Purification and partial characterisation of a novel 66-kDa seroreactive protein of Mycobacterium tuberculosis H37Rv.
A seroreactive protein (TB66) was purified from culture filtrate (CF) and cell sonicate (CS) of Mycobacterium tuberculosis H37Rv by immobilised metal affinity chromatography (IMAC) on a Ni-nitrilotriacetic acid (NTA) column. The TB66 preparations obtained by IMAC contained predominantly a 66-kDa protein with a pI of c. 5.5 as determined by two-dimensional electrophoresis. TB66 was detected in the CF as early as 1 week of growth of H37Rv. The NH2-terminal amino-acid sequence showed 85% homology with the N-terminal sequence of bovine serum albumin (BSA) and 80% homology with human serum albumin. Amino-acid analysis indicated a difference in the amino-acid content of TB66 when compared to BSA, with an abundance of acidic amino acids. A monoclonal antibody (MAb) OD4AG3, raised in this laboratory against an M. avium complex (MAC 101) sonicate cross-reacting with H37Rv sonicate, recognised a heat-stable and trypsin-sensitive epitope of this protein. TB66 was also recognised by MAbs IT1 and IT20 which also react with the 14-kDa antigen of the M. tuberculosis complex. Antibodies against TB66 were present in the sera of 62 of 64 patients with tuberculosis; sera from normal healthy individuals showed no significant reactivity. TB66 appears to be a predominant secretory protein of M. tuberculosis and could play an important role in the pathogenesis of this organism.
Deshpande
R G
RG
Department of Microbiology and Immunology, Morehouse School of Medicine, Atlanta, Georgia 30310.
Khan
M B
MB
Bhat
D A
DA
Navalkar
R G
RG
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000907:Antibodies, Bacterial / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology; D000942:Antigens, Bacterial / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D015153:Blotting, Western; D002846:Chromatography, Affinity; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000276:immunology*; D017386:Sequence Homology, Amino Acid; D012709:Serum Albumin / Q000737:chemistry; D012710:Serum Albumin, Bovine / Q000737:chemistry
D000596:Amino Acids; D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D007106:Immune Sera; D012709:Serum Albumin; D012710:Serum Albumin, Bovine