CONSTRUCTION OF A HUMAN CYTOCHROME-P450 1A1-RAT NADPH-CYTOCHROME P450REDUCTASE FUSION PROTEIN CDNA AND EXPRESSION IN ESCHERICHIA-COLI, PURIFICATION, AND CATALYTIC PROPERTIES OF THE ENZYME IN BACTERIAL-CELLS AND AFTER PURIFICATION

A plasmid (pCW) was modified to code for a fusion protein consisting o f the complete sequence of human cytochrome P450 (P450) 1A1 (with only the second amino acid changed) in the N-terminal portion connected by a Ser-Thr linker to the portion of rat NADPH-P450 reductase beginning at amino acid 57, This plasmid was used to express the fusion protein in Escherichia coli DH5 alpha cells and the protein was purified from detergent-solubilized bacterial membranes using DEAE and 2',5'-ADP ag arose chromatography. The purified fusion protein catalyzed benzo[a]py rene 3-hydroxylation, 7-ethoxyresorufin O-deethylation, and zoxazolami ne 6-hydroxylation, Catalytic activity was not increased in the presen ce of added NADPH-P450 reductase, cytochrome b(5), or phospholipid, Th e fusion protein could also transfer electrons to cytochromes c and b( 5) but not P450 1A2, The same oxidation products of benzo[a]pyrene wer e formed with the purified fusion protein and the fusion protein funct ioning in bacterial cells, The catalytic activity of the human P450 1A 1 fusion protein toward several substrates is markedly less than that of a similar fusion protein constructed with rat P450 1A1, in line wit h the reported differences in catalytic activities of the rat and huma n P450 1A1 enzymes, The purified fusion protein also oxidized (+)- and (-)-benzo[a]pyrene 7,8-dihydrodiols and eight aryl and heterocyclic a mines to ge-notoxic products, in the absence of added NADPH-P450 reduc tase, The demonstration of catalytic activities of the human fusion pr otein within bacterial cells suggests the prospect of utilizing such c ellular systems for production of human P450 metabolites. (C) 1996 Aca demic Press, Inc.