Cloning of cytochrome P-450 2C9 cDNA from human liver and its expression in CHL cells.
OBJECTIVE
Using bacterial, yeast, or mammalian cell expressing a human drug metabolism enzyme would seem good way to study drug metabolism-related problems. Human cytochrome P-450 2C9(CYP2C9) is a polymorphic enzyme responsible for the metabolism of a large number of clinically important drugs. It ranks among the most important drug metabolizing enzymes in humans. In order to provide a sufficient amount of the enzyme for drug metabolic research, the CYP2C9 cDNA was cloned and expressed stably in CHL cells.


METHODS
After extraction of total RNA from human liver tissue, the human CYP2C9 cDNA was amplified with reverse transcription-polymerase chain reaction (RT-PCR), and cloned into cloning vector pGEM-T. The cDNA fragment was identified by DNA sequencing and subcloned into a mammalian expression vector pREP9. A transgenic cell line was established by transfecting the recombinant vector of pREP9-CYP2C9 into CHL cells. The enzyme activity of CYP2C9 catalyzing oxidation of tolbutamide to hydroxy tolbutamide in S9 fraction of the cell was determined by high performance liquid chromatography(HPLC).


RESULTS
The amino acid sequence predicted from the cDNA segment was identical to that of CYP2C9*1, the wild type CYP2C9. However, there were two base differences, i.e. 21T>C, 1146C>T, but the encoding amino acid sequence was the same, L7, P382. The S9 fraction of the established cell line metabolizes tolbutamide to hydroxy tolbutamide; tolbutamide hydroxylase activity was found to be 0.465 +/- 0.109 micromol.min(-1).g(-1) S9 protein or 8.62 +/- 2.02mol.min(-1).mol(-1) CYP, but was undetectable in parental CHL cell.


CONCLUSIONS
The cDNA of human CYP2C9 was successfully cloned and a cell line of CHL- CYP2C9, efficiently expressing the protein of CYP2C9, was established.
Zhu
Ge-Jian
GJ
Department of Pathophysiology and Laboratory of Medical Molecular Biology, Zhejiang University School of Medicine, Hangzhou 310031, Zhejiang Province, China.
Yu
Ying-Nian
YN
Li
Xin
X
Qian
Yu-Li
YL
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics* / Q000378:metabolism*; D002458:Cell Fractionation; D002460:Cell Line; D002681:China; D003001:Cloning, Molecular; D065729:Cytochrome P-450 CYP2C9; D015870:Gene Expression; D006801:Humans; D007004:Hypoglycemic Agents / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D020033:Protein Isoforms; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014044:Tolbutamide / Q000378:metabolism
D007004:Hypoglycemic Agents; D020033:Protein Isoforms; D011994:Recombinant Proteins; D014044:Tolbutamide; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases