PURIFICATION OF MYCOBACTERIUM-BOVIS BCG TOKYO ANTIGENS BY CHROMATOFOCUSING, LECTIN-AFFINITY CHROMATOGRAPHY, AND HYDROPHOBIC INTERACTION CHROMATOGRAPHY

A combination of chromatofocusing, lectin-affinity chromatography, and hydrophobic interaction chromatography resulted in a simple purificat ion of protein antigens of Mycobacterium bovis BCG Tokyo culture filtr ate. Identification was established on the basis of chromatographic se paration, sodium dodecyl sulfate-polyacrylamide gel electrophoresis de termination of molecular weights, and N-terminal amino acid determinat ion. Chromatofocusing on PBE 94 accomplished the separation of BCG85B from other BCG85 complex antigens and partial separation of MPB64 and MPB70 antigens. Subsequently, MPB64 and MPB70 were completely separate d on a high-performance liquid chromatography TSK Phenyl 5PW hydrophob ic interaction chromatography column. This column also separated BCG85 B from a 17-kDa protein with an N-terminal amino acid sequence of A-V- P-I-T-G-K-L-G-S-E-L-T-M-T-D-( )-V-G-Q, which is similar to the sequenc e of MPT63. Concanavalin A-Sepharose-affinity chromatography separated MPB64 from a 43- and 47-kDa doublet with an amino acid sequence of D- P-E-P-A-P-P-V-P-P-V-P-A-( )-A-A-S-P, which is similar to the sequence of MPT32 and which appears to be glycosylated.