Expression of human cytochrome P450 2B6 in Escherichia coli: characterization of catalytic activity and expression levels in human liver.

Hanna IH, Reed JR, Guengerich FP, Hollenberg PF
Arch Biochem Biophys. 2000 376 (1): 206-16

PMID: 10729207 · DOI:10.1006/abbi.2000.1708

Expression of human cytochrome P450 (P450) 2B6 in Escherichia coli was achieved following supplementation of the expression medium with chloramphenicol. The recombinant protein was purified using Ni(2+)-nitrilotriacetate chromatography and was characterized with regard to its spectral properties and catalytic activities toward typical P450 substrates. The purified recombinant protein was also used to raise polyclonal antibodies in rabbits. Examination of a panel of human liver microsomal preparations revealed expression of P450 2B6 in most samples, with levels of <1 to 30 pmol 2B6/mg microsomal protein. Examination of purified P450 2B6 preparations revealed the presence of a protease-sensitive site located 126 residues away from the N-terminus. The identity of the cleavage boundary was verified by protein sequence analysis. Cleavage of P450 2B6 at that site results in the presence of a lower molecular weight fragment of approximately 35 kDa in purified preparations. An immunoreactive peptide of a similar molecular weight was consistently observed in some but not all human liver microsomal preparations suggesting cleavage at the same site. Examination of catalytic activities of the purified reconstituted protein indicated the potential utility of (S)-mephenytoin N-demethylation and testosterone 16beta-hydroxylation as markers for P450 2B6.

Copyright 2000 Academic Press.

MeSH Terms (20)

Animals Antibody Specificity Aryl Hydrocarbon Hydroxylases Binding Sites Cytochrome P-450 CYP2B6 Cytochrome P-450 Enzyme System Escherichia coli Gene Expression Humans Immunohistochemistry In Vitro Techniques Kinetics Mephenytoin Microsomes, Liver Oxidoreductases, N-Demethylating Rabbits Recombinant Proteins Spectrophotometry Substrate Specificity Testosterone

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