Human 11beta-hydroxysteroid dehydrogenase 1/carbonyl reductase: recombinant expression in the yeast Pichia pastoris and Escherichia coli.
Detoxification of aldehydes and ketones generally proceeds via reduction to their corresponding alcohols, which are then conjugated and eliminated. We focused our interest on 11beta-hydroxysteroid-dehydrogenase type 1 (11beta-HSD 1), a pluripotent enzyme which physiologically performs the interconversion of active and inactive glucocorticoid hormones, and which also participates in xenobiotic carbonyl compound detoxification. 11beta-HSD 1 belongs to the protein superfamily of the short-chain dehydrogenases/reductases (SDR), and has been structurally and functionally characterized. 11beta-HSD 1 is a glycosylated membrane protein which is very difficult to purify in an active state. In addition, expression levels in humans differ in a wide range. In order to facilitate biochemical and molecular studies on the significance of human 11beta-HSD 1 in detoxification processes, we have successfully performed the overexpression of recombinant human 11beta-HSD 1 in the yeast Pichia pastoris and in Escherichia coli. Recombinant 11beta-HSD 1 from E. coli was purified to homogeneity and used to generate a polyclonal antibody. The enzyme had no enzymatic activity, possibly due to the lack of glycosylation and/or incorrect folding in E. coli. In contrast, 11beta-HSD 1 overexpressed in P. pastoris was enzymatically active towards its physiological glucocorticoid substrates as well as towards xenobiotic carbonyl compounds. In western blot experiments the antibody crossreacted with both recombinant 11beta-HSD 1 forms and with the native enzyme from mouse and human liver. In conclusion, recombinant 11beta-HSD 1 from P. pastoris serves as a valuable tool for future studies on carbonyl compound detoxification.
Blum
A
A
Department of Pharmacology and Toxicology, School of Medicine, Philipps-University of Marburg, Karl-von-Frisch-Strasse 1, D-35033, Marburg, Germany.
Martin
H J
HJ
Maser
E
E
D043205:11-beta-Hydroxysteroid Dehydrogenase Type 1; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology*; D005656:Fungal Proteins / Q000096:biosynthesis; D006801:Humans; D006913:Hydroxysteroid Dehydrogenases / Q000096:biosynthesis* / Q000235:genetics; D007114:Immunization; D008861:Microsomes / Q000201:enzymology; D011994:Recombinant Proteins / Q000096:biosynthesis; D015262:Xenobiotics / Q000378:metabolism; D015003:Yeasts / Q000201:enzymology*
D018076:DNA, Complementary; D005656:Fungal Proteins; D011994:Recombinant Proteins; D015262:Xenobiotics; D006913:Hydroxysteroid Dehydrogenases; D043205:11-beta-Hydroxysteroid Dehydrogenase Type 1; C468386:HSD11B1 protein, human