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Expression of Human Cytochrome P450 2B6 in Escherichia coli: Characterization of Catalytic Activity and Expression Levels in Human Liver

https://doi.org/10.1006/abbi.2000.1708Get rights and content

Abstract

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 Ni2+–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 16β-hydroxylation as markers for P450 2B6.

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    This study was supported in part by USPHS Grants R35 CA44353 and P30 ES00267 (F.P.G.) and R01 CA16954 (P.F.H.). I.H.H. was supported in part by National Research Service Award F32 CA79162 from the National Institutes of Health.

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    To whom correspondence may be addressed at Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, 638 Medical Research Building I, 23rd and Pierce Avenues, Nashville, TN 37232-0146. Fax: (615) 322-3141. E-mail: [email protected].

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    To whom correspondence may be addressed at Department of Pharmacology, 2301 Medical Sciences Research Building III, 1150 West Medical Center Drive, Ann Arbor, MI 48109-0632. Fax: (734) 763-4450. E-mail: [email protected].

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