Expression and rapid purification of highly active hexahistidine-tagged guinea pig liver transglutaminase

被引：24

作者：

Gillet, SMFG ^{[1
]}

Chica, RA ^{[1
]}

Keillor, JW ^{[1
]}

Pelletier, JN ^{[1
]}

机构：

[1] Univ Montreal, Dept Chim, Montreal, PQ H3C 3J7, Canada

来源：

PROTEIN EXPRESSION AND PURIFICATION | 2004年 / 33卷 / 02期

关键词：

transglutaminase; hexahistidine tag; chaperone; IMAC; betaine;

D O I：

10.1016/j.pep.2003.10.003

中图分类号：

Q5 [生物化学];

学科分类号：

071010 [生物化学与分子生物学]; 081704 [应用化学];

摘要：

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.

引用

页码：256 / 264

页数：9

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