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 huma n liver. It is known to be capable of activating a number of carcinoge ns 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-kno wn E. coli strain for bacterial reverse mutagenicity assay. Expressed human P450 1A2 showed typical P450 hemoprotein spectra. Maximum expres sion was achieved at 48 hrs after incubating at 30 degrees C in terrif ic broth containing ampicillin, IPTG and other supplements. High level expression of P 450 1A2 in E. coli WP2 uvrA membranes was determined in SDS-PACE. The well-known mutagens 2-aminoanthracene and MelQ increa sed the revertant colonies of E. coli WP2 uvrA expressing human P450 1 A2 without an exogenous rat hepatic post-mitochondrial supernatant (59 fraction) in a dose-dependent manner. The results show that the funct ional expression of human P450 in bacterial mutagenicity tester strain will provide a useful tool for studying the mechanism of the mutagene sis and carcinogenesis of new drugs and environmental chemicals.