Expression and Purification of Glutathione‐S‐Transferase Fusion Proteins

Donald B. Smith1, Lynn M. Corcoran2

1 University of Edinburg, Edinburg, Scotland, 2 Walter & Eliza Hall Institute, Victoria, Australia
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 16.7
DOI:  10.1002/0471142727.mb1607s28
Online Posting Date:  May, 2001
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This unit describes how pGEX vectors can be used in bacterial systems to express foreign polypeptides as fusions with glutathione‐S‐transferase (GST). In general, such fusion proteins are soluble and are easily purified from lysed cells under nondenaturing conditions by absorption with glutathione‐agarose beads, followed by elution in the presence of free glutathione. Potential applications of the pGEX vectors include the expression and purification of individual polypeptides (including short peptides) for use as immunogens and as biochemical and biological reagents, and in the construction of cDNA expression libraries. This protocol describes production and screening of pGEX transformants and purification of milligram quantities of fusion proteins from 1‐liter cultures. The commentary describes several modifications to the expression and purification protocol that may be useful in cases where fusion proteins are insoluble or unstable.

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1:

  MaterialsFor recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 22; for suppliers, see appendix 44.
  • pGEX vector (pGEX1 from Amrad or pGEX2T and pGEX3X from Pharmacia Biotech)
  • Transformation‐competent Escherichia coli (unit 1.8)
  • LB plates containing 50 µg/ml ampicillin (unit 1.1)
  • LB medium containing 10 µg/ml ampicillin (unit 1.1)
  • 100 mM isopropyl‐1‐thio‐β‐D‐galactoside (IPTG), filter sterilized
  • Phosphate‐buffered saline (PBS; appendix 22), ice‐cold
  • recipeGlutathione‐agarose bead slurry (see recipe)
  • 2× SDS sample buffer (unit 10.210.2)
  • 10% (v/v) Triton X‐100
  • 50 mM Tris⋅Cl (pH 8.0)/5 mM reduced glutathione (freshly prepared; pH 7.5, final)
  • Glycerol
  • 37°C shaking incubator
  • Beckman JA‐10 and JA‐20 rotors (or equivalents)
  • Probe sonicator (with 2‐ and 5‐mm‐diameter probes)
  • Additional reagents and equipment for subcloning DNA fragments (units 1.4 & 3.16), SDS‐PAGE (unit 10.210.2), and Coomassie blue staining (unit 10.6)
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Literature Cited

   Frangioni, J.V. and Neel, B.G. 1993. Solubilization and purification or enzymatically active glutathione‐S‐transferase (pGEX) fusion proteins. Anal. Biochem. 210:179‐187.
   Smith, D.B. 1993. Purification of glutathione‐S‐transferase fusion proteins. Methods Mol. Cell Biol. 4:220‐229.
   Smith, D.B., Davern, K.M., Board, P.G., Tiu, W.U., Garcia, E.G., and Mitchell, G.F. 1986. Mr 26,000 antigen of Schistosoma japonicum recognized by resistant WEHI 129/J mice is a parasite glutathione S‐transferase. Proc. Natl. Acad. Sci. U.S.A. 83:8703‐8707.
Key Reference
   Smith et al., 1986. See above.
  Original description of the pGEX system.
   Smith, 1993. See above.
  Summary of modified pGEX vectors and alternative purification methods.
   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.
  First description of GST fusion system.
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