Isolation, characterisation and kinetic studies on SEVA TB ES-31 antigen, a metallo-serine protease of interest in serodiagnosis.
BACKGROUND
SEVA TB Excretory secretory-31 (ES-31) antigen, a glycoprotein isolated from M. tb H37Ra culture filtrate, was found to be useful in the serodiagnosis of pulmonary tuberculosis (TB), extrapulmonary TB and in HIV-TB coinfection. Further, it has been shown to be a zinc containing serine protease.


OBJECTIVE
To isolate and purify SEVA TB ES-31 antigen from M. tb H37Ra culture filtrate and study of its enzyme properties and peptide sequence.


METHODS
ES-31 antigen was purified from culture filtrate of M. tuberculosis H37Ra strain by ammonium sulphate precipitation, SDS-PAGE fractionation and FPLC. Protease activity of ES-31 antigen was studied using azocasein as substrate. ES-31 antigen was further fractionated by two dimensional polyacrylamide gel electrophoresis (2D PAGE) followed by LCMS-T analysis.


RESULTS
Mycobacterial metallo-serine protease was purified 3096 fold from M. tb H37Ra culture filtrate protein. Purified enzyme showed optimum activity at pH 7.0 at 37 degrees C. Of the four substrates explored, the enzyme has shown maximum activity with azocasein and had a Km value of 0.01 mM with specific activity of 6250 x 10(-6) U/mg protein. Further, analysis of ES-31 antigen by 2D PAGE showed two protein spots (A and B).


CONCLUSIONS
Kinetic studies on SEVA TB ES-31 protein, an immunogen with metallo serine protease activity are reported for the first time. Purified enzyme had a Km value of 0.01 mM with azocasein as substrate. Further, study on structure and biological role of serine protease will be of interest.
Upadhye
Vijay J
VJ
Jamnalal Bajaj Tropical Disease Research Centre, Mahatma Gandhi Institute of Medical Sciences, Sevagram, Wardha.
Gomashe
Ashok V
AV
Kumar
Satish
S
Harinath
Bhaskar C
BC
D000942:Antigens, Bacterial / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D015415:Biomarkers / Q000032:analysis / Q000378:metabolism; D018929:Cell Culture Techniques; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D013058:Mass Spectrometry; D009169:Mycobacterium tuberculosis / Q000276:immunology*; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D012698:Serologic Tests / Q000379:methods*; D014376:Tuberculosis / Q000175:diagnosis / Q000276:immunology
D000942:Antigens, Bacterial; D015415:Biomarkers; C541122:SEVA TB ES-31 antigen, Mycobacterium tuberculosis; D012697:Serine Endopeptidases