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 st eroids and exogenous alcohols. Here we report a method for prokaryotic expr ession and rapid purification of the recombinant hydroxysteroid sulfotransf erase STa, a major isoform of hydroxysteroid sulfotransferase in the rat. T he cDNA encoding STa was cloned into a pET-3c vector and expressed in Esche richia coli BL21 cells. After disruption of the cells by sonication, the en zyme 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 liv er. The expressed enzyme displayed similar substrate inhibition characteris tics with dehydroepiandrosterone as have been noted previously with the nat ive enzyme purified from rat liver. Furthermore, the catalytic efficiency i n sulfation of 7-hydroxymethyl-12-methylbenz[a]anthracene, as well as the s tereoselectivity of sulfation of the enantiomers of 1-phenyl-1-heptanol and 1-naphthyl-1-ethanol, catalyzed by the recombinant STa were consistent wit h characteristics of the STa isolated from rat liver. (C) 2001 Academic Pre ss.