Purification of full-length recombinant human and rat type 1 11beta-hydroxysteroid dehydrogenases with retained oxidoreductase activities

11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1) is a membrane-bound glycoprotein localized in the endoplasmic reticulum. This Enzyme has a key role in regulating local tissue glucocorticoid concentration, acting in vivo predominantly as an oxidoreductase. Previous attempts to purify the native Enzyme have yielded a protein without reductase activity. To facilitate detailed studies on its structure and regulation, we have developed a method to purify the full-length human and rat 11beta-HSD1 with retention of their natural oxidoreductase activities. This procedure involved recombinant expression of these histidine-tagged Enzymes in the yeast Pichia pastoris; large-scale culturing in a fermentor; and single-step purification by metal affinity chromatography. Both Enzymes were 90-95% pure and exhibited dehydrogenase and reductase activities with K(M) values in agreement with those reported in the literature.