Expression, purification and enzyme activity determination of pantothenate kinase from Mycobacterium tuberculosis

    ZHANG Lu1,2, WANG Qingª²Zhong2, XU Ying2, CHEN Jiaª²Zhen2, LU Fuª²Ping1, WANG Hongª²Hai2

    1College of Biotechnology, Tianjin University of Science and Technology, Tianjin Key Laboratory of Industrial Microbiology, Tianjin 300222, China, 2State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai 200433, China

    ¡¾Abstract¡¿ AIM: To obtain CoaA (pantothenate kinase) gene of Mycobacterium tuberculosis, express efficiently in E.coli, purify the target protein and detect its enzyme activity. METHODS: CoaA gene was amplified by PCR with sepecific primers from genome of Mycobacterium tuberculosis H37Rv, CoaA gene segments were inserted into pET28a and expressed in E.coli BL21 under the induction of IPTG (20¡æ, 10 h, 0.5 mmol/L). The recombinant (His)6 fusion protein were purified by Niª²NTA perification system. The purity of the recombinant CoaA was determined by high performance liquid chromatography (HPLC). CoaA MW was assayed by Mass spectrometry (MS). The enzyme activity was tested by spectrophotometry. RESULTS: HPLC showed CoaA was highly purified, its Mr were 39 168. CD spectrum showed Mycobacterium tuberculosis CoaA contained 39.3% ¦Áª²helix, 13.7% ¦Âª²sheet, 14.3% ¦Âª²turn, and 32.5% random coil. The kinetic parameters kcat and Km of Mycobacterium tuberculosis CoaA were found to be 273.81 kat/L, 35.27 ¦Ìmol/L for Pantothenate, and to be 169.10 kat/L, 94.43 ¦Ìmol/L for ATP respectively. CONCLUSION: The cloning, and expression of active Mycobacterium tuberculosis CoaA in E. coli systems is successful. These results provide the means for further studies on Mycobacterium tuberculosis CoaA as a potential antituberculosis drug. ......