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Human 17 beta-hydroxysteroid dehydrogenase: overproduction using a baculovirus expression system and characterization
- Source :
- The Journal of steroid biochemistry and molecular biology. 50(5-6)
- Publication Year :
- 1994
-
Abstract
- 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.
- Subjects :
- endocrine system
animal structures
17-Hydroxysteroid Dehydrogenases
Endocrinology, Diabetes and Metabolism
Clinical Biochemistry
Sf9
Biology
Spodoptera
Biochemistry
law.invention
Endocrinology
Western blot
law
Complementary DNA
medicine
Animals
Humans
Hydroxysteroid dehydrogenase
Molecular Biology
chemistry.chemical_classification
Molecular mass
medicine.diagnostic_test
Fast protein liquid chromatography
Cell Biology
Molecular biology
Nucleopolyhedroviruses
Recombinant Proteins
Molecular Weight
Kinetics
Enzyme
chemistry
Recombinant DNA
Molecular Medicine
Subjects
Details
- ISSN :
- 09600760
- Volume :
- 50
- Issue :
- 5-6
- Database :
- OpenAIRE
- Journal :
- The Journal of steroid biochemistry and molecular biology
- Accession number :
- edsair.doi.dedup.....2380ac2d5f10c0c88fba3453ddb64f6e