Cloning of cytochrome P-450 2C9 cDNA from human liver and its expression in CHL cells

World Journal of Gastroenterology : WJG
Ge-Jian ZhuYu-Li Qian

Abstract

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. 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). 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, ...Continue Reading

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Citations

Jan 13, 2004·World Journal of Gastroenterology : WJG·Jian ZhugeXiao-Dan Wu
Jul 27, 2006·Expert Opinion on Drug Metabolism & Toxicology·José V CastellMaría José Gómez-Lechón
Oct 6, 2006·Applied Microbiology and Biotechnology·Ri-He PengQuan-Hong Yao
Jan 13, 2005·Canadian Journal of Physiology and Pharmacology·Arwen L HunterDavid J Granville
Jun 12, 2003·World Journal of Gastroenterology : WJG·Si-Yuan WenSheng-Qi Wang
Dec 12, 2003·World Journal of Gastroenterology : WJG·Jian ZhugeYing-Nian Yu

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