ESCHERICHIA-COLI EXPRESSION AND CHARACTERIZATION OF CYTOCHROMES P450 2B11, 2B1, AND 2B5

Dog CYP2B11, rat CYP2B1, and rabbit CYP2B5 have been expressed in Esch erichia coli from cDNAs modified at the N-terminus (Barnes et al., 199 1, Proc. Natl. Acad. Sci. USA 88, 5597-5601). Using holamidopropyl)dim ethylammonio]-1-propanesulfonate (Chaps), solubilized membranes repres enting > 100 nmol of P450 2B11, > 35 nmol of P450 2B1, and > 7 nmol of P450 2B5 were efficiently extracted (40-70% yield) from a 1-liter cul ture. Chaps-solubilized preparations produced a reduced CO/reduced dif ference spectrum devoid of P420 and were used directly in a reconstitu ted system. The E. cell-expressed 2B enzymes retained the same functio nal characteristics as the purified hepatic enzymes or enzymes express ed in COS cells in terms of androstenedione metabolite profiles. Hydro xylation rates were determined under a variety of conditions, includin g two concentrations of NADPH-cytochrome P450 reductase (2 and 16 nmol /nmol P450) and the absence or presence of cytochrome b(5) (2 nmol/nmo l P450). The androstenedione hydroxylase activities of expressed 2B1 a nd 2B5 were stimulated by cytochrome b(5), whereas P450 2B11 was inhib ited slightly by cytochrome b(5). Purified expressed 2B11 (specific co ntent, 8 nmol/mg protein) had similar activities as the Chaps-solubili zed membrane preparation. The solubilized membranes containing 2B11 we re also tested with 2,2',4,4',5,5'-hexachlorobiphenyl (245-HCB). Three major metabolites, 2-hydroxy-4,5,2',4',5'-pentachlorobiphenyl, 3-hydr oxy-2,4,5,2',4',5'-hexachlorobiphenyl, and 2-hydroxy-3,4,5,2',4',5'-he xachlorobiphenyl were produced from 245-HCB. These metabolites are ide ntical to those produced by 2B11 purified from liver microsomes. The 2 45-HCB hydroxylation rates were similar for E. coli-expressed 2B11, do g liver microsomes, and purified liver 2B11. When only the secend codo n in the 2B1 was changed to GCT, > 25 nmol of P450 was extracted from a 1-liter culture, suggesting that the full Barnes ct al. modification scheme may not be necessary for high-level expression. An efficient m ethod of expressing, extracting, and analyzing different P450 2B enzym es has thus been achieved. In addition, rabbit P450 2B5, which has nev er been purified from liver, as well as different P450 2B mutants can now be expressed at much higher levels than previously reported. The a bility to express different 2B wild-type and mutant P450s in E. coli p rovides an excellent opportunity to study the molecular basis of speci es differences in substrate metabolism. (C) 1994 Academic Press, Inc.