Functional characterization of four allelic variants of human cytochrome P450 1A2
Section snippets
Chemicals
Glu-P-1, IQ, MeIQ, N-hydroxy-MeIQ, nitro-IQ, and PhIP were purchased from Toronto Research Chemicals (Toronto, ON). [Ring -3H]-Phenacetin was a gift of Dr. F.F. Kadlubar (National Center for Toxicological Research, Jefferson, AR). IPTG, δ-ALA, and ampicillin were obtained from Sigma Chemical (St. Louis, MO). Other chemicals were of the highest grades commercially available.
Bacterial culture medium and molecular biology reagents
Bacto agar, bacto yeast extract, and peptone were purchased from Difco (Detroit, MI). Luria–Bertani medium (capsule
Site-directed mutagenesis
All four P450 1A2 variants were successfully constructed by PCR site-directed mutagenesis of the bicistronic plasmid expression vector. In each case, the complete P450 1A2 open reading frame and the tac-tac promoter regions were sequenced, to ensure that no additional mutations had been introduced.
Recombinant protein expression
Immunoblot analysis (Fig. 1) revealed that the levels of expression of P450 1A2 variants C406Y, D348N, and I386F were lower than that of the wild-type enzyme. Variant R431W, however, was undetectable,
Acknowledgements
This study was supported by NIH Grants R01 CA90426 and P30 ES00267 and by the Natural Sciences and Engineering Research Council of Canada.
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