FUNCTIONAL COEXPRESSION OF CYP2D6 AND HUMAN NADPH-CYTOCHROME P450 REDUCTASE IN ESCHERICHIA-COLI

The polymorphic human CYP2D6 has been co-expressed with human NADPH-cy tochrome P450 oxidoreductase in Escherichia coli in order to generate a functional recombinant monooxygenase system for the study of xenobio tic metabolism. The two cDNAs were co-expressed from separate, compati ble plasmids with different antibiotic selection markers. The CYP2D6 c ould be detected in bacterial cells at levels up to 700 nmol l(-1) cul ture by Fe2+-CO versus Fe2+ difference spectroscopy, exhibiting the ch aracteristic absorbance peak at 450 nm. Immunoblotting demonstrated th e presence of both proteins in bacterial membranes, where they were ex pressed at levels significantly higher than those found in human liver microsomes. Membrane content was 150-200 pmol CYP2D6 (determined spec trally) and 100-230 pmol CYP-reductase (determined enzymatically) per mg protein. Critically, the two co-expressed proteins were able to cou ple to form a NADPH-dependent monooxygenase which metabolized the CYP2 D6 substrate bufuralol (V-max 3.30 nmol min(-1) mg(-1) protein; K-m 11 .1 mu M) in isolated membrane fractions. This K-m value was similar to the K-m determined in human liver microsomes. Activity could be inhib ited by the specific inhibitor quinidine. Of greater significance howe ver, was the finding that intact E. coli cells, even in the absence of exogenous NADPH, were able to metabolize bufuralol at rates almost as high as those measured in membranes (4.6 +/- 0.4 min(-1) versus 5.7 /- 0.2 min(-1) at 50 mu M substrate). Such recombinant strains will gr eatly facilitate the molecular characterization of isoenzymes. (C) 199 8 Chapman & Hall Ltd.