HIGH-LEVEL EXPRESSION IN ESCHERICHIA-COLI OF ENZYMATICALLY ACTIVE FUSION PROTEINS CONTAINING THE DOMAINS OF MAMMALIAN CYTOCHROMES-P450 AND NADPH-P450 REDUCTASE FLAVOPROTEIN
HIGH-LEVEL EXPRESSION IN ESCHERICHIA-COLI OF ENZYMATICALLY ACTIVE FUSION PROTEINS CONTAINING THE DOMAINS OF MAMMALIAN CYTOCHROMES-P450 AND NADPH-P450 REDUCTASE FLAVOPROTEIN
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DOI：
10.1073/pnas.89.22.10817
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发表时间：
1992-11-15
影响因子：
11.1
通讯作者：
ESTABROOK, RW
中科院分区：
文献类型：
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作者：
FISHER, CW;SHET, MS;ESTABROOK, RW
本报告描述了两种哺乳动物细胞色素P450的特性，它们在大肠杆菌中作为含有大鼠nadph -细胞色素P450还原酶黄蛋白结构域的酶活性融合蛋白高水平表达（EC 1.6.2.4）。我们利用大鼠肝脏NADPH-P450还原酶的cDNA与类固醇代谢的牛肾上腺P450 17A的cDNA进行基因工程，并引入Ser-Thr连接体，得到了我们命名为rF450[mBov17A/mRatOR]L1的融合蛋白。同样，将大鼠肝脏omega-羟化酶（P450 4A1） cDNA与大鼠肝脏NADPH-P450还原酶cDNA连接，得到rF450[mRat4A1/mRatOR]L1。通过超声波破坏转化的大肠杆菌，差速离心分离膜，用洗涤剂溶解，亲和层析，可以获得大量几乎等于118 kDa的纯化融合蛋白。在不添加磷脂的情况下，纯化的融合蛋白代谢类固醇（rF450[mBov17A/mRatOR]L1）或脂肪酸（rF450[mRat4A1/mRatOR]L1）的周转率为10 - 30/min。添加纯化的大鼠肝细胞色素b5刺激了17-羟基孕烯醇酮转化为脱氢表雄酮的17,20裂解酶反应，添加纯化的大鼠nadph -细胞色素P450还原酶增强了月桂酸和花生四烯酸的omega - 1代谢物的形成。NADPH氧化与纯化融合蛋白的底物羟基化紧密耦合。
This report describes the properties of two mammalian cytochromes P450 that have been expressed at high levels in Escherichia coli as enzymatically active fusion proteins containing the flavoprotein domain of rat NADPH-cytochrome P450 reductase (EC 1.6.2.4). Fusion proteins were prepared by engineering the cDNAs for the steroid-metabolizing bovine adrenal P450 17A with the cDNA for rat liver NADPH-P450 reductase with the introduction of a Ser-Thr linker to give a protein we have named rF450[mBov17A/mRatOR]L1. Similarly, the cDNA for the omega-hydroxylase of rat liver (P450 4A1) was linked with the cDNA for rat liver NADPH-P450 reductase to give rF450[mRat4A1/mRatOR]L1. A procedure involving disruption of transformed E. coli by sonication, isolation of membranes by differential centrifugation, solubilization with detergent, and affinity chromatography provided significant amounts of purified fusion proteins of almost-equal-to 118 kDa. The purified fusion proteins had turnover numbers for the metabolism of steroids (rF450[mBov17A/mRatOR]L1) or fatty acids (rF450[mRat4A1/mRatOR]L1) ranging from 10/min to 30/min in the absence of added phospholipid. Addition of purified rat liver cytochrome b5 stimulated the 17,20-lyase reaction for the conversion of 17-hydroxypregnenolone to dehydroepiandrosterone, and addition of purified rat NADPH-cytochrome P450 reductase enhanced the formation of omega - 1 metabolites from lauric and arachidonic acids. NADPH oxidation was tightly coupled to substrate hydroxylation with the purified fusion proteins.