Purification, partial characterization, and identification of a skin-reactive protein antigen of Mycobacterium bovis BCG

Abstract

An immunogenic and skin-reactive protein called P64 was purified from Sauton zinc-deficient culture filtrate of Mycobacterium bovis BCG by using successively hydrophobic chromatography on phenyl-Sepharose, ion exchange on DEAE-Sephacel, and molecular sieving on Sephadex G-200. The final P64 preparation was found to be homogeneous based on several analyses. Protein P64 was a constituent of BCG cells since it was present in soluble cellular extract from normally grown BCG cells. It represented 8 to 9% of the soluble proteins of the extract and appeared as the major soluble protein antigen of BCG. This protein was found to have a molecular weight of 64,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but in molecular sieving it eluted at a volume corresponding to a molecular weight of 246,000. An abnormal UV spectrum was observed for this protein. Its amino acid composition showed an abundance of acidic amino acids (or their amides). Aromatic amino acids represented only 3% of the total amino acid residues. The NH2-terminal amino acid sequence of this protein (10 amino acids) was determined. Its sugar content measured with the phenol-sulfuric acid test was lower than 0.3% (wt/wt). Isolated P64 was tested by various crossed-immunoelectrophoresis techniques and was shown to correspond to antigen 82 in the reference system for BCG antigens. The protein antigen P64 elicited a delayed cutaneous reaction in guinea pigs sensitized with either living or heat-killed BCG. Its potency in skin reaction was, respectively, two-and threefold that of the BCG purified protein derivative. The two types of sensitization used for skin test reactions promoted significant immunoglobulin G antibody production against the protein antigen P64 in guinea pigs 7 weeks after sensitization.

Heat shock response in Actinobacillus actinomycetemcomitans

“…In vitro observations indicate furthermore that members of the HSP60 family might have intrinsic capacity to induce release of proinflammatory cytokines from human monocytic cells [23], and this may further add to tissue destruction. Significant amounts of bacterial HSPs may be encountered in the subgingival milieu because the molecules have been found in bacterial culture supernatants [24]. In sum, HSPs from bacteria in dental plaque might also have the potential to contribute to the development of inflammation and the tissue damage found in periodontitis.…”

Cross‐Reactive Immune Responses Against Mycobacterium bovis BCG in Mice Infected with Non‐Tuberculous Mycobacteria Belonging to the MAIS‐Group

“…Briefly, culture filtrate (CF) and cytoplasmic extract were prepared from 2-week-old cultures of zinc-supplemented M. bovis BCG (GL2). Native Ag85 (30-32 kDa) and heat-shock protein 65 (hsp 65; GroEL related 65 kDa) were purified from zinc-supplemented and zinc-deprived M. bovis BCG culture filtrate, respectively, by sequential chromatography on phenyl-Sepharose, DEAE-Sephacel and Sephadex [11,12].…”

Multiple chaperonins in bacteria – why so many?

“…More unexpected was the finding that the Cpn60 proteins from mycobacteria appear not to exist as stable large oligomers. Early work on what subsequently proved to be the Cpn60.1 protein of M. bovis BCG showed that it had a subunit molecular mass of c. 65 kDa but purified as a complex of c. 250 kDa, corresponding roughly to a tetramer (de Bruyn et al, 1987). Moreover, both of the Cpn60 proteins from M. tuberculosis when purified formed only low molecular weight structures, probably monomers or dimers .…”