Expression and rapid purification of highly active hexahistidine-tagged guinea pig liver transglutaminase
Expression and rapid purification of highly active hexahistidine-tagged guinea pig liver transglutaminase
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DOI：
10.1016/j.pep.2003.10.003
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发表时间：
2004-02-01
影响因子：
1.6
通讯作者：
Pelletier, JN
中科院分区：
文献类型：
--
作者：
Gillet, SMFG;Chica, RA;Pelletier, JN
组织转谷氨酰胺酶已被确定为多种疾病的一个因素，包括白内障的形成和乳糜泻。豚鼠组织转谷氨酰胺酶具有非常广泛的底物特异性，因此对使用底物类似物进行动力学研究是有用的。在这里，我们报道了一种六组氨酸标记的豚鼠肝组织转谷氨酰胺酶（His(6)-tTGase）在大肠杆菌中的表达，这种酶可以通过固定化金属亲和层析快速纯化。使用此程序，我们获得了最高的报道比活性（17 U/mg）和高产量（22 mg/L培养），重组TGase使用一步纯化方案。通过两次独立的分光光度测定，我们确定了以Cbz-Gln-Gly为底物的重组酶的Km值与文献中报道的天然酶的值在相同的范围内。因此，我们开发了一种快速、可重复的方案，用于制备高质量的组织TGase。(C) 2003 Elsevier Inc.版权所有。
Tissue transglutaminase has been identified as a contributor to a wide variety of diseases, including cataract formation and Celiac disease. Guinea pig tissue transglutaminase has a very broad substrate specificity and therefore is useful for kinetic studies using substrate analogues. Here, we report the expression in Escherichia coli of a hexahistidine-tagged guinea pig liver tissue transglutaminase (His(6)-tTGase) allowing rapid purification by immobilized-metal affinity chromatography. Using this procedure we have obtained the highest reported specific activity (17 U/mg) combined with a high yield (22 mg/L of culture) for recombinant TGase using a single-step purification protocol. Using two independent spectrophotometric assays, we determined that the Km value of the recombinant enzyme with the substrate Cbz-Gln-Gly is in the same range as values reported in the literature for the native enzyme. We have thus developed a rapid and reproducible protocol for the preparation of high quality tissue TGase. (C) 2003 Elsevier Inc. All rights reserved.