title: Coexpression of mammalian cytochrome P450 and reductase in Escherichia coli.
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 in Escherichia coli was 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, and N-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 cytochrome b5 to preparations catalyzing p-nitrophenol or N-nitrosodimethylamine oxidation did not increase turnover, and, although activity could be shown with unsolubilized membranes, oxidation of these substrates in vivo could not be demonstrated. Nonetheless, the ability to coexpress P450 and reductase in E. coli so as to generate a functional monooxygenase system in vitro enhances the utility of this organism for the expression and characterization of cloned P450 isoforms.
authors.affiliation: Division of Pharmacology and Experimental Therapeutics, College of Pharmacy, University of Kentucky, Lexington, 40536-0082, USA.
authors.forename: J
authors.initials: J
authors.lastname: Dong
authors.forename: T D
authors.initials: TD
authors.lastname: Porter
mesh_terms: D000818:Animals; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D019392:Cytochrome P-450 CYP2E1; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008322:Mammals; D008969:Molecular Sequence Data; D009251:NADPH-Ferrihemoprotein Reductase / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D010089:Oxidoreductases, N-Demethylating / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013379:Substrate Specificity
chemical_list: D018076:DNA, Complementary; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D019392:Cytochrome P-450 CYP2E1; D010089:Oxidoreductases, N-Demethylating; D009251:NADPH-Ferrihemoprotein Reductase