Walker E A, Clark A M, Hewison M, Ride J P, Stewart P M
Division of Medical Sciences and the School of Biosciences, University of Birmingham, P. O. Box 363, Edgbaston, Birmingham B15 2TT.
J Biol Chem. 2001 Jun 15;276(24):21343-50. doi: 10.1074/jbc.M011142200. Epub 2001 Apr 6.
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 K(m) of 1.4 micrometer for cortisol and 9.5 micrometer 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.
11-β-羟基类固醇脱氢酶1型催化可的松转化为具有激素活性的皮质醇，并与包括胰岛素抵抗和肥胖在内的多种疾病的发病机制有关。该酶是一种糖基化的膜结合蛋白，已证明难以以活性状态纯化。提取的酶通常会丧失在完整细胞中所见的还原酶特性，主要表现为脱氢酶活性。已知C末端催化结构域含有一个二硫键，位于内质网腔内，通过单个N末端跨膜结构域锚定在膜上。我们在此报告人酶催化结构域（不含跨膜结构域和极端N末端）在大肠杆菌中的功能表达。使用与硫氧还蛋白的N末端融合和6xHis标签获得了中等水平的可溶性活性蛋白。相比之下，在C末端包含6xHis标签会对蛋白质的溶解度和活性产生不利影响。然而，使用表达无标签催化结构域的构建体获得了最高水平的活性蛋白。非还原电泳显示存在单体和二聚体二硫键结合形式；然而，一个非保守半胱氨酸残基的突变导致重组蛋白没有分子间二硫键但具有完整的酶活性。通过质粒构建体和大肠杆菌宿主菌株的最佳组合，重组蛋白通过单步亲和层析纯化至表观均一性。纯化的蛋白具有脱氢酶和还原酶活性，对皮质醇的K(m)为1.4微米，对可的松的K(m)为9.5微米。这项研究表明，糖基化、包括跨膜螺旋的N末端区域和分子间二硫键对于酶活性不是必需的，并且在细菌中的表达可以为未来的结构和功能研究提供活性重组蛋白。
