Functional expression, characterization, and purification of the catalyticdomain of human 11-beta-hydroxysteroid dehydrogenase type 1

11-beta -hydroxysteroid dehydrogenase type 1 catalyzes the conversion of co rtisone to hormonally active cortisol and has been implicated in the pathog enesis of a number of disorders including insulin resistance and obesity. T he enzyme is a glycosylated membrane-bound protein that has proved difficul t to purify in an active state. Extracted enzyme typically loses the reduct ase properties seen in intact cells and shows principally dehydrogenase act ivity. The C-terminal catalytic domain is known to contain a disulfide bond and is located within the lumen of the endoplasmic reticulum, anchored to the membrane by a single N-terminal transmembrane domain. We report here th e functional expression of the catalytic domain of the human enzyme, withou t the transmembrane domain and the extreme N terminus, in Escherichia coli, Moderate levels of soluble active protein were obtained using an N-termina l fusion with thioredoxin and a 6xHis tag. In contrast, the inclusion of a 6xHis tag at the C terminus adversely affected protein solubility and activ ity. However, the highest levels of active protein were obtained using a co nstruct expressing the untagged catalytic domain. Nonreducing electrophores is revealed the presence of both monomeric and dimeric disulfide bonded for ms; however, mutation of a nonconserved cysteine residue resulted in a reco mbinant protein with no intermolecular disulfide bonds but full enzymatic a ctivity. Using the optimal combination of plasmid construct and E, coli hos t strain, the recombinant protein was purified to apparent homogeneity by s ingle step affinity chromatography, The purified protein possessed both deh ydrogenase and reductase activities with a K-m of 1.4 muM for cortisol and 9.5 muM for cortisone. This study indicates that glycosylation, the N-termi nal region including the transmembrane helix, and intermolecular disulfide bonds are not essential for enzyme activity and that expression in bacteria can provide active recombinant protein for future structural and functiona l studies.