EXPRESSION OF MODIFIED HUMAN CYTOCHROME-P450 2E1 IN ESCHERICHIA-COLI,PURIFICATION, AND SPECTRAL AND CATALYTIC PROPERTIES

Human cytochrome P450 (P450) 2E1 is of interest because of its role in the oxidation of numerous drugs and carcinogens. The purification of the protein from human liver is difficult, and we report the developme nt of a system for relatively high-level expression in Escherichia col i. A cDNA was prepared from liver cDNA by polymerase chain reaction me thods and several variants with modified 5'-termini were constructed. Analysis of seven of these indicated that the highest levels of expres sion were found when the first 21 codons of the native sequence were d eleted and the Trp immediately following the resulting N-terminal Met was changed to Ala (GCT). Levels of 40-nmol membrane-bound P450 2E1 (l iter culture)(-1) mere routinely recovered. The recombinant P450 2E1 w as purified to electrophoretic homogeneity from the bacterial membrane s in two ion-exchange steps in >80% yield. Ferric P450 2E1 was isolate d in a mixed spin state. The enzyme was active in chlorzoxazone 6-hydr oxylation; the addition of human liver cytochrome b(5) lowered the K-m for the substrate and increased V-max. N-Terminal amino acid sequence analysis yielded the expected first 21 residues. The expression syste m should facilitate the availability of human P450 2E1 and antibodies for studies of the enzyme. (C) 1994 Academic Press, Inc.