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.
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References
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.
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.
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.
Singh, S. M. and Panda, A. K. (2005) Solubilization and refolding of bacterial inclusion body proteins. J Biosci Bioeng 99, 303–310.
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.
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.
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.
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.
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.
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.
Hakes, D. J. and Dixon, J. E. (1992) New vectors for high level expression of recombinant proteins in bacteria. Anal Biochem 202, 293–298.
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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Harper, S., Speicher, D.W. (2011). Purification of Proteins Fused to Glutathione S-Transferase. In: Walls, D., Loughran, S. (eds) Protein Chromatography. Methods in Molecular Biology, vol 681. Humana Press. https://doi.org/10.1007/978-1-60761-913-0_14
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DOI: https://doi.org/10.1007/978-1-60761-913-0_14
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