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Purification of Proteins Fused to Glutathione S-Transferase

  • Sandra Harper
  • David W. Speicher
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 681)

Abstract

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 analyses.

Key words

Glutathione S-transferase pGEX Protein expression Protein purification Thrombin Factor Xa Fusion tags 

Notes

Acknowledgments

This work was supported in part by the National Institutes of Health Grant HL038794 and institutional grants to The Wistar Institute, including an NCI Cancer Core Grant (CA10815) and grants from the Pennsylvania Department of Health.

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Sandra Harper
    • 1
  • David W. Speicher
    • 1
  1. 1.The Wistar InstitutePhiladelphiaUSA

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