovirus vectors expressed PA in insect cells; however, purification could not be possible due to low yields (14). Although PA has been expressed in Escherichia coli, attempts were not very successful in purifying the protein as it involved conventional purification strategies. The protein undergoes extensive degradation during the purification process (30,32,35). Our recent efforts are focused on developing a system for rapid and efficient purification of PA. We have expressed the full-length PA gene as a fusion protein with 63 Histidine residues in E. coli. Recombinant protein was purified using metal chelate affinity chromatography and anion exchange chromatography. The studies reported here are a part of continuing research that will lead to the development of more potent minimally reactogenic vaccine against anthrax. MATERIALS AND METHODS

Reagents and Supplies The enzymes and chemicals used for DNA manipulation were purchased from Boehringer-Mannheim (Germany); Life Technologies (U.S.A.); Amersham Inc. (UK); and New England Biolabs (U.S.A.). The oligonu-

cleotides were obtained from Monica Talmor (Critical Technologies for Molecular Medicine, Yale University Medical School, U.S.A.). The PCR was performed on Perkin–Elmer thermal cycler using DNA amplification kit from Perkin–Elmer (U.S.A.). DNA purification kit, gel extraction kit, expression vector pQE30, E. coli SG13009 cells, and Ni-NTA agarose were obtained from Qiagen (Germany). Agarose (Sea Kem GTG) was from FMC Corp. (U.S.A.). Mono-Q column was purchased from Pharmacia Biotech (Sweden). Cell culture plasticwares were obtained from Corning (U.S.A.). RPMI 1640, Dulbecco’s modified Eagle’s medium (DMEM), Hank’s balanced salt solution (HBSS), fetal calf serum (FCS), trypsin, 3-(4,5-dimethylthiazol-2-yl),5-diphenyltetrazolium bromide (MTT), bovine serum albumin (BSA) 3-{(3-cholamidopropyl) dimethyl ammonio}-1- propanesulfonic acid (Chaps), isopropyl-thio-bD-galactopyranoside (IPTG), and other chemicals were purchased from Sigma Chemical Co. (U.S.A.). J774A.1, a macrophage-like cell line was obtained from American Type Culture Collection (ATCC) (U.S.A.). Media components for bacterial growth were purchased from Hi-Media Laboratories (India). PA purified from B. anthracis was obtained as a generous gift from Dr. Stephen H. Leppla (NIDR, NIH, U.S.A.). Plasmid Construction PA was expressed as fusion protein with 63 Histidine affinity tag using the vector pQE30 (Qiaexpress QIAGEN). This vector contains T5 promoter for high level expression, ribosome binding site, 63 Histidine coding sequences, followed by a multiple cloning site. The vector also contains two lac operator sequences. For high level expression, SG13009 (pREP4) cells containing multiple copies of plasmid pREP4, which carries lacI q gene encoding the lac repressor, were used. The plasmid pREP4 contains a kanamycin resistance gene as a selection marker. Plasmids pXO1 and pQE30 were purified using DNA purification kit as described in the manual. The PA gene was amplified by polymerase chain reaction (PCR), using pXO1 (26) as a template and primers that added BamHI and KpnI sites to the 59 and 39 ends of the PCR product respectively (Fig. 1). The amplified PCR product and plasmid pQE30 were digested with restriction enzymes BamHI and KpnI. The digested products were separated on 1% agarose gel. The bands were excised and the DNA was eluted using the gel extraction kit. The digested PCR product and the vector were ligated overnight at 14°C and transformed into E. coli SG13009 (pREP4) competent cells. Preparation and transformation of competent E. coli SG13009 bacteria were performed according to procedures described by Maniatis et al. (34). The transformation mixture was plated on Luria agar plates containing 100 mg of ampicillin per milliliter and 25 mg of

