Bacterial expression, purification, and characterization of rat hydroxysteroid sulfotransferase STa.
Hydroxysteroid (alcohol) sulfotransferase catalyzes numerous reactions that are important to our understanding of the metabolism of both endogenous steroids and exogenous alcohols. Here we report a method for prokaryotic expression and rapid purification of the recombinant hydroxysteroid sulfotransferase STa, a major isoform of hydroxysteroid sulfotransferase in the rat. The cDNA encoding STa was cloned into a pET-3c vector and expressed in Escherichia coli BL21 cells. After disruption of the cells by sonication, the enzyme was purified in one step by affinity chromatography on adenosine 3',5'-diphosphate-agarose. The purified recombinant STa had a relative molecular mass on SDS-PAGE that was identical with the native hepatic STa in rat liver. The expressed enzyme displayed similar substrate inhibition characteristics with dehydroepiandrosterone as have been noted previously with the native enzyme purified from rat liver. Furthermore, the catalytic efficiency in sulfation of 7-hydroxymethyl-12-methylbenz[a]anthracene, as well as the stereoselectivity of sulfation of the enantiomers of 1-phenyl-1-heptanol and 1-naphthyl-1-ethanol, catalyzed by the recombinant STa were consistent with characteristics of the STa isolated from rat liver.
Sheng
J J
JJ
Division of Medicinal and Natural Products Chemistry, College of Pharmacy, Iowa City, IA 52242, USA.
Duffel
M W
MW
D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D016133:Polymerase Chain Reaction; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D015238:Sulfotransferases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism
D018076:DNA, Complementary; D007527:Isoenzymes; D011994:Recombinant Proteins; D015238:Sulfotransferases; C028152:alcohol sulfotransferase