CHARACTERIZATION OF A FUSION PROTEIN BETWEEN HUMAN CYTOCHROME-P450 1A1 AND RAT NADPH-P450 OXIDOREDUCTASE IN ESCHERICHIA-COLI

A cDNA of fusion protein between human cytochrome P450 1A1 and rat NAD PH-P450 reductase was genetically engineered and expressed in Escheric hia cell DH5 alpha cells under the control of an inducible tac promote r (Y. J. Chun, T. Shimada, and F. P. Guengerich, (1996) Arch. Biochem. Biophys. 330, 48-58). E. coli membranes of transformed cells showed m uch higher P450 1A1-dependent monooxygenase and NADPH-P450 reductase a ctivities than pCW control vector or P450 1A1 expression vector-transf ormed cells, Ethoxyresorufin O-deethylase and methoxyresorufin O-demet hylase were 22-fold and 11-fold higher than the control activity, resp ectively, alpha-Naphthoflavone and beta-naphthoflavone strongly inhibi ted P450 1A1 activity of the fusion protein, with alpha-naphthoflavone being more potent than beta-naphthoflavone. Divalent cations (e.g. Ca 2+ and Mg2+) increased P450 1A1 activity as well as NADPH-P450 reducta se activity. These results demonstrate that this fusion protein in E. coli membrane may be a useful model for elucidating details of protein -protein interactions between P450 and NADPH-P450 reductase in the end oplasmic reticulum of mammalian cells. (C) 1997 Academic Press