[Direct expression of the pig testicular 3 alpha/beta(20 beta)- hydroxysteroid dehydrogenase cDNA using baculovirus expression system].
The direct expression of the pig testicular 3 alpha/beta(20 beta)- hydroxysteroid dehydrogenase (HSD) cDNA using a baculovirus expression system was investigated. A minimum essential cDNA containing the coding region of 3 alpha/beta(20 beta)-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, 3 alpha/beta(20 beta)-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-20 beta) 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 3 alpha/beta(20 beta)-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 3 alpha/beta(20 beta)-HSD for 5 alpha-dihydrotestosterone and 20-keto reductase activity for 17 alpha-hydroxyprogesterone in the presence of NADPH.
Nakajin
S
S
Faculty of Pharmaceutical Sciences, Hoshi University, Tokyo, Japan.
Nakajima
T
T
Uchida
M
M
Shinoda
M
M
D015096:3-Hydroxysteroid Dehydrogenases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D016367:Baculoviridae; D001483:Base Sequence; D003349:Cortisone Reductase / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary / Q000032:analysis*; D005822:Genetic Vectors; D008297:Male; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D018411:Spodoptera; D013552:Swine; D013737:Testis / Q000201:enzymology*
D018076:DNA, Complementary; D015096:3-Hydroxysteroid Dehydrogenases; D003349:Cortisone Reductase