ISOLATION, CHARACTERISATION AND KINETIC STUDIES ON SEVA TB ES-31 ANTIGEN, A METALLO-SERINE PROTEASE OF INTEREST IN SERODIAGNOSIS

摘要

Background: SEVA TB Excretory secretory -31 (ES-31) antigen, a giycopratein isolated from M. tb H_(37)Ra culturefiltrate, was found to be useful in the serodiagnosis of pulmonary tuberculosis (TB), extrapulmonary TB and in HIV-TBcoinfection. Further, it has been shown to be a zinc containing serine protease.Aim: To isolate and purify SEVA TB ES-31 antigen from M. tb H_(37)Ra culture filtrate and study of its enzyme propertiesand peptide sequence.Methods: ES-31 antigen was purified from culture filtrate of M. tuberculosis H_(37)Ra strain by ammonium sulphateprecipitation, SDS-PAGE fractionation and FPLC. Protease activity of ES-31 antigen was studied using azocasein assubstrate. 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 H_(37)Ra culture filtrate protein.Purified enzyme showed optimum activity at pH 7.0 at 37 癈. Of the four substrates explored, the enzyme has shownmaximum 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).Conclusion: Kinetic studies on SEVA TB ES-31 protein, an immunogen with metallo serine protease activity arereported for the first time. Purified enzyme had a Km value of 0.01 mM with azocasein as substrate. Further, study onstructure and biological role of serine protease will be of interest.