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)-hydroxysteroid dehydrogenase was expressed in Escherichia coli by placing it under the control of an isopropylthiogalactoside (IPTG) inducible tac promoter. Production of 3 alpha/beta (20 beta)-HSD was demonstrated by Western blotting and by catalytic activity with 5 alpha-dihydrotestosterone as a substrate for 3 alpha/beta-HSD, and progesterone and 17 alpha-hydroxyprogesterone as substrates for 20 beta-HSD in the presence of NADPH. The 3 alpha/beta (20 beta)-HSD enzyme was detected in a soluble fraction 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 purified enzyme reduced not only steroids but also prostaglandins and other carbonyl compounds including aldehydes, ketones and quinones as demonstrated in native enzymes purified from pig testes. The amino terminus of the purified enzyme was serine which was coded next to the ATG start codon, and the sequence of the amino terminal 24 residues was identical with the coding amino acid in the cDNA; whereas, the amino terminus of the native 3 alpha/beta (20 beta)-HSD was not detected suggesting that the N-terminal amino acid was blocked.
Nakajin
S
S
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Hoshi University, Tokyo, Japan.
Nakajima
T
T
Uchida
M
M
Ohno
S
S
Shinoda
M
M
D015096:3-Hydroxysteroid Dehydrogenases / Q000096:biosynthesis* / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002845:Chromatography; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D008297:Male; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013552:Swine; D013737:Testis / Q000201:enzymology
D011994:Recombinant Proteins; D015096:3-Hydroxysteroid Dehydrogenases