The expression of the protective antigen of Bacillus anthracis in Bacillus subtilis.
The expression of Bacillus anthracis protective antigen (PA) in B. subtilis from the pag gene in pPA101-1 was explored in different genetic backgrounds in an attempt to identify opportunities to maximize expression. Introduction of AtxA, which positively regulates PA expression in B. anthracis did not improve expression levels in the protease-deficient strain WB600. Plasmid pPA101-1 was found to carry a deletion which created a new fusion point between vector and insert sequence, and which removed part of the AtxA binding site. The deletion may have occurred as a consequence of recombination between TCTAT sequences within both the vector and insert. Host mutations could influence expression; PA levels from pPA101-1 are threefold higher in a ccpA mutant than in an otherwise isogenic parent, and eightfold higher in an abrB mutant. These data demonstrate that the introduction of mutations affecting catabolite repression and growth phase regulation results in an increase in the yield of PA in this host-vector system. Combining these mutations with a multiply protease-negative background could potentially allow further improvements in PA yield.
Department of Molecular Biology and Biotechnology, University of Sheffield, UK.
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Baillie
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Moir
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Manchee
D000942:Antigens, Bacterial; D001408:Bacillus anthracis / Q000235:genetics* / Q000276:immunology*; D001412:Bacillus subtilis / Q000235:genetics*; D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005818:Genetic Engineering; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics; D014169:Transformation, Bacterial
C060762:AbrB protein, Bacillus subtilis; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; C030325:anthrax toxin; C069286:catabolite control proteins, bacteria