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High‐level expression of functional human cytochrome P450 1A2 in Escherichia coli
Author(s) -
Fisher Charles W.,
Caudle Deborah L.,
MartinWixtrom Cheryl,
Quattrochi Linda C.,
Tukey Robert H.,
Waterman Michael R.,
Estabrook Ronald W.
Publication year - 1992
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.6.2.1537466
Subject(s) - escherichia coli , complementary dna , cytochrome p450 , microbiology and biotechnology , open reading frame , hemeprotein , biology , biochemistry , hydroxylation , recombinant dna , chemistry , peptide sequence , heme , gene , enzyme
Enzymatically active human cytochrome P450 1A2 was expressed in Escherichia coli utilizing the pCWori+ vector containing a modified cDNA. The coding sequence for the NH 2 ‐terminal region of the protein was modified by the alignment and substitution of a 27 bp segment from a modified bovine P450 17A1 cDNA onto the 5′ end of the open reading frame of P450 1A2 at amino acid 21. The expressed chimeric P450 was produced at a high level in a functionally intact form, as assayed by the formation in vivo of the 449 nm absorbance band of the CO complex of the reduced hemoprotein. E. coli membrane preparations were shown to contain P450 1A2, which was active in the 2‐hydroxylation of estradiol, and the O‐deethylation of 7 ‐ethoxycoumarin and 7 ‐ethoxyresorufin, when reconstituted with recombinant rat liver NADPH‐cytochrome P450 reductase.—Fisher, C. W.; Caudle, D. L.; Martin‐Wixtrom, C.; Quattrochi, L. C.; Tukey, R. H.; Waterman, M. R.; Estabrook, R. W. High‐level expression of functional human cytochrome P450 1A2 in Escherichia coli . FASEB J. 6: 759‐764; 1992.