Baculovirus-directed expression of human prostatic steroid 5α-reductase 1 in an active form

• Published in: The Journal of steroid biochemistry and molecular biology Vol. 46; no. 2; pp. 177 - 182
• Main Authors: Iehlé, Catherine, Délos, Sylvie, Filhol, Odile, Martin, Pierre-Marie
• Format: Journal Article
• Language: English
• Published: Oxford Elsevier Ltd 01.08.1993; Elsevier Science
• Subjects: 3-Oxo-5-alpha-Steroid 4-Dehydrogenase - genetics; 3-Oxo-5-alpha-Steroid 4-Dehydrogenase - metabolism; Animals; Autographa californica; baculovirus; Biological and medical sciences; Cell Line; Cell Nucleus - enzymology; Cloning, Molecular; Fundamental and applied biological sciences. Psychology; Gene expression; Humans; Male; Microsomes - enzymology; Molecular and cellular biology; Molecular genetics; Nucleopolyhedrovirus - genetics; Prostate - enzymology; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Spodoptera; Spodoptera frugiperda; AcNPV; DHT; T; 4-MA; DNP; ATP; h5αR; Virus; Human; Baculoviridae; Enzyme; Cholestenone 5α-reductase; Baculovirus; Gene expression; Urogenital system; Prostate
• ISSN: 0960-0760; 1879-1220
• DOI: 10.1016/0960-0760(93)90292-5

In the human prostate, the enzyme steroid 5α-reductase (h5αR) catalyses the conversion of testosterone into the more potent androgen, dihydrotestosterone. Two distinct cDNAs coding for h5αR in the human prostate have been previously characterized. Enzyme h5αR1 shows a maximum activity at basic pH whereas h5αR2 has an acidic pH optimum activity. We report here the expression of the human steroid h5αR1 in a eukaryotic expression system : the baculovirus-directed-insect cell expression system. The full length cDNA was inserted into the Autographa californica nuclear polyhedrosis virus genome and expressed in Spodoptera frugiperda, Sf9, insect cells. Sf9 cells were infected with the recombinant baculovirus and homogenates used in h5αR activity assays by high pressure liquid chromatography showed that a catalytically active enzyme was produced. The recombinant enzyme showed an apparent Km for testosterone of 2.07 μM and a Vmax of 10.1 nmol of dihydrotestosterone/min/mg of protein. Recombinant h5αR1 activity was inhibited by specific h5αR inhibitors such as 4-MA (Ki = 2.6 nM). Subcellular distribution in Sf9 cells demonstrated that the enzyme was associated with the nuclear membrane.