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The major virulence factors of Bacillus anthracis, the causative organism of anthrax, are a poly-D-glutamic acid capsule and a three-component protein exotoxin. The genes coding for the toxin and the enzymes responsible for the capsule production are carried on B. anthracis plasmids pXO1 and pXO2, respectively (8, 19). The three proteins of the exotoxin are protective antigen (PA [83 kDa]), lethal factor (LF [90 kDa]), and edema factor (EF [89 kDa]). The toxins follow the A-B model, with the A moiety being the catalytic part and the B moiety being the receptor binding part. PA acts as the B moiety and binds to the cell surface receptor, while LF and EF compete for binding to PA (18, 25). LF and EF are individually nontoxic but in combination with PA form two distinct toxins causing different pathogenic responses in animals and cultured cells (6). In the process of cytotoxicity, PA binds to cell surface receptor and is cleaved at the sequence RKKR¹⁶⁷ by cell surface proteases such as furin, generating a cell-bound, C-terminal, 63-kDa protein (PA₆₃). PA₆₃ possesses a binding site to which LF or EF bind with high affinity (11, 28). The complex formed by EF bound to PA₆₃ is known as edema toxin, which causes edema when injected into the skin of animals (30). EF has calcium- and calmodulin-dependent adenylate cyclase activity and in combination with PA dramatically increases cyclic AMP levels of cells (13). LF bound to PA₆₃ produces lethal toxin, which causes death in experimental animals and lysis of mouse peritoneal macrophages and macrophage-like cell lines such as J774A.1 and RAW264.7 (6, 7, 30). After binding of LF (or EF) to PA₆₃, the complex is internalized by receptor-mediated endocytosis (3, 6). Following internalization of LF, there is an increase in macrophage permeability to Na⁺ and K⁺, which is followed by hydrolysis of ATP (9). Subsequently, there is influx of Ca²⁺ (3) and leakage of cytoplasmic lactate dehydrogenase, leading to cell death. Protein synthesis is required to express cytotoxicity in J774A.1 cells (4). However, the exact molecular mechanism of cell killing is not yet understood. LF has been proposed to be a zinc-dependent metalloprotease; however, its substrate is not yet known (12).

The genes for PA, LF, and EF have been cloned and sequenced (5, 24, 31). Protective antigen has been expressed and purified from Bacillus subtilis as well as from Escherichia coli (10, 27, 28). Attempts to express and purify LF from B. subtilis were not very successful (15, 16). The LF gene coding for amino acids 1 to 776 (LF^1-776) and LF^1-254 has been expressed in E. coli with various promoters (1, 2, 23). In these studies, the degree of expression of LF^1-254 has been obtained to significant levels. However, isolation of full-length LF did not yield significant amounts of LF due to extensive proteolytic degradation in the cytosol (22). Klimpel et al. expressed LF as a fusion protein along with 164 amino acids of PA in B. anthracis to enhance the expression of LF (12). The fusion protein required trypsin cleavage to obtain mature LF. To date, culture supernatants of B. anthracis have been the major source for the purification of LF (14, 21). Purification of LF from B. anthracis requires containment facilities because of the highly infectious nature of the organism. In addition, LF preparations from B. anthracis are invariably contaminated with other proteins with either similar charges or molecular masses such as PA, EF (30), and phospholipase C (2a). Currently, no simplified system to obtain full-length homogeneous preparations of LF in appreciable amounts from E. coli is available.

In this study, attempts have been made to express the full-length LF as a fusion protein with six histidine residues in E. coli. Recombinant LF (rLF) was purified by affinity chromatography, gel filtration, and finally ion-exchange chromatography.

