The Protein Journal

, Volume 30, Issue 8, pp 581–591 | Cite as

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

  • Yan Pan
  • Badrul Amini Abd-Rashid
  • Zakiah Ismail
  • Rusli Ismail
  • Joon Wah Mak
  • Chin Eng OngEmail author


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.


Cytochromes P450 Protein expression Dextromethorphan O-demethylation Testosterone 6β-hydroxylation HPLC 



δ-Aminolevulinic acid


Complementary deoxyribonucleic acid


Carbon monoxide


Cytochrome P450



E. coli

Escherichia coli


Ethylenediamine tetraacetic acid


Flavin adenine dinucleotide


Flavin mononucleotide


d-Glucose 6-phosphate


d-Glucose-6-phosphate dehydrogenase


High performance liquid chromatography


Immunoglobulin G




Potassium cyanide


Michaelis constant


Luria–Bertani media


β-Nicotinamide adenine dinucleotide phosphate


Reduced β-nicotinamide adenine dinucleotide phosphate


NADPH-cytochrome P450 oxidoreductase


Phenylemethanesulfonyl fluoride


Sodium dodecyl sulphate–polyacrylamide gel electrophoresis


Terrific broth


Tris–EDTA-sucrose solution


Maximum velocity



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.


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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Yan Pan
    • 2
  • Badrul Amini Abd-Rashid
    • 3
  • Zakiah Ismail
    • 3
  • Rusli Ismail
    • 4
  • Joon Wah Mak
    • 2
  • Chin Eng Ong
    • 1
    Email author
  1. 1.Jeffrey Cheah School of Medicine and Health SciencesMonash University Sunway CampusBandar SunwayMalaysia
  2. 2.School of Pharmacy and Health SciencesInternational Medical UniversityKuala LumpurMalaysia
  3. 3.Herbal Medicine Research CentreInstitute for Medical ResearchKuala LumpurMalaysia
  4. 4.Pharmacogenetics Research Group, Institute for Research in Molecular MedicineUniversiti Sains MalaysiaKubang KerianMalaysia

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