Purification, characterization and identification of a 32 kDa protein antigen of Mycobacterium bovis BCG

An immunogenic protein called P32 has been purified from Sauton zinc deficient culture filtrate of Mycobacterium bovis BCG using successively hydrophobic chromatography on Phenyl-Sepharose, ion exchange on DEAE-Sephacel and molecular sieving on Sephadex G-100. The final preparation was found to be homogeneous as based on several analyses. This P32 protein was a constituent of BCG cells grown in normal conditions. It represented about 3% of the soluble fraction of a cellular extract, and appeared as the major protein released in normal Sauton culture filtrate. This protein was found to have a molecular weight of 32,000 by SDS-polyacrylamide gel electrophoresis and in molecular sieving. Its amino acid composition showed an abundance of acidic amino acids (or their amides). The NH2-terminal amino acid sequence (6 amino acids) was determined. Purified P32 was tested by various crossed immunoelectrophoresis techniques, and was shown to belong to the antigen 85 complex in the reference system for BCG antigens. It was more precisely identified as antigen 85A. The protein antigen elicited a weak delayed hypersensitivity reaction in guinea pigs sensitized with heat-killed or living BCG. No delayed hypersensitivity reaction was observed in living BCG sensitized mice, however, it induced significant amounts of gamma interferon in cultured spleen cells from BCG-sensitized mice. Moreover, P32 either pure or as part of BCG soluble extract promoted substantial antibody levels when injected in rabbits.