cDNA-Directed expression of human cytochrome P450 CYP1A1 using baculovirus: Purification, dependency on NADPH-P450 oxidoreductase, and reconstitution of catalytic properties without purification

A recombinant baculovirus containing the human cytochrome P450 (CYP) 1A1 cDNA was constructed and used to express CYP1A1 in Spodoptera frugiperda (SF9) insect cells (0.14 ± 0.04 nmol/mg protein, 53 ± 14 nmol/liter, N = 30). The enzyme represented 1% of total cellular protein and was partially purified by a three-column procedure to a specific content of 5.0 nmol/mg protein. Catalytic activity was reconstituted with both the purified enzyme using lipid and NADPH-P450 oxidoreductase, and the SF9 insect cell membrane fraction without purification using NADPH-P450 oxidoreductase and small amounts of detergent. Catalytic activity of the enzyme after reconstitution was optimum using molar ratios of CYP1A1 to NADPH-P450 oxidoreductase of 1:8. Cytochrome b₅ had no additional stimulating effect. The enzyme metabolized substrates characteristic for CYP1A1: benzo[a]pyrene (4.0 ± 0.3 nmol/min/nmol CYP), 7-ethoxy-4-trifluoromethylcoumarin (36 ± 2), ethoxyresorufin (37 ± 1), but not pentoxyresorufin (0.77 ± 0.02). Recombinant baculovirus expresses the highest amounts of all expression systems published to date of catalytically active CYP1A1. Because human CYP1A1 has never been isolated in a catalytically active state from human tissue, nor has recombinant unmodified human CYP1A1, this system is an excellent alternative for the isolation and characterization of this CYP.