OVER-EXPRESSION OF HUMAN TYPE-I (PLACENTAL) 3-BETA-HYDROXY-5-ENE-STEROID DEHYDROGENASE ISOMERASE IN INSECT CELLS INFECTED WITH RECOMBINANT BACULOVIRUS

Human type I placental 3 beta-hydroxy-5-ene-steroid dehydrogenase/steroid 5 --> 4-ene-isomerase (3 beta-HSD/isomerase) synthesizes androstenedione from fetal dehydroepiandrosterone and progesterone from pregnenolone. The full length cDNA that encodes type I 3 beta-HSD/isomerase was inserted into the baculovirus, Autographa californica multiple nucleocapsid polyhedrosis virus, and expressed in Spodoptera fungiperda (Sf-9) insect cells. Western blots showed that the baculovirus-infected Sf-9 cells produced an immunoreactive protein that co-migrated with purified placental 3 beta-HSD/isomerase. Ultracentrifugation localized the expressed enzyme activities in all the membrane-associated organelles of the Sf-9 cell (nuclear, mitochondrial and microsomal). Kinetic studies showed that the expressed enzyme has 3 beta-HSD and isomerase activities. The Michaelis-Menton constant is very similar for the 3 beta-HSD substrate, 5 alpha-androstan-3 beta-ol-17-one, in the Sf-9 cell homogenate (K-m = 17.9 mu M) and placental microsomes (K-m = 16.7 mu M). The 3 beta-HSD activity (V-max = 14.5 nmol/min/mg) is 1.6-fold higher in the Sf-9 cell homogenate compared to placental microsomes (V-max = 9.1 nmol/min/mg). The K-m values are almost identical for the isomerase substrate, 5-androstene-3,17-dione, in the Sf-9 cell homogenate (K-m = 14.7 mu M) and placental microsomes (K-m = 14.4 mu M). The specific isomerase activity is 1.5-fold higher in the Sf-9 cells (V-max = 25.7 nmol/min/mg) relative to placenta (V-max = 17.2 nmol/min/mg). These studies show that our recombinant baculovirus system over-expresses fully active enzyme that is kinetically identical to native 3 beta-HSD/isomerase in human placenta.