PURIFICATION OF THE RECOMBINANT PROTECTIVE ANTIGEN

kanamycin per milliliter. The plates were incubated for 16 h at 37°C. Colonies appearing on the plate were screened for the recombinant plasmid pMW1 by minipreparations of plasmid DNA (34). The desired recombinant plasmid was confirmed by restriction enzyme digestion with BamHI and KpnI. Expression of Protective Antigen For localizing the expressed recombinant protein in the cell, the E. coli strain SG13009 (pREP4) carrying the recombinant plasmid pMW1 was grown at 37°C in Luria broth with 100 mg of ampicillin per milliliter and 25 mg of kanamycin per milliliter at 250 rpm in 500-ml flasks. When A 600 reached 1.0, IPTG was added to a final concentration of 1 mM. After 5 h of induction, cells were harvested by centrifugation at 4000g for 20 min. Periplasm, cytosol, and inclusion bodies were checked for the presence of PA. To check for periplasmic localization the pellet from 100 ml culture was resuspended in 10 ml 30 mM Tris–Cl, EDTA 1 mM, 20 % sucrose, pH 8.0, and incubated in ice for 10 –15 min in 50-ml tubes. Sample was centrifuged at 8000g at 4°C for 10 min. Supernatant was removed and the pellet was resuspended gently in 10 ml ice-cold 5 mM MgSO 4 and incubated in ice for 10 min. Tubes were centrifuged at 8000g at 4°C for 10 min. The supernatant (periplasmic extract) was collected. To check for cytosolic localization of the recombinant protein, the pellet from 100 ml culture was resuspended in 5 ml of sonication buffer (50 mM Na-phosphate, pH 7.8, 300 mM NaCl). Cells were sonicated at 4°C (1-min bursts/1-min cooling/ 200 –300 W) for 5 cycles. The lysate was centrifuged at 10,000g for 30 min. Supernatant (cytosolic extract) was collected while the pellet (insoluble matter) was solubilized in 5 ml of 8 M urea, 0.1 M Na-phosphate, 0.01 M Tris–Cl, pH 8.0, by stirring for 1 h at room temperature. Tubes were centrifuged at 10,000g for 30 min, and the supernatant (inclusion bodies) was collected. Expression of PA was established by SDS–PAGE and Western blot analyses of the samples collected.

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M Na-phosphate, pH 7.8, and 300 mM NaCl to facilitate the slow removal of the urea. The resin was then washed with a buffer containing 0.1 M Na-phosphate, pH 6.0, and 500 mM NaCl. The recombinant protein was eluted with a gradient of 0 –500 mM Imidazole in 0.1 mM Na-phosphate, pH 7.0, and 10% glycerol. The fractions were analyzed on SDS–PAGE and those containing the protein were pooled and dialyzed against T 10E 5 (Tris 10 mM and EDTA 5 mM, pH 8.0) buffer. The dialyzed sample was loaded onto the Mono-Q column. The protein was eluted with a gradient of 0 to 1 M NaCl in T 10E 5. The rPA was dialyzed against 10 mM Hepes overnight and stored in aliquots at 270°C. Quantitation of PA The fold purification of PA at different column stages was determined by calculating the amount of protein required to kill 50% of J774A.1 cells (EC 50) when incubated with LF (1 mg/ml) at 37°C. The protein was measured by the method of Lowry et al. (24). Receptor Binding Assay Receptor binding assay was performed with J774A.1 cells plated in 12-well plates using the radio-iodinated PA as described earlier (1). In brief, cells were cooled for 15 min and the medium was replaced with cold binding medium (DMEM containing 1% BSA and 25 mM Hepes, pH 7.4). Native PA ( 125I-PA, 0.95 3 10 7 cpm/mg)/recombinant PA ( 125I-PA, 1.12 3 10 7 cpm/mg) 1 mg/ml was added in the binding medium and cells were incubated for 12 h at 4°C. The cells were washed four times with cold HBSS, solubilized in 0.5 ml of 100 mM NaOH, and counts were taken in a gamma counter. Nonspecific binding of 125I-PA to cells was determined by incubating the cells with 100-fold excess of PA. Protein content of the cells/well was 1.12 6 0.05 mg. From the amount of radioactivity bound to cells, amount of PA bound per milligram of cell protein was determined.

