CYP2D6*1, CYP2D6*10 co-expressed with CYPOR in Bac-to-Bac expression system and activity determination.
- Author:
Ming-rong QIAN
1
;
Jing CHEN
;
Yao LIU
;
Lu-shan YU
;
Shu-qing CHEN
;
Su ZENG
Author Information
1. College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Baculoviridae;
enzymology;
genetics;
Catalysis;
Cells, Cultured;
Chromatography, High Pressure Liquid;
methods;
Cytochrome P-450 CYP2D6;
genetics;
metabolism;
Dextromethorphan;
metabolism;
Isoenzymes;
metabolism;
NADPH-Ferrihemoprotein Reductase;
genetics;
metabolism;
Plasmids;
Recombinant Proteins;
genetics;
metabolism;
Spectrometry, Mass, Electrospray Ionization;
Spodoptera;
cytology;
virology;
Transfection
- From:
Acta Pharmaceutica Sinica
2011;46(2):207-212
- CountryChina
- Language:Chinese
-
Abstract:
CYP2D6 is an important drug-metabolizing enzyme. The polymorphism of CYP2D6 leads to metabolism difference and the different reactions of drugs in the individuals and different races are normal phenomenon in clinical medication. CYP2D6*10 is an important subtype in Asian people and 51.3% Chinese are classified with this subtype. To obtain recombinant active CYP2D6*1/CYP2D6*10 in baculovirus system by optimizing coexpression with CYPOR, and detect their activity to catalyze dextromethorphan, three recombinants pFastBac-CYP2D6*1, pFastBac-CYP2D6*10 and pFastBac-CYPOR were constructed and transformed into DH10Bac cell to obtain the recombinant Bacmid-CYPOR, Bacmid-CYP2D6*1 and Bacmid-CYP2D6*10. And then the recombinant CYP2D6*1 and CYP2D6*10 virus were obtained by transfecting Sf9. Then homogenate protein activity was determined with dextromethorphan as substrate. The multiple of infection (MOI) and its ratio of recombinant CYP2D6 virus to CYPOR virus were adjusted by detecting the activity of the homogenate protein. The Km and Vmax are 26.67 +/- 2.71 micromol x L(-1) (n=3) and 666.7 +/- 56.78 pmol x nmol(-1) (CYP2D6) x min(-1) (n=3) for CYP2D6*1 to catalyze dextromethaphan. The Km and Vmax are 111.36 +/- 10.89 micromol x L(-1) (n=3) and 222.2 +/- 20.12 pmol x nmol(-1) (CYP2D6) x min(-1) (n=3) for CYP2D6*10 to catalyze dextromethorphan. There is significant difference between CYP2D6*1 and CYP2D6*10 for Vmax and Km (P < 0.01). The clearance ratio of CYP2D6*1 is 25.0 and the clearance ratio of CYP2D6*10 is 2.0. The expressed CYP2D6*1 and CYP2D6*10 are useful tools to screen the metabolism profile of many xenobiotics and endobiotics in vitro, which are benefit to understand individual metabolism difference.
