CDNA-DIRECTED EXPRESSION OF HUMAN CYTOCHROME-P450 CYP3A4 USING BACULOVIRUS

A recombinant baculovirus containing the human CYP3A4 cDNA was constru cted and used to express CYP3A4 in SF9 insect cells (0.46 +/- 0.13 nmo l/mg protein, 103 +/- 29 nmol/liter, N = 15). The enzyme represented s imilar to 2-3% of total cellular protein and could be purified by a tw o column procedure to a specific content of 12.7 nmol/mg protein. Cata lytic activity of the purified enzyme after reconstitution was optimum using molar ratios of CYP3A4 to cytochrome b(5) to NADPH-P450 oxidore ductase of 1:3:20, respectively. The enzyme metabolized cortisol, eryt hromycin, testosterone, and (R)-warfarin. Recombinant baculovirus expr esses the highest amounts of all expression systems published to date of catalytically intact CYP3A4. This system is an excellent alternativ e for the isolation and characterization of P450 forms from human live r.