UNIVERSAL APPROACH TO THE EXPRESSION OF HUMAN AND RABBIT CYTOCHROME P450S OF THE 2C SUBFAMILY IN ESCHERICHIA-COLI

Human cytochrome P450s 2C8, 2C9, 2C18, and 2C19 and rabbit cytochrome P450s 2C1, 2C2, 2C4, 2C5, and 2C16 were expressed from their respectiv e cDNAs in Escherichia coli as chimeric enzymes in which a portion of the N-terminal membrane anchor sequence was replaced with a modified s equence derived from P450 17A. For 2C1 and 2C2 removal of the extraneo us 3'-untranslated sequence allowed the successful expression of const ructs that were unproductive in its presence. The levels of expression varied from 180 to 1500 nmol/liter of culture and the addition of S-a minolevulinic acid to the culture media increased the amount of spectr ally detectable P450 for several of these enzymes 2- to 10-fold. The c atalytic properties of the modified human 2C P450s expressed in E. col i were concordant with previously published data for several marker su bstrates including (S)-mephenytoin for P450 2C19, tolbutamide and tetr ahydrocannabinol (THC) for P450 2C9, and taxol for P450 2C8. interesti ngly, P450 2C19 catalyzed the 21-hydroxylation of progesterone and, to a lesser extent, catalyzed the formation of 16 alpha-hydroxyprogester one. The rabbit enzyme P450 2C16 catalyzed the formation of 17 alpha- and 16 alpha-hydroxyprogesterone in addition to 21-hydroxylation. P450 2C19 also catalyzed the methylhydroxylation of tolbutamide and the 7- hydroxylation of THC at rates that were similar to or greater than tha t of P450 2C9. This work has identified important factors required for the high-level expression of 2C subfamily P450s in E. coli. The avail ability of these enzymes will facilitate detailed kinetic measurements for known and yet to be identified substrates. (C) 1995 Academic Pres s, Inc