Purification and characterization of three immunodominant proteins (38, 30, and 16 kDa) of Mycobacterium tuberculosis.
Specific mycobacterial antigens are an important prerequisite in the serodiagnosis of tuberculosis. Many studies have reported the use of both native and recombinant proteins. Even though recombinant proteins can form standardized reagents with unlimited supply, their diagnostic test characteristics were not satisfactory in some cases. In this study we have purified the 38-, 30- (antigen 85B), and 16-kDa native antigens of Mycobacterium tuberculosis by procedures with limited number of steps. Starting with the secreted antigens of M. tuberculosis H37Rv, the 38-kDa form was purified by preparative isoelectric focusing, followed by preparative electrophoresis. Separation of antigen 85 components was achieved by anion-exchange chromatography, followed by hydrophobic interaction chromatography. Gel-permeation chromatography was employed for the isolation of the 16-kDa form, from the cytosol fraction of M. tuberculosis H37Rv. By using a minimal number of steps, considerable yields of these proteins were obtained without loss of immunological activity. The native proteins purified were characterized by analytical two-dimensional electrophoresis, HPLC, and circular dichroism studies. Conformation of the native 38-kDa form purified in our laboratory was different from that of the recombinant 38-kDa form from the WHO Bank. The identities of these native antigens were established by immunoblotting with known monoclonal antibodies from the WHO Bank.
Devi
K R Uma
KR
Department of Immunology, Tuberculosis Research Centre (ICMR), Mayor V. R. Ramanathan Road, Chetput, Chennai 600 031, India.
Kumar
K S Senthil
KS
Ramalingam
B
B
Alamelu
Raja
R
D001426:Bacterial Proteins / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification*; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D015180:Electrophoresis, Gel, Two-Dimensional; D004797:Enzyme-Linked Immunosorbent Assay; D016056:Immunodominant Epitopes / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D007526:Isoelectric Point; D009169:Mycobacterium tuberculosis / Q000737:chemistry* / Q000276:immunology
D001426:Bacterial Proteins; D016056:Immunodominant Epitopes