结核分枝杆菌泛酸激酶的克隆表达及酶学性质  

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

作　　者：张鹭[1] 王庆忠[2] 徐颖[2] 陈嘉臻[2] 路福平[1] 王洪海[2] 

机构地区：[1]天津科技大学生物工程学院天津市工业微生物学重点实验室,天津300222 [2]复旦大学生命科学学院遗传工程国家重点实验室,遗传学研究所上海200433

出　　处：《第四军医大学学报》2006年第23期2135-2138,共4页Journal of the Fourth Military Medical University

基　　金：国家自然科学基金(30270072)

摘　　要：目的:获得具有生物学活性的结核分枝杆菌(Mtb)重组泛酸激酶蛋白.方法:以结核分枝杆菌模式菌株H37Rv基因组为模板,扩增泛酸激酶基因CoaA(Rv1092c),克隆入原核表达载体pET28a中,重组质粒转入大肠杆菌BL21(DE3)中进行IPTG诱导表达.在优化后的诱导条件(20℃,10h,0.5mmol/LIPTG)下,收集细菌进行超声破碎,低温高速离心,得到的上清液经镍离子螯合型亲和层析柱纯化重组蛋白.高效液相色谱及质谱鉴定重组蛋白.采用酶联法测定重组蛋白的酶学性质.结果:得到了高表达、高纯度的重组结核分枝杆菌的泛酸激酶,酶催化过程中对泛酸的kcat和Km值分别为273.81kat/L,35.27μmol/L;对ATP的kcat和Km值分别为169.10kat/L,94.43μmol/L.重组蛋白经高效液相色谱及质谱鉴定,测得Mr约为39168.在5mmol/LTris-HCl缓冲液,pH值7.5,25℃条件下,重组CoaA的二级结构中有39.3%α螺旋,13.7%β折叠,14.3%β转角,32.5%无规则卷曲.结论:成功克隆表达结核分枝杆菌泛酸激酶,酶学性质和二级结构鉴定表明重组泛酸激酶折叠正确,且具有生物学活性,为基于该酶筛选新型抗结核药物奠定了基础.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℃, 10 h, 0.5 mmol/L）. The recombinant （His） 6 fusion protein were purified by Ni-NTA 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% a-helix, 13.7% β-sheet, 14.3% β-turn, and 32.5% random coil. The kinetic parameters kcat and Km of Mycobacterium tuberculosis CoaA were found to be 273.81 kat/L, 35.27μmol/L for Pantothenate, and to be 169. 10 kat/L, 94.43 μmol/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.

关 键 词：结核分支杆菌 泛酸激酶 基因表达 酶类/分析 

分 类 号：R378.91[医药卫生—病原生物学]