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首页> 外文期刊>Protein Expression and Purification >Auto-induction and purification of a Bacillus subtilis transglutaminase (Tgl) and its preliminary crystallographic characterization
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Auto-induction and purification of a Bacillus subtilis transglutaminase (Tgl) and its preliminary crystallographic characterization

机译：枯草芽孢杆菌转谷氨酰胺酶（Tgl）的自动诱导和纯化及其初步晶体学表征

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摘要

Spores of Bacillus subtilis are covered by a multi-protein protective coat which is a key factor in their extreme environmental resilience. A fraction of the coat proteins undergoes covalent cross-linking following their assembly at the spore surface. Several types of covalent cross-links are found in the coat. These include epsilon-(gamma-glutamyl)lysine bonds whose formation is catalyzed by a transglutaminase, Tgl, itself a coat component. Tgl is the smallest known transglutaminase. It bears no sequence resemblance to other proteins in databases, except for its counterparts in other Bacillus and related species, suggesting a highly specialized role in coat assembly. It is not known to what degree are the Tgl-like proteins structural and mechanistically related to other transglutaminases. Here, we have fused the His(6) tag to the C-terminal end of Tgl, and shown that the fusion protein is functional in vivo. We have overproduced B. subtilis Tgl-His(6) by auto-induction with high yield and purified the protein to nearly homogeneity in a single chromatographic step. The purified protein, active as it catalyzed the cross-linking of bovine serum albumin, behaved as a monomer of about 33 kDa in solution. Lastly, Tgl was crystallized and X-ray diffraction data were collected using synchrotron radiation to 2.1 angstrom resolution. Crystals of Tgl belong to the tetragonal space group P4(1,3) and contain two molecules per asymmetric unit. (C) 2007 Elsevier Inc. All rights reserved.
机译：枯草芽孢杆菌的孢子被多蛋白保护层覆盖，这是它们极高的环境适应能力的关键因素。一部分外壳蛋白在它们在孢子表面组装之后经历共价交联。在涂层中发现了几种共价交联键。这些包括ε-（γ-谷氨酰胺基）赖氨酸键，其形成是由转谷氨酰胺酶Tgl（其本身是外壳组分）催化的。 Tgl是已知的最小的转谷氨酰胺酶。它与数据库中的其他蛋白质没有任何序列相似性，除了它与其他芽孢杆菌和相关物种中的对应物一样，表明它在外壳装配中具有高度专门化的作用。还不知道Tgl样蛋白与其他转谷氨酰胺酶在结构和机理上的关联程度。在这里，我们已经将His（6）标签融合到Tgl的C末端，并表明融合蛋白在体内具有功能。我们已经通过自动诱导以高收率过量生产了枯草芽孢杆菌Tgl-His（6），并在单个色谱步骤中将蛋白质纯化至几乎同质。纯化的蛋白质具有催化牛血清白蛋白交联的活性，在溶液中的分子量约为33 kDa。最后，使Tgl结晶，并使用同步加速器辐射至2.1埃的分辨率收集X射线衍射数据。 Tgl的晶体属于四边形空间群P4（1,3），每个不对称单元包含两个分子。 （C）2007 Elsevier Inc.保留所有权利。

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