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Originally published In Press as doi:10.1074/jbc.M011142200 on April 6, 2001

J. Biol. Chem., Vol. 276, Issue 24, 21343-21350, June 15, 2001
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Functional Expression, Characterization, and Purification of the Catalytic Domain of Human 11-beta -Hydroxysteroid Dehydrogenase Type 1*

Elizabeth A. WalkerDagger , Anya M. Clark§, M. HewisonDagger , Jon P. Ride§, and Paul M. StewartDagger

From the Dagger  Division of Medical Sciences and the § School of Biosciences, University of Birmingham, P. O. Box 363, Edgbaston, Birmingham B15 2TT

11-beta -hydroxysteroid dehydrogenase type 1 catalyzes the conversion of cortisone to hormonally active cortisol and has been implicated in the pathogenesis of a number of disorders including insulin resistance and obesity. The enzyme is a glycosylated membrane-bound protein that has proved difficult to purify in an active state. Extracted enzyme typically loses the reductase properties seen in intact cells and shows principally dehydrogenase activity. 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 the functional expression of the catalytic domain of the human enzyme, without the transmembrane domain and the extreme N terminus, in Escherichia coli. Moderate levels of soluble active protein were obtained using an N-terminal fusion with thioredoxin and a 6xHis tag. In contrast, the inclusion of a 6xHis tag at the C terminus adversely affected protein solubility and activity. However, the highest levels of active protein were obtained using a construct expressing the untagged catalytic domain. Nonreducing electrophoresis revealed the presence of both monomeric and dimeric disulfide bonded forms; however, mutation of a nonconserved cysteine residue resulted in a recombinant protein with no intermolecular disulfide bonds but full enzymatic activity. Using the optimal combination of plasmid construct and E. coli host strain, the recombinant protein was purified to apparent homogeneity by single step affinity chromatography. The purified protein possessed both dehydrogenase and reductase activities with a Km of 1.4 µM for cortisol and 9.5 µM for cortisone. This study indicates that glycosylation, the N-terminal 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 functional studies.

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 44-121-627-2380; Fax: 44-121-627-2384; E-mail: p.m.stewart@bham.ac.uk.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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