title: Over-expression of human type I (placental) 3 beta-hydroxy-5-ene-steroid dehydrogenase/isomerase in insect cells infected with recombinant baculovirus.
abstract: 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 (Km = 17.9 microM) and placental microsomes (Km = 16.7 microM). The 3 beta-HSD activity (Vmax = 14.5 nmol/min/mg) is 1.6-fold higher in the Sf-9 cell homogenate compared to placental microsomes (Vmax = 9.1 nmol/min/mg). The Km values are almost identical for the isomerase substrate, 5-androstene-3,17-dione, in the Sf-9 cell homogenate (Km = 14.7 microM) and placental microsomes (Km = 14.4 microM). The specific isomerase activity is 1.5-fold higher in the Sf-9 cells (Vmax = 25.7 nmol/min/mg) relative to placenta (Vmax = 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.
authors.affiliation: Department of Obstetrics and Gynecology, Washington University School of Medicine, St Louis, Missouri 63110.
authors.forename: W E
authors.initials: WE
authors.lastname: Nash
authors.forename: R W
authors.initials: RW
authors.lastname: Mercer
authors.forename: G
authors.initials: G
authors.lastname: Blanco
authors.forename: R C
authors.initials: RC
authors.lastname: Strickler
authors.forename: J I
authors.initials: JI
authors.lastname: Mason
authors.forename: J L
authors.initials: JL
authors.lastname: Thomas
mesh_terms: D000818:Animals; D016367:Baculoviridae; D002451:Cell Compartmentation; D002460:Cell Line; D003001:Cloning, Molecular; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000737:chemistry*; D011375:Progesterone Reductase / Q000737:chemistry*; D011994:Recombinant Proteins; D018411:Spodoptera; D013251:Steroid Isomerases / Q000737:chemistry*
chemical_list: C021425:3 beta-hydroxysteroid oxidoreductase-delta(5) 3-ketosteroid isomerase; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D011375:Progesterone Reductase; D013251:Steroid Isomerases