Plasmid construction and expression of LF. Plasmid pLF7, a generous gift from Stephen H. Leppla, National Institute for Dental Research, National Institutes of Health, contains the entire native LF (nLF) gene. Expression vector pQE30 (Qiagen) contains the T5 promoter for high-level expression, a ribosome binding site, and × histidine coding sequences followed by a multiple cloning site. The vector also contains two lac operator sequences. Plasmids pLF7 and pQE30 were purified with the DNA purification kit (Qiagen), as described in the manufacturer's manual. The LF gene was amplified by PCR with pLF7 (23) as a template and primers that added BamHI and SalI sites to the 5' and 3' ends of the PCR product, respectively. Sequences of the forward and reverse oligonucleotides were 5' GTA CAG GGA TCC GCG GGC GGT 3' and 5' GAA AAT TTT TAA TAG TCG ACT TAT GAG 3', respectively. The amplified PCR product and plasmid pQE30 were digested with restriction enzymes BamHI and SalI. The digested products were separated on a 1% agarose gel. The bands were excised, and the DNA was eluted with 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. (26). The transformation mixture was plated on Luria agar plates containing 100 µg of ampicillin per ml and 25 µg of kanamycin per ml. The plates were incubated for 16 h at 37°C. Colonies appearing on the plate were screened for the recombinant plasmid pPG-LF1 by minipreparations of plasmid DNA (26). The desired recombinant plasmid was confirmed by restriction enzyme digestion with BamHI and SalI. For high-level expression of the gene, SG13009(pREP4) cells containing multiple copies of the plasmid pREP4, which carries lacI^q gene encoding the lac repressor, were used. Multiple copies of pREP4 present in the host cell ensured high levels of lac repressor and tight regulation of protein expression. Expression of rLF was established by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. Cells carrying the plasmid pPG-LF1 were grown and induced with 1 mM isopropyl-1-thio--D-galactopyranoside. Periplasm, cytosol, and inclusion bodies were checked for the presence of LF. LF was found to be mainly localized in the cytosol (Fig. 1).

View larger version (81K): [in this window] [in a new window]   FIG. 1.   Electrophoretic analysis of E. coli-expressed LF. (a) Proteins separated by SDS-10% PAGE and stained with Coomassie blue; (b) Western blot of the E. coli proteins containing LF, developed with a rabbit polyclonal LF antibody. Lanes: A, E. coli SG13009 cells without the vector; B, cells containing the vector pQE30 without the LF gene; C, cells containing the construct pPG-LF1 (uninduced); D, cells expressing LF; E, periplasmic proteins of cells expressing LF; F, cytosolic proteins of cells expressing LF; G, inclusion bodies of cells expressing LF; H, LF purified from B. anthracis.

