Expression Of Recombinant Anthrax Toxin & Study On Toxin Inhibitors

Bacillus anthracis is the etiologic agent of anthrax. Two plasmid-encoded virulence factors, a capsule and an exotoxin, have been described for the bacteria. The exotoxin is composed of three proteins: protective antigen (PA), edema factor (EF), and lethal factor (LF). Toxic activity is expressed only when PA is combined with EF or LF. A combination of PA with EF or LF forms edema toxin or lethal toxin respectively. PA binds EF and LF to target cells and transports these catalytic subunits into the host cell cytosol where they carry out their enzymatic functions.Antibiotics can tackle anthrax at the early stage of the disease. But toxins of the bacterial may be present in large amounts in the host later and contribute directly to the death of the infected organism. So we need effective anti-toxin agents to treat the bacterium's toxic effect.In this study, an expression plasmid carrying PA gene was constructed, which has an OmpA signal sequence attached to the 5' end of the gene. The plasmid was transformed into E.coli and induced to express recombinant PA (rPA). The recombinant protein, about 10% of the total bacterial protein in volume, was secreted to the periplasmic space of the cell. After a purification procedure including ion-exchange, hydrophobic interaction chromatography, and gel filtration, about 15 mg of 95% pure rPA was obtained from 1-liter culture. Data from N-terminal sequencing suggests that the amino acid sequences of rPA and natural PA were identical. The bioactivity of rPA was proved by in vitro cytotoxicity assay.The gene encoding LF was cloned into a similar secretory expression plasmid and then expressed in periplasmic space of E.coli. The recombinant LF (rLF) expressed was about 4% of the total proteins. About 3 mg electrophoresis purity rLF could be obtained after the purification of 1 liter culure using ion exchange chromatography and gel filtration. The N-terminal amino acid sequence of rLF was identical to that of natural LF. In vitro toxicity analysis also shows that rLF has an excellent biological activity.The gene encoding EF was cloned into an expression plasmid based on pQE30. Recombinant EF (rEF) expressed in E.coli M15 was purified to homogeneity by a three-step procedure involving metalchelate affinity chromatography, cation exchange and gel filtration. From 1 liter of culture, 5mg of biologically active rEF was easily purified. And it was found that rEF can compete with LF for the binding site on PA in two different cellular models.The purified rPA was used to immunize BALB/c mice and monoclonal antibodies to PA were prepared by hybridoma fusions. The antibodies were screened for their ability to neutralize lethal toxin activities by in vitro cytotoxicity assay. Three toxin-neutralizing monoclonal antibodies which recognize two different epitope of PA were identified.The gene fragment encoding ATR(CMG2)-EXCELL (excellular portion of anthrax toxin receptor/capillary morphogenesis factor) was cloned into a secretory expression plasmid and then expressed in Pichia pastoris. The recombinant ATR(CMG2)-EXCELL expressed was about 20% of the total proteins in media supernatant. In vitro binding activity analysis and cell protection experiments have shown that the recombinant protein has an excellent biological activity. So the recombmant ATR(CMG2)-EXCELL obtained may be a potential inhibitor of anthrax toxin.Signal sequence of Omp A was attached to the 5' end of the gene encoding PA receptor-binding domain (the fourth domain of PA, PA-D4). The plasmid carrying the fusion gene was then transformed into E.coli and induced to express recombinant PA-D4 by IPTG The recombinant protein, about 10% of the total bacterial protein, was secreted to the periplasmic space of the cell. After a purification procedure including ion-exchange chromatography and gel filtration, about 10 mg of homogeneous recombinant PA-D4 was obtained from 1-liter culture. N-terminal sequencing suggested that the amino acid sequence of recombinant PA-D4 was identical with its natural counterpart. And western blot showed the recombinant protein could bind with anti-PA serum from rabbit.The results reported here may be helpful to study mechanism of anthrax toxin's effect and develop potential therapeutic agents and new vaccines against anthrax.