Park Hyoung-Goo, Lim Young-Ran, Han Songhee, Jeong Dabin, Kim Donghak
Department of Biological Sciences, Konkuk University, Seoul 05025, Republic of Korea.
J Microbiol Biotechnol. 2017 May 28;27(5):983-989. doi: 10.4014/jmb.1701.01028.
NADPH-P450 reductase (NPR) transfers electrons from NADPH to cytochrome P450 and heme oxygenase enzymes to support their catalytic activities. This protein is localized within the endoplasmic reticulum membrane and utilizes FMN, FAD, and NADPH as cofactors. Although NPR is essential toward enabling the biochemical and pharmacological analyses of P450 enzymes, its production as a recombinant purified protein requires a series of tedious efforts and a high cost due to the use of NADP in the affinity chromatography process. In the present study, the rat NPR clone containing a 6× Histidine-tag (NPR-His) was constructed and heterologously expressed. The NPR-His protein was purified using Ni-affinity chromatography, and its functional features were characterized. A single band at 78 kDa was observed from SDS-PAGE and the purified protein displayed a maximum absorbance at 455 nm, indicating the presence of an oxidized flavin cofactor. Cytochrome and nitroblue tetrazolium were reduced by purified NPR-His in an NADPH-dependent manner. The purified NPR-His successfully supported the catalytic activities of human P450 1A2 and 2A6 and fungal CYP52A21, yielding results similar to those obtained using conventional purified rat reductase. This study will facilitate the use of recombinant NPR-His protein in the various fields of P450 research.
NADPH - 细胞色素P450还原酶（NPR）将电子从NADPH转移至细胞色素P450和血红素加氧酶，以支持它们的催化活性。该蛋白定位于内质网膜内，并利用黄素单核苷酸（FMN）、黄素腺嘌呤二核苷酸（FAD）和NADPH作为辅因子。尽管NPR对于细胞色素P450酶的生化和药理学分析至关重要，但由于在亲和层析过程中使用了烟酰胺腺嘌呤二核苷酸磷酸（NADP），其作为重组纯化蛋白的生产需要一系列繁琐的工作且成本高昂。在本研究中，构建了含有6×组氨酸标签（NPR - His）的大鼠NPR克隆并进行了异源表达。使用镍亲和层析法纯化了NPR - His蛋白，并对其功能特性进行了表征。从十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）中观察到一条78 kDa的单条带，纯化后的蛋白在455 nm处显示出最大吸光度，表明存在氧化型黄素辅因子。纯化后的NPR - His以NADPH依赖的方式还原细胞色素和硝基蓝四唑。纯化后的NPR - His成功支持了人细胞色素P450 1A2和2A6以及真菌细胞色素P450 52A21的催化活性，产生的结果与使用传统纯化的大鼠还原酶所获得的结果相似。本研究将促进重组NPR - His蛋白在细胞色素P450研究的各个领域中的应用。
