A universal approach to the expression of human and rabbit cytochrome P450s of the 2C subfamily in Escherichia coli

Toby H. Richardson, Frank Jung, Keith J. Griffin, Mike Wester, Judy L. Raucy, Byron Kemper, Lester M. Bornheim, Christopher Hassett, Curtis J. Omiecinski, Eric F. Johnson

Research output: Contribution to journalArticlepeer-review

Abstract

Human cytochrome P450s 2C8, 2C9, 2C18, and 2C19 and rabbit cytochrome P450s 2C1, 2C2, 2C4, 2C5, and 2C16 were expressed from their respective cDNAs in Escherichia coli as chimetic enzymes in which a portion of the N-terminal membrane anchor sequence was replaced with a modified sequence derived from P450 17A. For 2C1 and 2C2 removal of the extraneous 3'-untranslated sequence allowed the successful expression of constructs that were unproductive in its presence. The levels of expression varied from 180 to 1500 nmol/liter of culture and the addition of δ-aminolevulinic acid to the culture media increased the amount of spectrally detectable P450 for several of these enzymes 2- to 10-fold. The catalytic properties of the modified human 2C P450s expressed in E. coli were concordant with previously published data for several marker substrates including (S)-mephenytoin for P450 2C19, tolbutamide and tetrahydrocannabinol (THC) for P450 2C9, and taxol for P450 2C8. Interestingly, P450 2C19 catalyzed the 21-hydroxylation of progesterone and, to a lesser extent, catalyzed the formation of 16α-hydroxyprogesterone. The rabbit enzyme P450 2C16 catalyzed the formation of 17α- and 16α- 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 that of P450 2C9. This work has identified important factors required for the high-level expression of 2C subfamily P450s in E. coli. The availability of these enzymes will facilitate detailed kinetic measurements for known and yet to be identified substrates.

Original languageEnglish (US)
Pages (from-to)87-96
Number of pages10
JournalArchives of Biochemistry and Biophysics
Volume323
Issue number1
DOIs
StatePublished - Oct 1995

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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