Molecular Cloning and Expression of Human Placental 3β-Hydroxysteroid Dehydrogenase

The present studies were performed to obtain 3β-hydroxysteroid dehydrogenase (3β-HSD) protein in human placenta. For cloning of 3β-HSD cDNA, reverse transcription-polymerase chain reaction (RT-RCR) with the total RNA of human placenta was performed and the product was confirmed by sequence analysis and enzyme digestion with Pvu I and Hin III. After cloning 3β-HSD cDNA to pGEX 4T-2 as expression vector, the recombinant DNA was transformed to E. coli (Bl 21) and glutathion-s-transferase (GST) fusion protein was inducted with isoprophyl thiogalactoside. Induced GST fusion protein was detected at 67 KDa by sodium dodesyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting with polyclonal antibody for 3β-HSD. Form these results, overexpressed 3β-HSD in GST fusion protein was identified as human 3β-HSD.