title: Human 11beta-hydroxysteroid dehydrogenase 1/carbonyl reductase: recombinant expression in the yeast Pichia pastoris and Escherichia coli.
abstract: 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.
authors.affiliation: Department of Pharmacology and Toxicology, School of Medicine, Philipps-University of Marburg, Karl-von-Frisch-Strasse 1, D-35033, Marburg, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Blum
authors.forename: H J
authors.initials: HJ
authors.lastname: Martin
authors.forename: E
authors.initials: E
authors.lastname: Maser
mesh_terms: 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*
chemical_list: 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