FERMENTATION, PURIFICATION, AND CHARACTERIZATION OF PROTECTIVE ANTIGEN FROM A RECOMBINANT, AVIRULENT STRAIN OF BACILLUS-ANTHRACIS

Bacillus anthracis, the etiologic agent for anthrax, produces two bipa rtite, AB-type exotoxins, edema toxin and lethal toxin, The B subunit of both exotoxins is an M-r 83,000 protein termed protective antigen ( PA), The human anthrax vaccine currently licensed for use in the Unite d States consists primarily of this protein adsorbed onto aluminum oxy hydroxide, This report describes the production of PA from a recombina nt, asporogenic, nontoxigenic, and nonencapsulated host strain of B, a nthracis and the subsequent purification and characterization of the p rotein product, Fermentation in a high-tryptone, high-yeast-extract me dium under nonlimiting aeration produced 20 to 30 mg of secreted PA pe r liter, Secreted protease activity under these fermentation condition s was low and was inhibited more than 95% by the addition of EDTA, A p urity of 88 to 93% was achieved for PA by diafiltration and anion-exch ange chromatography, while greater than 95% final purity was achieved with an additional hydrophobic interaction chromatography step, The pu rity of the PA product was characterized by reversed-phase high-pressu re liquid chromatography, sodium dodecyl sulfate (SDS)-capillary elect rophoresis, capillary isoelectric focusing, native gel electrophoresis , and SDS-polyacrylamide gel electrophoresis, The biological activity of the PA, when combined with excess lethal factor in the macrophage c ell lysis assay, was comparable to previously reported values.