Establishment of a transgenic cell line stably expressing human cytochrome P450 2C18 and identification of a CYP2C18 clone with exon 5 missing

AIM: The human cytochrome P-450 2C18(CYP2C18) hasbeen characterized. However, the protein has not beenpurified from liver and very little is known regarding thespecific substrate of CYP2C18. In order to study its enzymaticactivity for drug metabolism, the CYP2C18cDNA was clonedand a stable CHL cell line expressing recombinant CYP 2C18was established.METHODS: The human CYP2C18cDNA was amplified withreverse transcription-polymerase chain reaction (RT-PCR)from total RNAs extracted from human liver and cloned intopGEM-T vector. The cDNA segment was identified by DNAsequencing and subcloned into a mammalian expressionvector pREP9. A transgenic cell line was established bytransfecting the recombinant plasmid of pREPg-CYP2C18toChinese hamster lung (CHL) cell. The enzyme activity ofCYP2C18 catalyzing oxidation of tolbutamide tohydroxytolbutamide in postmitochondrial supernant(Sg)fraction of the cell was determined by high performanceliquid chromatography(HPLC).RESULTS: The amino acid sequence predicted from thecloned cDNA segment was identical to that of reported byRomkes et al(GenBank accession number: M61856,J05326).The S9 fraction of the established cell line metabolizestolbutamide to hydroxytolbutamide. Tolbutamide hydroxylaseactivity was found to be 0.509±0.052 μmol.min-1.g-1 S9protein or 8.82±0.90 mol.min-1.mol-1 CYP, but wasundetectable in parental CHL cell. In addition, we haveidentified a CYP2C18cDNA clone with exon 5 missing.CONCLUSION: The cDNA of human CYP2C18 wassuccessfully cloned and a cell line, CHL-CYP2C18, efficientlyexpressing the protein of CYP2C18, was established. Aspliced variant of CYP2C18 with exon 5 missing was identifiedin the cloning process.

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 CYP2C18cDNA 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-CYP2C18to 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 etal(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 CYP2C18cDNA 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.