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Coexpression of Mammalian Cytochrome P450 and Reductase inEscherichia coli

https://doi.org/10.1006/abbi.1996.0118Get rights and content

Abstract

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.

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