DIRECT EXPRESSION OF PIG TESTICULAR 3-ALPHA BETA-(20-BETA)-HYDROXYSTEROID DEHYDROGENASE IN ESCHERICHIA-COLI/

The cDNA coding for pig testicular 3 alpha/beta (20 beta)-hydroxystero id dehydrogenase was expressed in Escherichia coli by placing it under the control of an isopropylthiogalactoside (IPTG) inducible tac promo ter. Production of 3 alpha/beta (20 beta)-HSD was demonstrated by West ern blotting and by catalytic activity with 5 alpha-dihydrotestosteron e as a substrate for 3 alpha/beta-HSD, and progesterone and 17 alpha-h ydroxyprogesterone as substrates for 20 beta-HSD in the presence of NA DPH. The 3 alpha/beta (20 beta)-HSD enzyme was detected in a soluble f raction of the lysate of E. coli added to IPTG to induce the synthesis of the protein. Its molecular weight was estimated to be 30.5 kDa by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Recombinant 3 alpha /beta (20 beta)-HSD was purified to apparent homogeneity as determined by SDS-PAGE by column chromatography using DEAE-cellulose. The purifi ed enzyme reduced not only steroids but also prostaglandins and other carbonyl compounds including aldehydes, ketones and quinones as demons trated in native enzymes purified from pig testes. The amino terminus of the purified enzyme was serine which was coded next to the ATG star t codon, and the sequence of the amino terminal 24 residues was identi cal with the coding amino acid in the cDNA; whereas, the amino terminu s of the native 3 alpha/beta (20 beta)-HSD was not detected suggesting that the N-terminal amino acid was blocked.