Purification of Protective Antigen As PA was mainly localized in the inclusion bodies, the protein was purified under denaturing conditions. The pellet from 2 liters of culture was resuspended in 50 ml of buffer containing 6 M GuHCl, 0.1 M Naphosphate, pH 7.8, and 300 mM NaCl. Cells were stirred at room temperature for 1 h. Lysate was centrifuged at 10,000g for 30 min at 4°C. The supernatant was mixed with 8 ml of 50% slurry of Ni-NTA resin and allowed to stir at room temperature for 45 min, and then the resin was loaded carefully into a 1.6-cm diameter column. The column was washed with 10 column vol of buffer containing 8 M urea, 0.1 M Na-phosphate, pH 7.8, and 300 mM NaCl. The resin was then washed with a gradient of 8 to 0 M urea in buffer containing 0.1

In Vitro Binding of Recombinant PA to LF To study the binding of PA to LF, PA molecule was cleaved with trypsin. PA (native as well as recombinant) at 1.0 mg/ml was incubated with trypsin (1 ng/mg protein) for 30 min at 25°C in 25 mM Hepes, 1 mM CaCl 2, and 0.5 mM EDTA (11). Trypsin was inactivated by adding PMSF (1 mM). Trypsin nicked PA (1 mg/ml) was incubated with LF (1 mg/ml) in Tris, pH 9.0, containing 2 mg/ml Chaps for 15 min. Samples were applied to 4 –15% polyacrylamide gradient Phast gels (Pharmacia LKB Biotechnology Ltd., native buffer strips). Gels were stained in Coomassie brilliant blue R-250, destained, and dried (11).

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Cell Culture and Cytotoxicity Assay The biological activity of the rPA was determined by the cytotoxicity assay (2). Cytotoxicity was determined by percentage viability of J774A.1 cells after incubation with anthrax toxin using MTT dye. Macrophagelike cell line J774A.1 was maintained in RPMI 1640 medium containing 10% heat-inactivated FCS. The cell suspension was plated at 150 ml/well in 96-well flatbottom plates, and cells were allowed to adhere by incubation at 37°C for 16 h (95% humidity and 5% CO 2). The next day medium and detached cells were removed by gentle aspiration and replaced (100 ml/ well) with RPMI containing 1.0 mg/ml LF and varying concentrations of PA purified from either B. anthracis or E. coli and incubated for 3 h at 37°C in a humidified CO 2 incubator. All experiments were done in triplicates. After 3 h MTT dye dissolved in RPMI was added to the cells to a final concentration of 0.5 mg/ml and the cells were incubated for 30 min at 37°C to allow uptake and oxidation of the dye by viable cells. The medium was replaced by 100 ml of 0.5% (w/v) sodium dodecyl sulfate, 25 mM HCl in 90% isopropyl alcohol, and vortexed to dissolve the precipitate. The absorption was read at 540 nm using a microplate reader (Nunc.GMBH) and percentage cytotoxicity was calculated (2). RESULTS AND DISCUSSION

The main objective of this study was to express and purify functionally as well as biologically active PA from E. coli. To date culture supernatants of B. anthracis have been the major source for purification of anthrax toxin proteins (21,22,23). Although PA has been purified from other sources such as B. subtilis and Baculovirus etc. (17,23); however, purification of PA from E. coli has not been very successful due to extensive degradation of PA and the presence of large amount of E. coli proteins in comparison to PA. Singh et al. tried purification of PA by guiding it to the periplasm (35). Their purification procedure involved time-consuming multistep conventional chromatographic techniques and their yield was 0.5 mg/liter. In this study we have described two-step purification involving metal chelate affinity chromatography and anion exchange Mono-Q column on FPLC for rapid and efficient purification of PA and the yield from this procedure is 2 mg/liter of the culture. Expression of Recombinant PA The structural gene for the protective antigen was cloned in the BamHI and KpnI sites of the vector pQE30 to generate the construct pMW1 (Fig. 1). The expression vector pQE30 has a six-histidine coding sequence added upstream of the multiple cloning sites. E. coli SG13009 (pREP4) cells were transformed with

FIG. 1. Construction of plasmid used for the expression of PA. Left panel: plasmid pQE30, an expression vector containing P, T5 polymerase promoter; R, synthetic ribosome binding site; ■, 63 Histidine affinity tag sequence; MCS, multiple cloning site; T, transcriptional terminator; O, origin of replication and amp r, ampicillin resistance gene. BKXSPH in MCS are sites for restriction endonucleases BamHI, KpnI, XmnI, SalI, PstI, and HindIII, respectively. Right panel: plasmid pXO1 containing the entire native PA gene (pag). The PA gene was amplified by PCR with new restriction sites added at the ends by primers P1 and P2 and cloned into the BamHI and KpnI sites of pQE30 to generate construct pMW1. Sequence of the primers P1 and P2 are 59 GCG CAG GCC GGA TCC GAA GTT AAA CAG 39 and 59 CCT AGA GGT ACC TTA TCC 39, respectively.

