Zhu-Ge Jian, Yu Ying-Nian, Qian Yu-Li, Li Xin
Department of Pathophysiology and Laboratory of Medical Molecular Biology, Zhejiang University School of Medicine, Hangzhou 310031, Zhejiang Province, China.
World J Gastroenterol. 2002 Oct;8(5):888-92. doi: 10.3748/wjg.v8.i5.888.
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 CYP2C18 was established.
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).
The amino acid sequence predicted from the cloned cDNA segment was identical to that of reported by Romkes et al (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 micromol x min(-1) x g(-1) S9 protein or 8.82+/-0.90 mol x min(-1) x mol(-1) CYP, but was undetectable in parental CHL cell. In addition, we have identified a CYP2C18 cDNA clone with exon 5 missing.
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.
人类细胞色素P-450 2C18（CYP2C18）已被鉴定。然而，该蛋白尚未从肝脏中纯化出来，且关于CYP2C18的特异性底物知之甚少。为了研究其在药物代谢中的酶活性，克隆了CYP2C18 cDNA，并建立了表达重组CYP2C18的稳定CHL细胞系。
用人肝脏提取的总RNA通过逆转录-聚合酶链反应（RT-PCR）扩增人CYP2C18 cDNA，并克隆到pGEM-T载体中。通过DNA测序鉴定cDNA片段，并亚克隆到哺乳动物表达载体pREP9中。通过将pREP9-CYP2C18重组质粒转染到中国仓鼠肺（CHL）细胞中建立转基因细胞系。通过高效液相色谱（HPLC）测定细胞线粒体后上清液（S9）组分中CYP2C18催化甲苯磺丁脲氧化为羟基甲苯磺丁脲的酶活性。
从克隆的cDNA片段预测的氨基酸序列与Romkes等人报道的序列相同（GenBank登录号：M61856，J05326）。建立的细胞系的S9组分将甲苯磺丁脲代谢为羟基甲苯磺丁脲。发现甲苯磺丁脲羟化酶活性为0.509±0.052微摩尔·分钟-1·克-1 S9蛋白或8.82±0.90摩尔·分钟-1·摩尔-1 CYP，但在亲代CHL细胞中未检测到。此外，我们鉴定出一个缺失外显子5的CYP2C18 cDNA克隆。
成功克隆了人CYP2C18 cDNA，并建立了高效表达CYP2C18蛋白的细胞系CHL-CYP2C18。在克隆过程中鉴定出一种缺失外显子5的CYP2C18剪接变体。
