中国红豆杉细胞色素P450还原酶的基因克隆、表达与活性分析

细胞色素P450还原酶(Cytochrome P450 reductase,CPR)是细胞色素P450羟基化酶电子传递链的组成部分,在生物体内起着重要的电子传递作用。文章从中国红豆杉(Taxuswallichiana var. Chinensis)愈伤组织细胞中克隆CPR基因(TchCPR),TchCPR含有一个2154bp碱基的阅读框,编码717个氨基酸残基;在氨基酸水平上它与裸子植物细胞色素P450还原酶的同源性(82%)高于其他被子植物的细胞色素P450还原酶(74%)。在大肠杆菌BL21(DE3)中诱导表达了全长和从N-端截短不同数目氨基酸残基的6个融合肽段,经亲和层析纯化,分析了表达的不同长度融合蛋白的电子传递效率。结果表明截短长度大于61个氨基酸残基肽段的胞色素P450还原酶都能够诱导表达,在表达水平上无显著差异,而截短61个氨基酸的CPR融合蛋白电子传递的催化活性(1.6057nmol Cyt Cred/min/μg TchCPR融合蛋白)高于其他4个融合蛋白。

cDNA cloning, heterologous overexpression and activity analysis of cytochrome P450 reductase of Taxus chinensis

NADPH-cytochrome P450 reductase (CPR), a partner for P450 monooxygenases, serves as the electron donor to almost all eukaryotic cytochrome P450s. One cDNA (TchCPR) encoding cytochrome P450 reductase of T. chinensis was isolated from callus cells. The cDNA contains an open reading frame of 2154 nucleotides which encodes a protein of 717 amino acid residues. The TchCPR has higher similarity to other CPRs of gumnosperms (>82%) than that of angiosperms (<74%). The recombinant full-length TchCPR and a series of N-terminal truncated constructs with N-terminal fusion of His Tag were obtained and induced to express in E. coli B121(DE3), and then purified using affinity chromatography. The truncated forms of N-terminal more than 61 amino acid residues could be efficiently expressed while the truncated mutant of N-terminal 48 amino acid residues and the wild-type TchCPR were not successfully expressed in E. coli cells. The activity of the truncated TchCPR was assayed by measuring the reduction of cytochrome C. The electron transfer activity of the recombinantly purified CPRT61 was 1.6057 nmol of cytochrome C reduced per min per microg TchCPR reductase, and it is higher than that of the other four truncated forms.