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

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 CYP2C18 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 (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.

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.

Figure 1

Scheme of recombinants of pGEM-CYP2C18 and pREP9-CYP2C18

Figure 2

Electrophoresis identification of recombinants of pGEM-CYP2C18 and pREP9-CYP2C18. Lane 1: Marker (λ/EcoR I and Hind III); 2: pGEM-T vector; 3: Recombinant of pGEM-CYP2C18 digested by Nhe I and Xho I (incompleted digestion); 4: PCR products of CYP2C18 (1.67 kb); 5: Recombinant of pREP9-CYP2C18 digested by Nhe I and Xho I; 6: pREP9 vector.

Figure 3

Electrophoresis and sequencing identification of a CYP2C18 clone with exon 5 missing. A: Electrophoresis identification of a CYP2C18 clone with an exon 5 being skipped; Lane 1: Marker (λ/EcoR I and Hind III); 2: Recombinant of pGEM-CYP2C18 digested by Nhe I and Xho I (incompleted digestion); B: Partial sequencing of an CYP2C18 cDNA clone with an exon 5 being skipped. The upper sequence represent the sense strand and the underside sequence represent the anti-sense strand being sequenced.

Figure 4

Representative chromatogram of extracts. A Shim-pack CLC-ODS column (15 × 0.6 cm i.d.) was used. The mobile phase was constituted with 0.05% phosphoric acid (pH2.6), acetonitrile (6:4/V:V) with the flow rate of 1 mL. min^-1. Hydroxytolbutamide was monitored at 230 nm. A: hydroxytolbutamide; B: tolbutamide