Construction and evaluation of recombinant plasmids expressing CYP3A4 and GSTA1

Objective To create a recombinant plasmid that is capable of expressing the drug metabolism enzymes, cytochrome P450 family member 3A4 (CYP3A4) and glutathione S-transferase alpha 1, (GSTA1) in eukaryotic cells.Methods The CYP3A4 and GSTA1 genes were amplified by PCR.The fragments were cloned by means of digestion and ligation into the pBudCE4.1 expression vector, which supports simultaneous expression of two genes.The recombinant plasmid was designated as pBudCE4.1-CYP3A4-GSTA1 and confirmed by sequencing and basic local alignment search tool (BLAST) analysis.After transfection into the human hepatoma-derived HepG2 cell line, C3A, the activity of CYP3A4 and expression of GSTA1 were assessed by midazolam (MDZ) 1'-hydroxylation and 4-hydroxylation assay and immunohistochemistry, respectively.Results The recombinant pBudCE4.1-CYP3A4-GSTA1 plasmid was constructed successfully and expressed GSTA1 and CYP3A4 in mammalian cells in vitro.Conclusion The recombinant pBudCE4.1-CYP3A4-GSTA1 plasmid can increase expression of the drug metabolizing enzymes, CYP3A4 and GSTA1, in mammalian liver cells.Future studies may develop this tool into a novel therapy to improve metabolic activity and liver detoxification of drugs in humans.