Use of a Baculovirus-Mammalian Cell Expression-System for Expression of Drug-Metabolizing Enzymes: Optimization of Infection With a Focus on Cytochrome P450 3A4

DOI 機関リポジトリ (HANDLE) Web Site PubMed 参考文献49件 オープンアクセス
  • 宮内, 優
    崇城大学薬学部衛生化学研究室 九州大学大学院薬学府細胞生物薬学分野
  • 木村, 茜
    九州大学大学院薬学府細胞生物薬学分野
  • Sawai, Madoka
    崇城大学薬学部衛生化学研究室
  • 藤本, 景子
    九州大学大学院薬学研究院細胞生物薬学分野
  • 廣田, 有子
    九州大学大学院薬学研究院細胞生物薬学分野
  • 田中, 嘉孝
    九州大学大学院薬学研究院細胞生物薬学分野
  • 武知, 進士
    崇城大学薬学部衛生化学研究室
  • Mackenzie, Peter I.
    Clinical Pharmacology, College of Medicine and Public Health, Flinders Medical Centre and Flinders University
  • 石井, 祐次
    九州大学大学院薬学研究院細胞生物薬学分野

説明

Heterologous expression systems are important for analyzing the effects of genetic factors including single nucleotide polymorphisms on the functions of drug-metabolizing enzymes. In this study, we focused on a baculovirus-mammalian cell (Bac-Mam) expression system as a safer and more efficient approach for this purpose. The baculovirus-insect cell expression system is widely utilized in large-scale protein expression. Baculovirus has been shown to also infect certain mammalian cells, although the virus only replicates in insect cells. With this knowledge, baculovirus is now being applied in a mammalian expression system called the Bac-Mam system wherein a gene-modified baculovirus is used whose promotor is replaced with one that can function in mammalian cells. We subcloned open-reading frames of cytochrome P450 3A4 (CYP3A4), UDP-glucuronosyltransferase (UGT) 1A1, and UGT2B7 into a transfer plasmid for the Bac-Mam system, and prepared recombinant Bac-Mam virus. The obtained virus was amplified in insect Sf9 cells and used to infect mammalian COS-1 cells. Expression of CYP3A4, UGT1A1, and UGT2B7 in COS-1 cell homogenates were confirmed by immunoblotting. Optimum infection conditions including the amount of Bac-Mam virus, culture days before collection, and concentration of sodium butyrate, an enhancer of viral-transduction were determined by monitoring CYP3A4 expression. Expressed CYP3A4 showed appropriate activity without supplying hemin/5-aminolevulinic acid or co-expressing with NADPH-cytochrome P450 reductase. Further, we compared gene transfer efficiency between the Bac-Mam system and an established method using recombinant plasmid and transfection reagent. Our results indicate that the Bac-Mam system can be applied to introduce drug-metabolizing enzyme genes into mammalian cells that are widely used in drug metabolism research. The expressed enzymes are expected to undergo appropriate post-translational modification as they are in mammalian bodies. The Bac-Mam system may thus accelerate pharmacogenetics and pharmacogenomics research.

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