title: Expression of recombinant human cytochrome P450 1A2 in Escherichia coli bacterial mutagenicity tester strain.
abstract: Human cytochrome P450 1A2 is one of the major cytochrome P450s in human liver. It is known to be capable of activating a number of carcinogens such as arylamines and heterocyclic amines. In order to develop the new bacterial mutagenicity test system with human P450, a full length of human P450 1A2 cDNA inserted into pCW bacterial expression vector was introduced to Escherichia coli WP2 uvrA strain which is a well-known E. coli strain for bacterial reverse mutagenicity assay. Expressed human P450 1A2 showed typical P450 hemoprotein spectra. Maximum expression was achieved at 48 hrs after incubating at 30 degrees C in terrific broth containing ampicillin, IPTG and other supplements. High level expression of P 450 1A2 in E. coli WP2 uvrA membranes was determined in SDS-PAGE. The well-known mutagens 2-aminoanthracene and MelQ increased the revertant colonies of E. coli WP2 uvrA expressing human P450 1A2 without an exogenous rat hepatic post-mitochondrial supernatant (S9 fraction) in a dose-dependent manner. The results show that the functional expression of human P450 in bacterial mutagenicity tester strain will provide a useful tool for studying the mechanism of the mutagenesis and carcinogenesis of new drugs and environmental chemicals.
authors.affiliation: College of Pharmacy, Chung Ang University, Seoul, Korea.
authors.forename: Y J
authors.initials: YJ
authors.lastname: Chun
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019388:Cytochrome P-450 CYP1A2 / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000187:drug effects; D009152:Mutagenicity Tests / Q000379:methods*; D009153:Mutagens / Q000633:toxicity; D010957:Plasmids / Q000276:immunology; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D009153:Mutagens; D011994:Recombinant Proteins; D019388:Cytochrome P-450 CYP1A2