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) 1 A1 cDNA was constructed and used to express CYP1A1 in Spodoptera frugi perda (SF9) insect cells (0.14+/-0.04 nmol/mg protein, 53+/-14 nmol/li ter, N=30). The enzyme represented approximate to 1% of total cellular protein and was partially purified by a three-column procedure to spe cific content of 5.0 nmol/mg protein. Catalytic activity was reconstit uted with both the purified enzyme using lipid and NADPH-P450 oxidored uctase, and the SF9 insect cell membrane fraction without purification using NADPH-P450 oxidoreductase and small amounts of detergent. Catal ytic activity of the enzyme after reconstitution was optimum using mol ar ratios of CYP1A1 to NADPH-P450 oxidoreductase of 1:8. Cytochrome b( 5) had no additional stimulating effect. The enzyme metabolized substr ates characteristic for CYP1A1: benzoapyrene (4.0+/-0.3 nmol/min/nmo l CYP), 7-ethoxy-4-trifluoromethylcoumarin (36+/-2), ethoxyresorufin ( 37+/-1), but not pentoxyresorufin (0.77+/-0.02). Recombinant baculovir us expresses the highest amounts of all expression systems published t o date of catalytically active CYP1A1. Because human CYP1A1 has never been isolated in a catalytically active state from human tissue, nor h as recombinant unmodified human CYP1A1, this system is an excellent al ternative for the isolation and characterization of this CYP.