Human 11β-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 11 beta-hydroxysteroid-dehydrogenase type 1 (11 beta-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. 11 beta-HSD 1 belongs to the protein superfamily of the short-chain dehydrogenases/reductases (SDR), and has been structurally and functionally characterized. 11 beta-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 11 beta-HSD 1 in detoxification processes, we have successfully performed the overexpression of recombinant human 11 beta-HSD 1 in the yeast Pichia pastoris and in Escherichia coli. Recombinant 11 beta-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, 11 beta-HSD 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 11 beta-HSD 1 forms and with the native enzyme from mouse and human liver. In conclusion, recombinant 11 beta-HSD 1 from P. pastoris serves as a valuable tool for future studies on carbonyl compound detoxification. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.