Purification of proteins fused to glutathione S-transferase.
Purification of proteins fused to glutathione S-transferase.
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DOI：
10.1007/978-1-60761-913-0_14
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发表时间：
2011
期刊：
影响因子：
--
通讯作者：
Speicher, David W
中科院分区：
文献类型：
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作者：
Harper, Sandra;Speicher, David W
本章介绍了使用谷胱甘肽S-转移酶（GST）基因融合蛋白作为一种方法诱导，高水平的蛋白表达和纯化细菌细胞裂解物。该蛋白在pGEX载体中表达，GST部分位于N-末端，随后是靶蛋白。使用GST作为融合标签是合乎需要的，因为它可以充当分子伴侣以促进蛋白质折叠，并且融合蛋白通常可以表达为可溶性蛋白而不是在包涵体中。此外，GST融合蛋白可以在不变性或使用温和洗涤剂的情况下轻松亲和纯化。融合蛋白被固定的谷胱甘肽捕获，杂质被洗掉。然后在温和的非变性条件下使用还原型谷胱甘肽洗脱融合蛋白。如果需要，通过使用位于GST部分和靶蛋白之间的位点特异性蛋白酶识别序列来实现GST亲和标记的去除。纯化的蛋白质已成功地用于免疫学研究、结构测定、疫苗生产、蛋白质-蛋白质和蛋白质-DNA相互作用研究以及其他生物化学分析。
This chapter describes the use of glutathione S-transferase (GST) gene fusion proteins as a method for inducible, high-level protein expression and purification from bacterial cell lysates. The protein is expressed in a pGEX vector, with the GST moiety located at the N-terminus followed by the target protein. The use of GST as a fusion tag is desirable because it can act as a chaperone to facilitate protein folding, and frequently the fusion protein can be expressed as a soluble protein rather than in inclusion bodies. Additionally, the GST fusion protein can be affinity purified facilely without denaturation or use of mild detergents. The fusion protein is captured by immobilized glutathione and impurities are washed away. The fusion protein then is eluted under mild, non-denaturing conditions using reduced glutathione. If desired, the removal of the GST affinity tag is accomplished by using a site-specific protease recognition sequence located between the GST moiety and the target protein. Purified proteins have been used successfully in immunological studies, structure determinations, vaccine production, protein-protein, and protein-DNA interaction studies and other biochemical analysis.