the recombinant plasmid pMW1. To establish the expression of the recombinant protein, cells were grown and induced by 1 mM IPTG. The presence of PA was determined by SDS–PAGE and Western blotting analyses of periplasm, cytosol, and inclusion bodies. The protein was getting hyperexpressed and aggregating to form inclusion bodies (Fig. 2). Purification of rPA The recombinant PA was purified from inclusion bodies under denaturing conditions. Cells containing the recombinant construct were grown to an OD 600 0.7 to 1.0 and induced with 1 mM IPTG. After 5 h of induction, cells were harvested and the cell pellet was lysed by 6 M Gu.HCl. The inclusion bodies are soluble in 6 M Gu.HCl or 8 M urea. Cell lysate was removed by centrifugation and the supernatant was stirred with Ni-NTA slurry for 45 min at room temperature. The slurry was loaded onto a column and the denaturant was changed to 8 M urea by washing the slurry with 10

373

PURIFICATION OF THE RECOMBINANT PROTECTIVE ANTIGEN

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

FIG. 2. Electrophoretic analysis of E. coli-expressed PA. Proteins were separated by 12% SDS–PAGE and stained with Coomassie blue (a) and Western blot of the E. coli proteins containing PA, developed with a rabbit polyclonal PA antibody (b). Lane A, E. coli SG13009 cells without the vector; Lane B, cells containing the vector pQE30 without the PA gene; Lane C, cells containing the construct pMW1 (uninduced); Lane D, cells expressing PA; Lane E, periplasmic proteins of cells expressing PA; Lane F, cytosolic proteins of cells expressing PA; Lane G, inclusion bodies of cells expressing PA; Lane H, PA purified from B. anthracis; and Lane M, molecular weight standards.

column vol of 8 M urea. The protein was then renatured by the slow and gradient removal of the urea while immobilized on the Ni-NTA column. The slurry was washed with a gradient of 8 – 0 M urea in sodium phosphate buffer, pH 7.8. The advantage of using such a refolding approach where the proteins are immobilized on a column is that aggregation is prevented. Proteins are immobilized so that the hydrophobic faces exposed in partially folded and folding proteins are then not free to associate with one another. The renatured protein was then washed with sodium-phosphate buffer of pH 6.0. At pH 6.0 most of the contaminating proteins were removed without affecting the binding of the recombinant protein to the Ni-NTA. The protein was then eluted with a gradient of imidazole. The protein eluted at 100 –200 mM Imidazole concentration. The protein after affinity chromatography was about 70% pure with small amount of degraded prod-

ucts and other impurities. The pooled fractions containing affinity purified PA were dialyzed against T 10E 5 buffer and subjected to anion exchange chromatography using Mono-Q column on FPLC. The protein was eluted with a gradient of NaCl and was 95% pure after anion exchange chromatography (Fig. 3). The rPA was purified 3023-fold compared to the protein in the cell lysate (Table 1). Meanwhile we also tried various strategies to avoid the formation of inclusion bodies such as growing the cultures at 28°C, reducing the incubation period after induction, and induction by low IPTG concentrations (0.1– 0.5 mM). To purify the protein under native conditions, the cell pellet was resuspended in sonication buffer and sonicated at 4°C (1min bursts/1-min cooling/200 –300 Watts) for five cycles. The lysate was centrifuged at 10,000g and mixed with 5 ml of Ni-NTA slurry. The slurry was packed into a column (5.0 3 1.6 cm) and allowed to settle. The matrix was washed with a Na-phosphate, TABLE 1 Purification of PA from Escherichia coli

Fractions

Volume (ml)

Protein (mg/ml)

Activity (EC 50) a

Purification (fold) b

Cell lysate c Affinity purification FPLC

50 10 2

115.84 0.65 2.0

75.580 0.040 0.025

1 1890 3023

EC 50 is defined as the concentration of PA (mg/ml) along with LF (1 mg/ml) required to kill 50% of the J774A.1 cells. After 3 h of incubation, viability was determined by MTT dye. The results represent the mean of three experiments. b Purification fold was determined by dividing EC 50 for cell lysate with EC 50 for fractions obtained from different columns. c Cell lysate prepared from 2 liters of culture. a

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GUPTA, WAHEED, AND BHATNAGAR

