Expression of recombinant human cytochrome P450 1A2 in Escherichia coli bacterial mutagenicity tester strain.
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.
Chun
Y J
YJ
College of Pharmacy, Chung Ang University, Seoul, Korea.
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
D009153:Mutagens; D011994:Recombinant Proteins; D019388:Cytochrome P-450 CYP1A2