Korean J Anat.  1998 Oct;31(5):771-778.

Molecular cloning and expression of 3beta-hydroxy steroid dehydrogenase in E-Coli

• Oh JM

Abstract

This study was carried out to obtain the 3beta-hydroxy-5-ene steroid dehydrogenase (3beta-HSD) which is contained in human trophoblasts. To produce 3beta-HSD cDNA, reverse transcription-polymerase chain reaction (RT-PCR) with the total RNA of human placenta was performed and RT-PCR products was detected at 1.2 Kb. After cloning 3beta-HSD cDNA in pGEX 4T-2 as expression vector, the recombinant DNA was transformed to E-coli and glutathione-s-transferase (GST) fusion protein was inducted with isoprophyl thiogalactoside (IPTG). Inducted GST fusion protein was overexpressed in cytoplasm of E-coli. The obtained GST fusion protein was characterized with antibody against 3beta-HSD by Western blot analysis and purified by affinity chromatography using glutathione sepharose 4B column. And then the GST fusion protein was cleaved by thrombin and The cleaved protein, 3beta-HSD, was detected at 43 KDa by SDS-PAGE.

Keyword

3beta-HSD; Trophoblast; GST fusion protein; Antibody; Cloning

MeSH Terms

Blotting, Western
Chromatography, Affinity
Clone Cells
Cloning, Molecular*
Cloning, Organism
Cytoplasm
DNA, Complementary
DNA, Recombinant
Electrophoresis, Polyacrylamide Gel
Glutathione
Humans
Oxidoreductases*
Placenta
RNA
Sepharose
Thrombin
Trophoblasts
DNA, Complementary
DNA, Recombinant
Glutathione
Oxidoreductases
RNA
Sepharose
Thrombin