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Human 17 beta-hydroxysteroid dehydrogenase: overproduction using a baculovirus expression system and characterization
1MRC Group in Molecular Endocrinology, CHUL Research Center, Quebec, Canada.
The Journal of Steroid Biochemistry and Molecular Biology
|September 1, 1994
Summary
Researchers produced active estrogenic 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) using a baculovirus system. This method yields large quantities of the enzyme, identical to the natural form, suitable for further physico-chemical studies.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Estrogenic 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) is crucial for estrogen synthesis.
- Studying the enzyme mechanism requires a reliable source of active 17 beta-HSD.
Purpose of the Study:
- To overproduce human placental estrogenic 17 beta-HSD using a baculovirus expression system.
- To obtain sufficient quantities of functional enzyme for physico-chemical studies.
Main Methods:
- Human 17 beta-HSD cDNA was inserted into the baculovirus genome.
- Expression was carried out in Spodoptera frugiperda (Sf9) insect cells.
- Recombinant protein was purified using affinity fast liquid protein chromatography.
Main Results:
- Active 17 beta-HSD was expressed in Sf9 cells, with maximal expression at 60 hours post-infection.
- Intracellular enzyme activity was approximately 70 times higher than in human placenta.
- Purified recombinant enzyme exhibited identical molecular mass and specific activity to the native enzyme, forming a dimer.
Conclusions:
- The baculovirus expression system successfully produced active, functional estrogenic 17 beta-HSD.
- The recombinant enzyme is suitable for physico-chemical studies due to its yield and identity to the native form.