Coexpression of mammalian cytochrome P450 and reductase in Escherichia coli.
Coexpression of mammalian cytochrome P450 and reductase in Escherichia coli.
复制标题
哺乳动物细胞色素 P450 和还原酶在大肠杆菌中的共表达。
DOI：
10.1006/abbi.1996.0118
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Porter,TD
中科院分区：
文献类型：
--
作者：
Dong,J;Porter,TD
将人细胞色素P450 2 E1和大鼠NADPH-细胞色素P450还原酶的cDNA分别串联克隆到细菌表达载体中，并通过免疫印迹、光谱和催化分析监测这两种蛋白在大肠杆菌中的表达。共表达质粒上的cDNA由22个核苷酸分开，其中P450 cDNA位于还原酶cDNA之前。无论是单独表达还是与P450还原酶共表达，溶解的细胞膜中的P450含量约为0.11 nmol/mg蛋白质，每升培养物可获得约0.8 nmol。还原酶的含量是5 - 6倍大于P450的含量时，共表达，但几倍小于没有上游P450的cDNA表达时获得的，表明两种蛋白质之间的稳定性和可翻译性的差异。从细胞表达两种蛋白质的溶解膜催化苯胺羟基化，对硝基苯酚羟基化，andN-亚硝基二甲胺脱甲基化的速率相当于那些通过结合P450和还原酶制剂;此外，纯化的还原酶，这些膜没有增加的活动。然而，与其他异源系统中表达的P450 2 E1所获得的结果相反，在催化对硝基苯酚或N-亚硝基二甲胺氧化的制剂中加入兔肝细胞色素b 5并没有增加营业额，而且，尽管不溶性膜可以显示活性，但这些底物在体内的氧化不能得到证实。尽管如此，在E. coliso体外产生功能性单加氧酶系统增强了该生物体用于克隆的P450同种型的表达和表征的效用。
cDNAs for human cytochrome P450 2E1 and rat NADPH–cytochrome-P450 reductase were cloned separately and in tandem into bacterial expression vectors, and expression of the two proteins inEscherichia coliwas monitored by immunoblotting, spectroscopy, and catalytic assays. The cDNAs were separated on the coexpression plasmid by 22 nucleotides, with the P450 cDNA preceding the reductase cDNA. P450 content in solubilized cell membranes, whether expressed alone or coexpressed with P450 reductase, was approximately 0.11 nmol/mg of protein, and approximately 0.8 nmol could be obtained per liter of culture. Reductase content was five- to sixfold greater than P450 content when coexpressed, but severalfold less than that obtained when expressed without the upstream P450 cDNA, indicating differences in both stability and translatability between the two proteins. Solubilized membranes from cells expressing both proteins catalyzed aniline hydroxylation,p-nitrophenol hydroxylation, andN-nitrosodimethylamine demethylation at rates equivalent to those obtained by combining P450 and reductase preparations; addition of purified reductase to these membranes did not augment the activity. However, in contrast to results obtained with P450 2E1 expressed in other heterologous systems, addition of rabbit liver cytochromeb5to preparations catalyzingp-nitrophenol orN-nitrosodimethylamine oxidation did not increase turnover, and, although activity could be shown with unsolubilized membranes, oxidation of these substratesin vivocould not be demonstrated. Nonetheless, the ability to coexpress P450 and reductase inE. coliso as to generate a functional monooxygenase systemin vitroenhances the utility of this organism for the expression and characterization of cloned P450 isoforms.