Constitutive expression of protective antigen gene of Bacillus anthracis in Escherichia coli.
The fatal bacterial infection caused by inhalation of the Bacillus anthracis spores results from the synthesis of protein toxins-protective antigen (PA), lethal factor (LF), and edema factor (EF)--by the bacterium. PA is the target-cell binding protein and is common to the two effector molecules, LF and EF, which exert their toxic effects once they are translocated to the cytosol by PA. PA is the major component of vaccines against anthrax since it confers protective immunity. The large-scale production of recombinant protein-based anthrax vaccines requires overexpression of the PA protein. We have constitutively expressed the protective antigen protein in E. coli DH5alpha strain. We have found no increase in degradation of PA when the protein is constitutively expressed and no plasmid instability was observed inside the expressing cells. We have also scaled up the expression by bioprocess optimization using batch culture technique in a fermentor. The protein was purified using metal-chelate affinity chromatography. Approximately 125 mg of recombinant protective antigen (rPA) protein was obtained per liter of batch culture. It was found to be biologically and functionally fully active in comparison to PA protein from Bacillus anthracis. This is the first report of constitutive overexpression of protective antigen gene in E. coli.
Chauhan
V
V
Centre For Biotechnology, Jawaharlal Nehru University, New Delhi, 110067, India.
Singh
A
A
Waheed
S M
SM
Singh
S
S
Bhatnagar
R
R
D000818:Animals; D000881:Anthrax / Q000209:etiology; D000942:Antigens, Bacterial / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D001408:Bacillus anthracis / Q000235:genetics* / Q000276:immunology* / Q000472:pathogenicity; D001427:Bacterial Toxins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology*; D001709:Biotechnology; D002352:Carrier Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D011956:Receptors, Cell Surface / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D013604:T-Phages / Q000235:genetics
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D002352:Carrier Proteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C030325:anthrax toxin