Purification of full-length recombinant human and rat type 1 11β-hydroxysteroid dehydrogenases with retained oxidoreductase activities |
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Authors: | C. Stefan I. Nobel, Finn Dun s,Lars. B. Abrahms n |
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Affiliation: | C. Stefan I. Nobel, Finn Dunås,Lars. B. Abrahmsén, |
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Abstract: | 11β-Hydroxysteroid dehydrogenase type 1 (11β-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 11β-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 KM values in agreement with those reported in the literature. |
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