TABLE 2 Binding of PA to Cell Surface Receptors

a

Protein

CPM

PA (ng)

PA/Cell Protein b (ng/mg)

nPA rPA

76608 6 1254 86464 6 1616

8.06 6 0.12 7.72 6 0.15

7.20 6 0.12 c 6.89 6 0.15

J774A.1 cells were incubated with 1 mg of 125I-PA (nPA, native PA, or rPA recombinant PA) for 12 h at 4°C. Cells were washed with HBSS and solubilized in 100 mM NaOH. Radioactivity was counted in a gamma counter. b Protein content of the cells per well was 1.12 6 0.05 mg as determined by the method of Lowry et al. (24). c The values are means 6 SD and are representative of three individual experiments done in triplicate. a

pH 6.0, buffer. Protein was eluted with gradients of 0 and 500 mM imidazole chloride. Fractions containing the protein were further purified using anion exchange Mono-Q column on FPLC. However, the yields after purification were considerably reduced as the protein underwent extensive degradation and we could only purify 0.7– 0.8 mg of rPA per liter of the bacterial culture. Thus we had to revert purifying the protein under denaturing conditions.

nPA or rPA at 4°C for 12 h. The cells were washed with HBSS, solubilized in 100 mM NaOH, and the radioactivity was measured. The protein content of the cells per well was 1.12 6 0.05 mg. From the amount of radioactivity bound per milligram cell protein, PA bound per milligram was also calculated. About 7.2 ng of the native PA bound per milligram cell protein, while the binding of recombinant PA was 6.9 ng/mg cell protein (Table 2). PA cleaved by trypsin has the ability to bind to LF in vitro. Recombinant as well as native PA was digested with trypsin and incubated with LF in a buffer containing Chaps. Samples were analyzed on 4 to 15% polyacrylamide gradient phast gels. It was observed that like nPA, rPA could bind to LF and the mobility of PA63 LF complex was retarded on nondenaturing phast gels (Fig. 4). To determine whether the rPA is biologically active or not we performed the cytotoxicity assay. Macrophage-like cell line J774A.1, which is sensitive to anthrax lethal toxin, was used (5). Various concentrations of rPA or nPA were added to

Comparison of rPA with nPA The rPA was compared to PA from B. anthracis by receptor binding assay, by determining in vitro binding of trypsin-nicked PA to LF, and finally by macrophage lysis assay. To determine whether the rPA binds to cell surface receptors, J774A.1 cells were incubated with

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

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

PURIFICATION OF THE RECOMBINANT PROTECTIVE ANTIGEN

the cells in combination with LF (1 mg/ml) and incubated for 3 h. After 3 h, viability was determined by adding MTT dye. Live cells oxidized the dye to formazon crystals while the dead cells did not. The precipitate was solubilized and optical density was read at 540 nm from which percentage cytotoxicity was determined (Fig. 5). It was observed that rPA along with LF were able to lyse the macrophages and showed biological activity comparable to that of PA obtained from B. anthracis. The currently available vaccine is unsatisfactory as its composition is not very well defined and the immunity is not long lasting and it has reactogenicity due to the presence of contaminants such as LF or EF. Research shows that these vaccines may not give protection against all natural strains of anthrax. For this reason there is a need to focus attention to address these problems and the present work is an attempt in this direction. The work has been focused to obtain highly purified, minimally reactogenic recombinant protective antigen for an attractive alternative in the future anthrax vaccine. ACKNOWLEDGMENT

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This work was supported by Department of Biotechnology, Government of India, New Delhi.

