Prokaryotic expression, purification and identification of Mycobacterium tuberculosis Ag85B/ILª²2 fusion protein and detection of its biological activity

    YANG Dengª²Ke, JIN Fengª²Shuo, ZHANG Yong

    1Department of Urology, PLA 159 Hospital, Zhumadian 463000, China

    2Department of Urology, Research Institute of Field Surgery, Daping Hospital, Third Military Medical University, Chongqing 400042, China

    ¡¾Abstract¡¿ AIM£º To express Mycobacterium tuberculosis Ag85B/ ILª²2 fusion protein in E.coli, to purify and identify it, and to detect its biological activity. METHODS£º By using genetic engineering techniques, we constructed a recombinant expression plasmid pGEXª²Ag85B/ILª²2, and expressed Ag85B/ILª²2ª²GST fusion protein in E.coli BL21 under the induction of IPTG. Moreover, we also studied the optimal expression conditions concerning IPTG concentration and induction time. After renatured by urea in a concentration gradient, and purified by GSTª²Sepharose affinity chromatography and digested by thrombin, the Ag85B/ILª²2 fusion protein was further purified by anionª²exchange chromatography and RPª²HPLC and identified with Western blot. Its Nª²terminal amino acid sequence and ILª²2 bioª²activity were measured as well. RESULTS£º A novel fusion protein Ag85B/ILª²2ª²GST was expressed in E.coli in a way of inclusion body,accounting for 30% of total lysate protein of bacteria. After purified by GSTª²Sepharose affinity chromatography, anionª²exchange chromatography and RPª²HPLC, the desired Ag85B/ILª²2 fusion protein with purification degree of 98.32% was acquired and confirmed by Western blot. Its Nª²terminal amino acid sequence was identical to the anticipation, and its specific ILª²2 bioactivity was 2500 u/mg. CONCLUSION£º Ag85B/ILª²2 fusion protein was successfully expressed in E.coli and purified. This results established a groundwork for the further researches of Ag85B/ILª²2 fusion protein in the immunotherapy of bladder tumor. ......