Heterologous Expression of Human Cytochromes P450 2D6 and CYP3A4 in Escherichia coli and Their Functional Characterization

被引:20
作者
Pan, Yan [2 ]
Abd-Rashid, Badrul Amini [3 ]
Ismail, Zakiah [3 ]
Ismail, Rusli [4 ]
Mak, Joon Wah [2 ]
Ong, Chin Eng [1 ]
机构
[1] Monash Univ, Jeffrey Cheah Sch Med & Hlth Sci, Bandar Sunway 46150, Selangor, Malaysia
[2] Int Med Univ, Sch Pharm & Hlth Sci, Kuala Lumpur 57000, Malaysia
[3] Inst Med Res, Herbal Med Res Ctr, Kuala Lumpur 50588, Malaysia
[4] Univ Sains Malaysia, Pharmacogenet Res Grp, Inst Res Mol Med, Kubang Kerian 16150, Kelantan, Malaysia
关键词
Cytochromes P450; Protein expression; Dextromethorphan O-demethylation; Testosterone; 6; beta-hydroxylation; HPLC; N-DEMETHYLATION; LIVER-MICROSOMES; DRUG-METABOLISM; IN-VITRO; REDUCTASE; CYP2D6; DEXTROMETHORPHAN; PURIFICATION; COEXPRESSION; SYSTEM;
D O I
10.1007/s10930-011-9365-6
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
This study aimed to express two major drug-metabolizing human hepatic cytochromes P450 (CYPs), CYP2D6 and CYP3A4, together with NADPH-cytochrome P450 oxidoreductase (OxR) in Escherichia coli and to evaluate their catalytic activities. Full length cDNA clones of both isoforms in which the N-terminus was modified to incorporate bovine CYP17 alpha sequence were inserted into a pCWori(+) vector. The modified CYP cDNAs were subsequently expressed individually, each together with OxR by means of separate, compatible plasmids with different antibiotic selection markers. The expressed proteins were evaluated by immunoblotting and reduced CO difference spectral scanning. Enzyme activities were examined using high performance liquid chromatography (HPLC) assays with probe substrates dextromethorphan and testosterone for CYP2D6 and CYP3A4, respectively. Results from immunoblotting demonstrated the presence of both CYP proteins in bacterial membranes and reduced CO difference spectra of the cell preparations exhibited the characteristic absorbance peak at 450 nm. Co-expressed OxR also demonstrated an activity level comparable to literature values. Kinetic parameters, K-m and V-max values determined from the HPLC assays also agreed well with literature values. As a conclusion, the procedures described in this study provide a relatively convenient and reliable means of producing catalytically active CYP isoforms suitable for drug metabolism and interaction studies.
引用
收藏
页码:581 / 591
页数:11
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