REFERENCES 1. Bhatnagar, R., Singh, Y., Leppla, S. H., and Friedlander, A. M. (1989) Calcium is required for the expression of anthrax lethal toxin activity in the macrophage like cell line J774A.1. Infect. Immun. 57, 2107–2114. 2. Bhatnagar, R., and Friedlander, A. M. (1994) Protein synthesis is required for expression of anthrax lethal toxin cytotoxicity. Infect. Immun. 62, 2958 –2962. 3. Brachman, P. S., Gold, H., Plotkin, S. A., Fekety, F. R., Werrin, M., and Ingraham, N. R. (1962) Field evaluation of human anthrax vaccine. Am. J. Public Health 52, 632– 645. 4. Coulson, M., Fulop, M., and Tidball, R. W. (1994) B. anthracis protective antigen, expressed in Salmonella typhimurium SL 3261, affords protection against anthrax spore challenge. Vaccine 12, 1395–1401. 5. Duesbery, N. S., Craig, P. W., Leppla, S. H., Gordon, V. M., Klimpel, K. R., Copeland, T. W., Ahn, N. G., Oskarsson, M. K., Fukasawa, K., Paull, K. D., and Woude, G. F. V. (1998) Proteolytic inactivation of MAP Kinase Kinase by anthrax lethal factor. Science 280, 734 –737. 6. Farchaus, J. W., Ribot, W. J., Jendrek, S., and Little, S. F. (1998) Fermentation, purification and characterization of protective antigen from a recombinant, avirulent strain of B. anthracis. Appl. Environ. Microbiol. 64, 982–991. 7. Friedlander, A. M. (1986) Macrophages are sensitive to anthrax toxin through an acid dependent process. J. Biol. Chem. 261, 7123–7126. 8. Friedlander, A. M., Bhatnagar, R., Leppla, S. H., and Singh, Y. (1993) Characterization of macrophage sensitivity and resistance to anthrax lethal toxin. Infect. Immun. 61, 245–252. 9. Gladstone, G. P. (1946) Immunity to anthrax. Protective antigen present in cell free filtrates. Br. J Exp. Pathol. 27, 349 – 418. 10. Green, B. D., Battisti, L., Koehler, T. M., Thorne, C. B., and

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

375

Ivins, B. E. (1985) Demonstration of a capsule plasmid in B. anthracis. Infect. Immun. 49, 291–297. Gupta, P., Batra, S., Chopra, A. P., Singh, Y., and Bhatnagar, R. (1998) Expression and purification of the recombinant lethal factor of Bacillus anthracis. Infect Immun. 66, 862– 865. Hambleton, P., Craman, J. A., and Melling, J. (1984) Anthrax: the disease in relation to vaccines. Vaccine 2, 125–132. Hanna, P. C., Kochi, S., and Collier, R. F. (1992) Biochemical and physiological changes induced by anthrax lethal toxin in J774A.1 macrophage like cells. Mol. Biol. Cell 3, 1269 –1277. Iacono-Connors, L. C., Schmaljohn, C. S., and Dalrymple, J. M.. (1990) Expression of Bacillus anthracis protective antigen by baculovirus and vaccinia virus recombinants. Infect. Immun. 58, 366 –372. Ivins, B. I., Ezzel, J. W., Jemski, J., Ristroph, J. D., and Leppla, S. H. (1986) Immunization studies with attenuated strains of Bacillus anthracis. Infect. Immun. 52, 454 – 458. Ivins, B. E., Fellows, P. F., and Nelson, G. O. (1994) Efficacy of standard human anthrax vaccine against B. anthracis spore challenge in guinea pigs. Vaccine 12, 872– 874. Ivins, B. E., and Welkos, S. L. (1987) Recent advances in the development of an improved human anthrax vaccine. Eur. J. Epidemol. 4, 12–19. Ivins, B. E., and Welkos, S. L. (1986) Cloning and expression of the B. anthracis protective antigen gene in B. subtilis. Infect. Immun. 54, 537–542. Klimpel, R. K., Molloy, S. S., Thomas, G., and Leppla, S. H. (1992) Anthrax toxin protective antigen is activated by a cell surface protease with the sequence specificity and catalytic properties of furin. Proc. Natl. Acad. Sci. USA 89, 10277–10281. Leppla, S. H. (1982) Anthrax toxin edema factor: A bacterial adenylate cyclase that increases cAMP conc. in eukaryotic cells. Proc. Natl. Acad. Sci. USA 79, 3162–3166. Leppla, S. H. (1988) Production and purification of anthrax toxin, in “Methods in Enzymology” (Harshman, S., Ed.), Vol. 165, pp. 103–116, Academic Press, San Diego. Leppla, S. H. (1991) The anthrax toxin complex, in “Sourcebook of Bacterial Protein Toxins” (Alouf, J. E., and Freer, J. H., Eds.), pp. 277–302, Academic Press, London. Leppla, S. H. (1995) Anthrax toxins, in “Bacterial Toxins and Virulence Factors in Disease. Handbook of Natural Toxins” (Moss, J., Iglewski, B., Vaughan, M., and Tu, A. T., Eds.), Vol. 8, pp. 543–567, Dekker, New York. Lowry, O. H., Rosenbrough, N. J., Farr, A. L., and Randall, R. J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275. McBride, B. W., Mogg, A., Telfer, J. L., Lever, M. S., Miller, J., Turnbull, P. C. B., and Baillie, L. (1998) Protective efficacy of a recombinant protective antigen against B. anthracis challenge and assessment of immunological markers. Vaccine 16, 810 – 817. Mikesell, O. P., Ivins, B. E., Ristroph, J. D., and Dreir, T. M. (1983) Evidence for plasmid mediated toxin production in B. anthracis. Infect. Immun. 39, 371–376. Mikesell, O. P., and Vodkin, M. (1985) Plasmids of Bacillus anthracis, in “Microbiology” (Leive, L., Ed.). pp. 52–55, Am. Soc. Microbiol., Washington, DC. Miller, J., McBride, B. W., Manchee, R. J., Moore, P., and Baille L. W. J. (1998) Production and purification of recombinant protective antigen and protective efficacy against Bacillus anthracis. Lett. Appl. Microbiol. 26, 56 – 60. Milne, J. C., Furlong, D., Hanna, P. C., Wall, J. S., and Collier, R. J. (1994) Anthrax protective antigen forms oligomers during

