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.
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The authors declare that they have no conflict of interest.
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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