Recombinant human cytochrome P450 1B1 expression in Escherichia coli

Human cytochrome P450 (P450) 1B1 was expressed in Escherichia coli at a level of 200 nmol/liter culture using a pCW vector by removal of codons 2-4 and modification of the nucleotide sequence of the resulting N-terminal seven codons; a similar level of expression was found with a bicistronic constru...

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Bibliographic Details
Published in:Archives of biochemistry and biophysics 1998-09, Vol.357 (1), p.111
Main Authors: Shimada, T, Wunsch, R M, Hanna, I H, Sutter, T R, Guengerich, F P, Gillam, E M
Format: Article
Language:English
Subjects:
Aryl Hydrocarbon Hydroxylases
Carcinogens - metabolism
Catalysis
Cell Membrane - enzymology
Cell Membrane - metabolism
Cytochrome P-450 CYP1A1 - metabolism
Cytochrome P-450 CYP1A2 - metabolism
Cytochrome P-450 CYP1B1
Cytochrome P-450 Enzyme System - biosynthesis
Cytochrome P-450 Enzyme System - genetics
Cytochrome P-450 Enzyme System - isolation & purification
Escherichia coli - enzymology
Escherichia coli - genetics
Genetic Vectors - chemical synthesis
Humans
Recombinant Proteins - biosynthesis
Recombinant Proteins - isolation & purification
Spectrophotometry
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Summary:Human cytochrome P450 (P450) 1B1 was expressed in Escherichia coli at a level of 200 nmol/liter culture using a pCW vector by removal of codons 2-4 and modification of the nucleotide sequence of the resulting N-terminal seven codons; a similar level of expression was found with a bicistronic construct that also expressed human NADPH-P450 reductase. P450 1B1 was purified (from the monocistronic system) to electrophoretic homogeneity and a specific content of 9.2 nmol P450/mg protein using DEAE, CM, and hydroxylapatite chromatography. The absolute spectra showed a considerable fraction of high-spin iron and little cytochrome P420. The catalytic activity of the purified enzyme was considerably enhanced in the presence of cholate. Both reconstituted P450 1B1 and the bacterial membranes prepared from the bicistronic vector system had similar7-ethoxyresorufin O-deethylation activities; as expected, 17beta-estradiol was hydroxylated primarily at the 4-position. The ability of human P450 1B1 to activate several heterocyclic amines and polycyclic hydrocarbon dihydrodiols was confirmed with reconstituted P450 1B1 and the P450 1B1 membranes in which NADPH-P450 reductase was coexpressed.
ISSN:0003-9861
DOI:10.1006/abbi.1998.0808