重组NADPH-细胞色素P450还原酶在大肠杆菌中的表达和纯化  

Expression and purification of recombinant human NADPH-cytochrome P450 reductase in E.coli

作　　者：王志刚[1] 王秀宏[1] 滕悦秋[1] 刘明[1] 周虹[1] 

机构地区：[1]哈尔滨医科大学生物化学与分子生物学教研室,黑龙江哈尔滨150086

出　　处：《哈尔滨医科大学学报》2004年第2期99-101,106,共4页Journal of Harbin Medical University

基　　金：国家自然科学基金资助项目 ( 3 0 0 0 0 0 3 0 )

摘　　要：目的　在大肠杆菌中表达重组NADPH -细胞色素P4 5 0还原酶 (CPR)并纯化获得有活性的CPR蛋白。方法　将已构建的NADPH -细胞色素P4 5 0还原酶重组质粒pMW1 72a CPR采用温和转化法转入大肠杆菌BL 2 1中 ,依次使用超速离心、DE5 2纤维素阴离子交换层析和 2′ 5′ADP亲和层析的方法纯化CPR ,并检测酶的活性。结果　获得了纯度达 99%以上的可溶性CPR蛋白 ,纯化倍数 5 .2 0倍 ,检测酶的比活性为每分钟 1 0 4 5 .4 9nmol mg。结论　获得了纯度高、有活性的CPR蛋白 ,可作为工具酶用于某些相关酶的研究 。Objective To express and purify NADPH cytochrome P450 reductase in E.coli . BL 21.Methods pMW172a CPR, the reconstructed plasmid with NADPH cytochrome P450 reductase was transformed into E.coli . BL 21 by mild method. Then ultracentrifugation, DE52 cellulose anion exchange chromatography and 2′ 5′ ADP affinity chromatography were applied in turn to purify CPR. Meanwhile, the catalytic activities of the purified enzyme were tested.Results The soluble CPR which purity was more than 99% was acquired. The specific activity was 1 045.49 nmol/(mg·min).Conclusion CPR with high purity and strong activity is acquired, which could be used for the research of the related enzymes. What's more, the whole protocol could provide a feasible reference course for CPR industrial purification.

关 键 词：重组NADPH-细胞色素 P450还原酶 大肠杆菌 基因表达 CPR 生物医学 

分 类 号：R378.21[医药卫生—病原生物学]