376

30.

31.

32.

33.

34.

35.

36.

GUPTA, WAHEED, AND BHATNAGAR

intoxication of mammalian cells. J. Biol. Chem. 269, 20607– 20612. Quinn, P. C., Singh, Y., Klimpel, R. K., and Leppla, S. H., (1991) Functional mapping of anthrax toxin lethal factor by in frame insertion mutagenesis. J. Biol. Chem. 266, 20124 –20130. Quinn, P. C., Shone, C. C., Turnbell, T. B., and. Melling, J. (1988) Purification of anthrax toxin components by high performance anion-exchange, gel filtration and hydrophobic interaction chromatography. Biochem. J. 252, 753–758. Radha, C., Salotra, P., Bhat, R., and Bhatnagar, R. (1996) Thermostabilization of protective antigen - the binding component of anthrax lethal toxin. J. Biotech. 50, 235–242. Saelinger, C. B. (1990) Toxin structure and function, in “Trafficking of Bacterial Toxins” (Saelinger, C. B., Ed.), pp. 1–14, CRC Press, Boca Raton, FL. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) “Molecular Cloning: A Laboratory Manual,” Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Sharma, M., Swain, P. K., Chopra, A. P., Chaudhary, V. K., and Singh Y (1996) Expression and purification of anthrax toxin protective antigen from E. coli. Protein Exp. Purif. 7, 33–38. Shlyakhov, E. N., and Rubinstein, E. (1993) Human live anthrax vaccine in the former USSR. Vaccine 12, 727–730.

37. Singh, Y., Chaudhary, V. K., and Leppla, S. H. (1989) A deleted variant of B. anthracis protective antigen is non toxic and blocks anthrax toxin action in vivo. J. Biol. Chem. 264, 19103–19107. 38. Smith, H., and Keppie, J. (1954) Observations on experimental anthrax: demonstration of a specific lethal factor produced in vivo by Bacillus anthracis. Nature 173, 869 – 870. 39. Smith, H., Keppie, J., and Stanley, J. L. (1955) The chemical basis of virulence of Bacillus anthracis V. The specific toxin produced by B. anthracis in vivo. Br. J. Exp. Pathol. 36, 460 – 472. 40. Stanley, J. L., and Smith, H. (1961) Purification of factor I and recognition of a third factor of anthrax toxin. J. Gen. Microbiol. 26, 49 – 66. 41. Turnbull, P. C. B. (1992) Anthrax vaccines: past, present and future. Vaccine 9, 533–539. 42. Vodkin, M. H., and Leppla, S. H. (1983) Cloning of the protective antigen gene of B. anthracis. Cell 34, 693– 697. 43. Welkos, S. L., and Friedlander, A. M. (1988) Comparative safety and efficacy against Bacillus anthracis of protective antigen and live vaccines in mice. Microb. Pathogen. 5, 127–139. 44. Welkos, S. L., Lowe, J. R., Eden-McCutchan, F., Vodkin, M., and Leppla, S. H. (1988) Sequence and analysis of the DNA encoding protective antigen of Bacillus anthracis. Gene 69, 287–300.