ブタ精巣3α/β(20β)-ヒドロキシステロイドデヒドロゲナーゼcDNAのバキュロウイスル発現系を利用したタンパク質直接発現

概要

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The direct expression of the pig testicular 3α/β (20β)-hydroxysteroid dehydrogenase (HSD) cDNA using a baculovirus expression system was investigated. A minimum essential cDNA containing the coding region of 3α/β (20β)-HSD was amplified by polymerase chain reaction (PCR) to extend the DNA linker including the Bam HI restriction site in the both ends of the cDNA. As the template, 3α/β (20β)-HSD cDNA, pBS 52,which was subcloned to Bluescript II was used. The PCR fragment was ligated to Bam HI-cut transfer plasmid (pBlueBac III). Recombinant transfer plasmid (pBlueBac-20β) constructed was cotransfected into Spodoptera frugiperda Sf-9 cells with the baculovirus. After transfection, the recombinant virus was detected by the plaque assay with color selection. The expression of 3α/β (20β)-HSD cDNA was detected by Western blotting and enzyme assay. The expressed protein showed the same molecular weight and immunochemical cross-reaction to the native enzyme. Furthermore, it had 3-keto reductase activity of 3α/β (20β)-HSD for 5α-dihydrotestosterone and 20-keto reductase activity for 17α-hydroxyprogesterone in the presence of NADPH.
社団法人日本薬学会の論文
1995-04-25