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

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Abstract

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 CYP17α 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, Km and Vmax 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.

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Abbreviations

δ-ALA:

δ-Aminolevulinic acid

cDNA:

Complementary deoxyribonucleic acid

CO:

Carbon monoxide

CYP:

Cytochrome P450

DTT:

Dithiothreitol

E. coli :

Escherichia coli

EDTA:

Ethylenediamine tetraacetic acid

FAD:

Flavin adenine dinucleotide

FMN:

Flavin mononucleotide

G6P:

d-Glucose 6-phosphate

G6PDH:

d-Glucose-6-phosphate dehydrogenase

HPLC:

High performance liquid chromatography

IgG:

Immunoglobulin G

IPTG:

Isopropyl-β-d-thiogalactopyranoside

KCN:

Potassium cyanide

Km :

Michaelis constant

LB:

Luria–Bertani media

NADP:

β-Nicotinamide adenine dinucleotide phosphate

NADPH:

Reduced β-nicotinamide adenine dinucleotide phosphate

OxR:

NADPH-cytochrome P450 oxidoreductase

PMSF:

Phenylemethanesulfonyl fluoride

SDS–PAGE:

Sodium dodecyl sulphate–polyacrylamide gel electrophoresis

TB:

Terrific broth

TES:

Tris–EDTA-sucrose solution

Vmax :

Maximum velocity

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Acknowledgments

We express our gratitude to the International Medical University, Malaysia (Grant: IMU 091-05), as well as the Malaysian Ministry of Science, Technology and Innovation (Grant: eScienceFund 02-02-09-SF0005) for kind funding and support given to this project.

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Authors and Affiliations

  1. Jeffrey Cheah School of Medicine and Health Sciences, Monash University Sunway Campus, Jalan Lagoon Selatan, 46150, Bandar Sunway, Selangor, Malaysia

    Chin Eng Ong

  2. School of Pharmacy and Health Sciences, International Medical University, 126, Jalan 19/155B, Bukit Jalil, 57000, Kuala Lumpur, Malaysia

    Yan Pan & Joon Wah Mak

  3. Herbal Medicine Research Centre, Institute for Medical Research, Jalan Pahang, 50588, Kuala Lumpur, Malaysia

    Badrul Amini Abd-Rashid & Zakiah Ismail

  4. Pharmacogenetics Research Group, Institute for Research in Molecular Medicine, Universiti Sains Malaysia, 16150, Kubang Kerian, Kelantan, Malaysia

    Rusli Ismail

Authors
  1. Yan Pan
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  2. Badrul Amini Abd-Rashid
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  3. Zakiah Ismail
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  4. Rusli Ismail
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  5. Joon Wah Mak
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Correspondence to Chin Eng Ong.

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Pan, Y., Abd-Rashid, B.A., Ismail, Z. et al. Heterologous Expression of Human Cytochromes P450 2D6 and CYP3A4 in Escherichia coli and Their Functional Characterization. Protein J 30, 581–591 (2011). https://doi.org/10.1007/s10930-011-9365-6

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  • DOI: https://doi.org/10.1007/s10930-011-9365-6

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