EXPRESSION OF MODIFIED HUMAN CYTOCHROME-P450-1A1 IN ESCHERICHIA-COLI - EFFECTS OF 5' SUBSTITUTION, STABILIZATION, PURIFICATION, SPECTRAL CHARACTERIZATION, AND CATALYTIC PROPERTIES

Human cytochrome P450 (P450) 1A1 is primarily an extrahepatic enzyme and is important because of its roles in the activation of polycyclic hydrocarbons and other xenobiotic chemicals. Purification of active enzyme from human tissues has not been successful. We report the expression and purification of the recombinant enzyme from Escherichia coli. A full-length cDNA of human cytochrome P450 1A1 and several modified constructs were engineered into a pCW vector and used to transform E. coli cells. Little expression was observed with the native sequence and several modified constructs, but successful expression (20-25 nmol membrane-bound P450 1A1 per liter of culture) was achieved with a construct in which the Ala codon GCT was placed in the second position and the 5'-terminal codons were maximized for AT content and minimized for the potential of secondary structure formation of the mRNA transcript, alpha-Naphthoflavone was found to protect against denaturation by detergents during solubilization and was added to buffers used for purification. The recombinant P450 1A1 was purified to electrophoretic homogeneity after two ion-exchange chromatography steps in similar to 50% yield. N-Terminal amino acid sequence analysis verified the expected first 21 residues, with the exception of the terminal Met. The isolated human ferric P450 1A1 was predominantly in the high spin state, in contrast to the orthologous rat and rabbit enzymes. Recombinant P450 1A1 catalyzed 7-ethoxyresorufin O-deethylation and benzo[a]pyrene 3-hydroxylation with K-m values of 0.58 and 15 mu M and V-max values of 8.3 and 2.5 nmol min(-1) (nmol P450 1A1)(-1), respectively. The successful expression and purification of human P450 1A1 should increase the availability of this enzyme and the generation of antibodies for further biochemical and other biological studies. (C) 1994 Academic Press, Inc.