期刊
PROTEIN EXPRESSION AND PURIFICATION
卷 87, 期 1, 页码 41-46出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2012.10.002
关键词
Enzyme; Tissue transglutaminase; Recombinant expression; GST fusion; PreScission (TM) Protease
类别
资金
- Natural Sciences and Engineering Research Council of Canada (NSERC)
- NSERC
- Fonds Quebecois de Recherche sur la Nature et les Technologies (FQRNT)
The expression of soluble recombinant transglutaminase (TGase) has proven to be a challenge for many research groups. Herein, we report a complementary method for the expression, in BL21(DE3) Escherichia coli, of recombinant human tissue transglutaminase (hTG2) whose solubility is enhanced through N-terminal fusion to glutathione S-transferase (GST). Moreover, we report the cleavage of the GST tag using PreScission (TM) Protease (PSP) and purification of hTG2 in its untagged form, distinctively suitable for subsequent studies of its remarkable conformational equilibrium. The effects of co-solvents and storage conditions on stability of purified hTG2 are also reported. Furthermore, we demonstrate for the first time the use of a convenient chromogenic assay to measure the activity of the human enzyme. The utility of this assay was demonstrated in the measurement of the kinetic parameters of a wide variety of substrates and inhibitors of both hTG2 and the extensively studied guinea pig liver TGase. Finally, comparison of these results provides further evidence for the functional similarity of the two enzymes. (C) 2012 Elsevier Inc. All rights reserved.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据