【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 ,同时也为CPR的工业纯化提供了可行的参考路线更多还原

【Abstract】 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.更多还原