Purification of LF. E. coli SG13009(pREP4) carrying the recombinant plasmid pPG-LF1 was grown at 37°C in Luria broth with 100 µg of ampicillin and 25 µg of kanamycin per ml at 250 rpm. When the A₆₀₀ reached 1.0, isopropyl-1-thio--D-galactopyranoside was added to a final concentration of 1 mM. After 5 h of induction, the cells were harvested by centrifugation at 4,000 × g for 20 min. For the purification of protein from 2 liters of culture, the pellet was resuspended in 50 ml of sonication buffer (50 mM Na phosphate [pH 7.8], 300 mM NaCl). Lysozyme (1 mg/ml) was added to the slurry and incubated on ice for 30 min. Phenylmethylsulfonyl fluoride was added to a final concentration of 1 mM. Cells were sonicated at 4°C (1-min bursts, 1 min of cooling, 200 to 300 W) for five cycles. The lysate was centrifuged at 10,000 × g for 30 min. The supernatant was mixed with 8 ml of a 50% Ni-nitriloacetic acid resin previously equilibrated with sonication buffer. The slurry was packed into a column (5.0 by 1.6 cm) and allowed to settle. The matrix was washed first with sonication buffer followed by wash buffer (50 mM Na phosphate [pH 6.0], 500 mM NaCl, 10% glycerol). The column was washed until the A₂₈₀ of the flowthrough was less than 0.01 (approximately 50 ml). Protein was eluted with a linear gradient of 15 ml each of 0 and 500 mM imidazole chloride in elution buffer (50 mM Na phosphate [pH 7.0], 100 mM NaCl, 10% glycerol). Fractions of 1 ml were collected and analyzed on an SDS-10% PAGE gel. rLF eluted at a gradient of 100 to 250 mM imidazole chloride. Affinity-purified protein possessed full-length rLF, and approximately 40% degraded LF as determined by SDS-PAGE and Western blotting with anti-LF antibodies (Fig. 2). The fractions containing LF were pooled and dialyzed against T₁₀E₅ buffer (10 mM Tris and 5 mM ETDA [pH 8.0]) overnight. The dialyzed sample was concentrated by Centricon-30 (Amicon) to a volume of 1 ml. Concentrated sample was loaded onto a Sephacryl S-200 (Pharmacia) gel filtration column (100 by 1.6 cm) previously equilibrated with T₁₀E₅ buffer. Void volume was allowed to pass through, and 1-ml fractions were then collected. The rLF eluted immediately after the void volume, while the mobility of the degraded products was retarded. This resulted in approximately 90% pure rLF with a very small amount of degraded LF. The fractions containing LF were pooled and loaded onto the Resource-Q (Pharmacia) anion-exchange column previously equilibrated with T₁₀E₅ buffer. Protein was eluted with a linear gradient of 0 to 1 M NaCl in T₁₀E₅ buffer (15 ml each). Fractions of 1 ml each were collected. The protein eluted at a gradient of 250 to 300 mM NaCl. The purified LF was dialyzed against 10 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer (pH 7.0) containing 50 mM NaCl and was frozen at 70°C in aliquots. The fold purification of LF at different column stages was determined by calculating the amount of protein required to kill 50% of J774A.1 cells (50% effective concentration [EC₅₀]) when they were incubated with PA (1 µg/ml) at 37°C (Table 1). The protein was measured by the method described by Lowry et al. (17). By using this procedure, rLF was purified to homogeneity with 3,054-fold purification compared to the cytosolic preparation.

View larger version (55K): [in this window] [in a new window]   FIG. 2.   Purification of E. coli-expressed LF. (a) Proteins analyzed on an SDS-10% PAGE gel and stained with Coomassie blue; (b) Western blot of LF proteins developed with polyclonal rabbit LF antibody. Lanes: A, E. coli SG13009 cells expressing the LF gene; B, cytosolic preparation of cells expressing LF; C, proteins after Ni-nitriloacetic acid affinity purification; D, protein after passing through a Sephacryl S-200 gel filtration column; E, protein after passing through a Resource-Q column on FPLC; F, LF purified from B. anthracis; M, molecular weight standards (103).

Comparison of rLF with nLF. The biological activity of rLF was compared with that of nLF purified from B. anthracis by the receptor binding assay, by determining in vitro binding of LF to trypsin-nicked PA, and, finally, by the macrophage lysis assay. To examine whether LF binds to receptor-bound PA, J774A.1 cells were incubated for 12 h at 4°C with PA (1 µg/ml) and radioiodinated nLF or rLF (1 µg/ml) in a 12-well plate as described earlier (3, 29). The binding of rLF to PA was 3.55 ± 0.15 ng compared to 3.7 ± 0.13 ng of nLF per mg of cell protein. Nonspecific binding in the absence of PA was 0.18 ng of the LF per mg of cell protein (Table 2).

View larger version (46K): [in this window] [in a new window]   FIG. 3.   Binding of LF to PA proteins in solution. LF (1 µg) was incubated with trypsin-nicked PA (1 µg) for 15 min, and the samples were analyzed on a nondenaturing 8 to 25% Phast gradient gel. The gel was stained with Coomassie blue and dried (a), then 125I-labeled LF (100,000 cpm) was incubated with trypsin-nicked PA (100 ng) and, after electrophoresis, the gel was dried and autoradiographed (b). Lanes: A, PA; B, LF from B. anthracis; C, LF from E. coli; D, PA nicked with trypsin incubated with LF from B. anthracis; E, PA nicked with trypsin incubated with LF from E. coli.