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 purification of protein antigens of Mycobacterium bovis BCG Tokyo culture filtrate. Identification was established on the basis of chromatographic separation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis determination of molecular weights, and N-terminal amino acid determination. 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 separated on a high-performance liquid chromatography TSK Phenyl 5PW hydrophobic interaction chromatography column. This column also separated BCG85B 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 sequence 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.
Sugden
E A
EA
Agriculture and Agri-Food Canada, Animal Diseases Research Institute, Nepean, Ontario, Canada. sugdene@em.agr.ca
Stilwell
K
K
Watson
D C
DC
Rohonczy
E B
EB
Martineau
P
P
D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000302:isolation & purification* / Q000378:metabolism*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D008969:Molecular Sequence Data; D009163:Mycobacterium bovis / Q000276:immunology*; D011485:Protein Binding / Q000276:immunology
D000942:Antigens, Bacterial