Expression of Recombinant 3-Beta Hydroxysteroid Dehydrogenase Protein in E. coli

Document Type: Research Paper

Authors

1 Division of Genetics, Department of Biology, Faculty of Science, Isfahan University. Isfahan, I.R. Iran

2 Molecular Medicine Department, King´s College School of Medicine and Dentistry, King´s College, London, UK.

Abstract

3-beta hydroxysteroid dehydrogenase (3BHSD) is secreted by the cortex of adrenal gland functioning in
stress conditions. The gene encoding the 3-beta hydroxysteroid dehydrogenase protein was PCRamplified from a λgt11 cDNA library using specific primers. The amplified PCR product was then cloned into pGEX-4T-1 expression vector under Ptac promoter and the expression of the enzyme was examined in E. coli (BL21). Upon optimization of the expression condition, the enzyme was produced as a glutathione S-transferase (GST) fusion protein, which was purified by affinity chromatography using glutathione sepharose column. The GST part was then removed by selective proteolytic digestion with thrombin. The purified recombinant enzyme could be used in construction of diagnostic kits for screening the patients with premature ovarian failure (POF) for the presence of autoantibodies against 3BHSD as an important molecular target.

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