Human 17 beta-hydroxysteroid dehydrogenase: overproduction using a baculovirus expression system and characterization.
Estrogenic 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) plays a pivotal role in the synthesis of estrogens. We overproduced human placental estrogenic 17 beta-HSD using a baculovirus expression system for the study of the enzyme mechanism. A cDNA encoding the entire open reading frame of human 17 beta-HSD was inserted into the genome of Autographa californica nuclear polyhedrosis virus and expressed in Spodoptera frugiperda (Sf9) insect cells. Metabolic labeling and Western blot analysis using polyclonal antibodies raised against native human 17 beta-HSD indicated that a molecule with an apparent mass of 35 kDa was maximally expressed 60 h after infection. At that time interval, intracellular 17 beta-HSD activity reached 0.26 U/mg of protein in crude homogenate, about 70 times the level measured in human placenta. Purification of recombinant 17 beta-HSD was achieved by a single affinity fast liquid protein chromatography step yielding 24 mg of purified 17 beta-HSD protein per liter of suspension culture, with a specific activity of about 8 mumol/min/mg of protein for conversion of estradiol into estrone, at pH 9.2. In addition, the recombinant protein purified from infected Sf9 cells was assembled as a dimer with molecular mass and specific activity identical to those of the enzyme purified directly from placenta. The present data show that the baculovirus expression system can provide active 17 beta-HSD that is functionally identical to its natural counter-part and easy to purify in quantities suitable for its physico-chemical studies.
Breton
R
R
MRC Group in Molecular Endocrinology, CHUL Research Center, Quebec, Canada.
Yang
F
F
Jin
J Z
JZ
Li
B
B
Labrie
F
F
Lin
S X
SX
D015067:17-Hydroxysteroid Dehydrogenases / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D017924:Nucleopolyhedroviruses; D011994:Recombinant Proteins; D018411:Spodoptera
D011994:Recombinant Proteins; D015067:17-Hydroxysteroid Dehydrogenases