Purification of Proteins Fused to Glutathione S-Transferase

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


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 



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.


  1. 1.
    Smith, D. B. and Johnson, K. S. (1988) Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene 67, 31–40.PubMedCrossRefGoogle Scholar
  2. 2.
    Frangioni, J. V. and Neel, B. G. (1993) Solubilization and purification of enzymatically active glutathione S-transferase (pGEX) fusion proteins. Anal Biochem 210, 179–187.PubMedCrossRefGoogle Scholar
  3. 3.
    Grieco, F., Hay, J. M., and Hull, R. (1992) An improved procedure for the purification of protein fused with glutathione S-transferase. Biotechniques 13, 856–858.PubMedGoogle Scholar
  4. 4.
    Singh, S. M. and Panda, A. K. (2005) Solubilization and refolding of bacterial inclusion body proteins. J Biosci Bioeng 99, 303–310.PubMedCrossRefGoogle Scholar
  5. 5.
    Singh, S. M., Eshwari, A. N., Garg, L. C., and Panda, A. K. (2005) Isolation, solubilization, refolding, and chromatographic purification of human growth hormone from inclusion bodies of Escherichia coli cells: a case study. Methods Mol Biol 308, 163–176.PubMedGoogle Scholar
  6. 6.
    Davies, A. H., Jowett, J. B., and Jones, I. M. (1993) Recombinant baculovirus vectors expressing glutathione-S-transferase fusion proteins. Biotechnology (N Y) 11, 933–936.CrossRefGoogle Scholar
  7. 7.
    Mitchell, D. A., Marshall, T. K., and Deschenes, R. J. (1993) Vectors for the inducible overexpression of glutathione S-transferase fusion proteins in yeast. Yeast 9, 715–722.PubMedCrossRefGoogle Scholar
  8. 8.
    Grossman, T. H., Kawasaki, E. S., Punreddy, S. R., and Osburne, M. S. (1998) Spontaneous cAMP-dependent derepression of gene expression in stationary phase plays a role in recombinant expression instability. Gene 209, 95–103.PubMedCrossRefGoogle Scholar
  9. 9.
    Pan, S. H. and Malcolm, B. A. (2000) Reduced background expression and improved plasmid stability with pET vectors in BL21 (DE3). Biotechniques 29, 1234–1238.PubMedGoogle Scholar
  10. 10.
    Guan, K. L. and Dixon, J. E. (1991) Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. Anal Biochem 192, 262–267.PubMedCrossRefGoogle Scholar
  11. 11.
    Hakes, D. J. and Dixon, J. E. (1992) New vectors for high level expression of recombinant proteins in bacteria. Anal Biochem 202, 293–298.PubMedCrossRefGoogle Scholar

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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