BACULOVIRUS-DIRECTED EXPRESSION OF HUMAN PROSTATIC STEROID 5-ALPHA-REDUCTASE-1 IN AN ACTIVE FORM

In the human prostate, the enzyme steroid 5alpha-reductase (h5alphaR) catalyses the conversion of testosterone into the more potent androgen, dihydrotestosterone. Two distinct cDNAs coding for h5alphaR in the human prostate have been previously characterized. Enzyme h5alphaR1 shows a maximum activity at basic pH whereas h5alphaR2 has an acidic pH optimum activity. We report here the expression of the human steroid h5alphaR1 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 h5alphaR activity assays by high pressure liquid chromatography showed that a catalytically active enzyme was produced. The recombinant enzyme showed an apparent K(m) for testosterone of 2.07 muM and a V(max) of 10.1 nmol of dihydrotestosterone/min/mg of protein. Recombinant h5alphaR1 activity was inhibited by specific h5alphaR inhibitors such as 4-MA (K(i) = 2.6 nM). Subcellular distribution in Sf9 cells demonstrated that the enzyme was associated with the nuclear membrane.