Jw. Farchaus et al., FERMENTATION, PURIFICATION, AND CHARACTERIZATION OF PROTECTIVE ANTIGEN FROM A RECOMBINANT, AVIRULENT STRAIN OF BACILLUS-ANTHRACIS, Applied and environmental microbiology, 64(3), 1998, pp. 982-991
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.