UNIT 6.6 Expression and Purification of GST Fusion Proteins

  1. Sandra Harper,
  2. David W. Speicher

Published Online: 1 MAY 2008

DOI: 10.1002/0471140864.ps0606s52

Current Protocols in Protein Science

Current Protocols in Protein Science

How to Cite

Harper, S. and Speicher, D. W. 2008. Expression and Purification of GST Fusion Proteins. Current Protocols in Protein Science. 52:6.6:6.6.1–6.6.26.

Author Information

  1. The Wistar Institute, Philadelphia, Pennsylvania

Publication History

  1. Published Online: 1 MAY 2008
  2. Published Print: MAY 2008


This unit describes the use of the glutathione-S-transferase (GST) gene fusion system as a method for high-level protein expression and purification from bacterial lysates. Several pGEX vectors are available with multiple cloning sites to allow for unidirectional insertion of the coding-region DNA into the pGEX vector. The GST fusion protein is easily purified by affinity chromatography using a glutathione-Sepharose matrix under mild conditions. Removal of the GST moiety from the protein of interest is accomplished through a specific protease cleavage site located between the GST moiety and the recombinant polypeptide. For solution digestions, GST is easily removed by a second round of chromatography on the glutathione column. Removal of proteases is facilitated by the use of a benzamidine-Sepharose column or a gel-filtration step. Purified protein has been used successfully in structural determinations, immunological studies, vaccine production, and structure-function analysis of protein-protein or DNA-protein interactions. Curr. Protoc. Protein Sci. 52:6.6.1-6.6.26. © 2008 by John Wiley & Sons, Inc.


  • glutathione S-transferase (GST);
  • pGEX;
  • protein expression;
  • protein purification;
  • thrombin;
  • Factor Xa;
  • fusion tags