Expression, purification, and characterization of a bacterial GTP-dependent PEP carboxykinase

被引：16

作者：

Aich, S ^{[1
]}

Imabayashi, F ^{[1
]}

Delbaere, LTJ ^{[1
]}

机构：

[1] Univ Saskatchewan, Dept Biochem, Saskatoon, SK S7N 5E5, Canada

来源：

PROTEIN EXPRESSION AND PURIFICATION | 2003年 / 31卷 / 02期

基金：

加拿大健康研究院;

关键词：

kinase; monomeric; PEP carboxykinase; phosphotransfer; enzyme; catalysis; GTP-dependent;

D O I：

10.1016/S1046-5928(03)00189-X

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

The Corynebacterium glutamicum (C glutamicum) phosphoenolpyruvate carboxykinase (PCK) gene (pckA) was cloned into an Escherichia coli expression vector with a glutathione S-transferase (GST) tag. This recombinant DNA can produce highly overexpressed tagged protein in soluble form. This is the first report of the production of C glutamicum PCK overexpressed in E coli. The GST-fused PCK was purified using the glutathione-Sepharose 4B affinity column and the GST tag was removed in one-step. This one-step, easy purification method would be very useful for future mutational and structural studies. The molecular mass of the purified protein is similar to68 kDa as confirmed by mass spectrometry and it is a monomeric enzyme. Also, the enzyme assays revealed that C. glutamicum PCK has a GTP-specific activity and that its activity is maximal in the presence of both Mn2+ and Mg2+. (C) 2003 Elsevier Science (USA). All rights reserved.

引用

页码：298 / 304

页数：7

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