Basic Research
Copyright ©The Author(s) 2002. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Oct 15, 2002; 8(5): 888-892
Published online Oct 15, 2002. doi: 10.3748/wjg.v8.i5.888
Establishment of a transgenic cell line stably expressing human cytochrome P450 2C18 and identification of a CYP2C18 clone with exon 5 missing
Jian Zhu-Ge, Ying-Nian Yu, Yu-Li Qian, Xin Li
Jian Zhu-Ge, Ying-Nian Yu, Department of Pathophysiology and Laboratory of Medical Molecular Biology, Zhejiang University School of Medicine, Hangzhou 310031, Zhejiang Province, China
Yu-Li Qian, Present address: Center of laboratory, Women's hospital, School of Medicine, Zhejiang University, Hangzhou 310031, Zhejiang Province, China
Xin Li, Department of pharmaceutical analysis & drug metabolism, College of Pharmacology Science, Zhejiang University, Hangzhou 310031, Zhejiang Province, China
Author contributions: All authors contributed equally to the work.
Supported by National Natural Science Foundation of China, No.39770868 and Natural Science Foundation of Zhejiang Province, No.397490.
Correspondence to: Prof. Ying-Nian Yu, Department of Pathophysiology and Laboratory of Medical Molecular Biology, Zhejiang University School of Medicine, Hangzhou 310031, Zhejiang Province, China. ynyu@mail.hz.zj.cn
Telephone: +86-571-87217149 Fax: +86-571-87217149
Received: March 30, 2002
Revised: April 15, 2002
Accepted: April 20, 2002
Published online: October 15, 2002
Abstract

AIM: The human cytochrome P-450 2C18 (CYP2C18) has been characterized. However, the protein has not been purified from liver and very little is known regarding the specific substrate of CYP2C18. In order to study its enzymatic activity for drug metabolism, the CYP2C18 cDNA was cloned and a stable CHL cell line expressing recombinant CYP 2C18 was established.

METHODS: The human CYP2C18 cDNA was amplified with reverse transcription-polymerase chain reaction (RT-PCR) from total RNAs extracted from human liver and cloned into pGEM-T vector. The cDNA segment was identified by DNA sequencing and subcloned into a mammalian expression vector pREP9. A transgenic cell line was established by transfecting the recombinant plasmid of pREP9-CYP2C18 to Chinese hamster lung (CHL) cell. The enzyme activity of CYP2C18 catalyzing oxidation of tolbutamide to hydroxytolbutamide in postmitochondrial supernant (S9) fraction of the cell was determined by high performance liquid chromatography (HPLC).

RESULTS: The amino acid sequence predicted from the cloned cDNA segment was identical to that of reported by Romkes et al[3] (GenBank accession number: M61856, J05326). The S9 fraction of the established cell line metabolizes tolbutamide to hydroxytolbutamide. Tolbutamide hydroxylase activity was found to be 0.509 ± 0.052 μmol·min-1·g-1 S9 protein or 8.82 ± 0.90 mol·min-1·mol-1 CYP, but was undetectable in parental CHL cell. In addition, we have identified a CYP2C18 cDNA clone with exon 5 missing.

CONCLUSION: The cDNA of human CYP2C18 was successfully cloned and a cell line, CHL-CYP2C18, efficiently expressing the protein of CYP2C18, was established. A spliced variant of CYP2C18 with exon 5 missing was identified in the cloning process.

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