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.
Nobel
C Stefan I
CS
Biovitrum AB, Division of Pharmaceuticals, Department of Assay Development and Screening, S-112 87 Stockholm, Sweden.
Dunås
Finn
F
Abrahmsén
Lars B
LB
D043205:11-beta-Hydroxysteroid Dehydrogenase Type 1; D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D006913:Hydroxysteroid Dehydrogenases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017385:Sequence Homology; D013379:Substrate Specificity
D018076:DNA, Complementary; D011994:Recombinant Proteins; D010088:Oxidoreductases; D006913:Hydroxysteroid Dehydrogenases; D043205:11-beta-Hydroxysteroid Dehydrogenase Type 1; C468386:HSD11B1 protein, human