Expression and Purification of GST Fusion Proteins

Sandra Harper1, David W. Speicher1

1 The Wistar Institute, Philadelphia, Pennsylvania
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 6.6
DOI:  10.1002/0471140864.ps0606s52
Online Posting Date:  May, 2008
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Abstract

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.

Keywords: glutathione S‐transferase (GST); pGEX; protein expression; protein purification; thrombin; Factor Xa; fusion tags

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Expression of Glutathione‐S‐Transferase Fusion Protein
  • Basic Protocol 2: Affinity Chromatography Purification of a Soluble GST Fusion Protein
  • Alternate Protocol 1: Affinity Chromatography Purification of GST Fusion Protein from Inclusion Bodies
  • Alternate Protocol 2: Batch Purification of GST Fusion Protein
  • Basic Protocol 3: Protease Cleavage of Fusion Protein in Solution to Remove GST Affinity Tag
  • Alternate Protocol 3: Protease Cleavage of Fusion Protein Bound to a Glutathione‐Sepharose Column
  • Alternate Protocol 4: Batch Protease Cleavage and Separation of Fusion Protein on Glutathione Resin
  • Support Protocol 1: Affinity Chromatography Purification of Polypeptides after Enzymatic Cleavage
  • Support Protocol 2: Further Purification of Cleaved Recombinant Protein Using HPLC Gel Filtration
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Expression of Glutathione‐S‐Transferase Fusion Protein

  Materials
  • LB medium, pH 7.2 (see recipe)
  • 5 mg/ml ampicillin (see recipe)
  • Glycerol culture of transformed E. coli cells expressing GST fusion protein of interest in a pGEX vector (see )
  • 100 mM isopropyl‐1‐thio‐β‐D‐galactopyranoside (IPTG; see recipe)
  • 2‐liter culture flasks
  • 500‐ml culture flasks
  • Inoculating loops
  • Refrigerated environmental shaker (refrigeration is necessary for proteins that need to be expressed at temperatures below room temperature; e.g., New Brunswick Scientific Innova 4330 Refrigerated Environmental Shaker)
  • UV/visible light spectrophotometer
  • Large centrifuge bottles (e.g., 1‐liter capacity)
  • 50‐ml centrifuge tubes
  • Low‐speed refrigerated centrifuge (e.g., Beckman J6‐B and JS‐4.2 rotor or equivalent), 4°C
  • Additional reagents and equipment for SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE; unit 10.1)

Basic Protocol 2: Affinity Chromatography Purification of a Soluble GST Fusion Protein

  Materials
  • Glutathione‐Sepharose 4B resin (GE Healthcare)
  • Phosphate‐buffered saline (PBS; appendix 2E)
  • Glutathione buffer (see recipe)
  • PBS/EDTA/PMSF buffer (see recipe)
  • Pelleted E. coli culture expressing fusion protein (see protocol 1, step 14)
  • Lysis buffer (see recipe), ice cold
  • Wash buffer (see recipe), ice cold
  • PBS/EDTA (see recipe)
  • 2.5 × 8–cm glutathione‐Sepharose 4B column (e.g., Bio‐Rad Econo)
  • Peristaltic pump
  • Sonicator equipped with microtip probe (e.g., Branson)
  • 50‐ml centrifuge tubes
  • High‐speed refrigerated centrifuge (e.g., Beckman J2‐21M centrifuge and JA‐18 rotor or equivalent), 4°C
  • Dounce homogenizer
  • 60‐ml centrifuge bottles (capable of handling force of 48,000 × g)
  • Additional reagents and equipment for pouring chromatographic columns (unit 8.3) and SDS‐PAGE (unit 10.1)

Alternate Protocol 1: Affinity Chromatography Purification of GST Fusion Protein from Inclusion Bodies

  • U buffer (see recipe)
  • Triton X‐100
  • PBS/glycerol buffer (see recipe)
  • Low‐speed refrigerated centrifuge (e.g., Beckman J6‐B and JS‐4.2 rotor or equivalent), 4°C
  • Additional reagents and equipment for dialysis ( appendix 3B)

Alternate Protocol 2: Batch Purification of GST Fusion Protein

  Materials
  • Solution of affinity‐purified fusion protein (see protocol 2, step 15, or protocol 4, step 10)
  • Thrombin (Sigma) reconstituted in water at 0.5 U/µl or 1 µg/µl factor Xa (Boehringer Mannheim)
  • 0.15 M PMSF in isopropanol
  • PBS/EDTA/PMSF buffer (see recipe)
  • 37°C water bath (for thrombin cleavage) or 25°C water bath (for factor Xa cleavage)
  • Beckman J6‐B centrifuge and JS‐4.2 rotor (or equivalent), 4°C
  • Additional reagents and equipment for dialysis ( appendix 3B) and SDS‐PAGE (unit 10.1)

Basic Protocol 3: Protease Cleavage of Fusion Protein in Solution to Remove GST Affinity Tag

  • PBS ( appendix 2E)
  • Glutathione‐Sepharose 4B column containing bound fusion protein (see protocol 2, step 13)
  • Glutathione buffer (see recipe)

Alternate Protocol 3: Protease Cleavage of Fusion Protein Bound to a Glutathione‐Sepharose Column

  • Glutathione‐Sepharose 4B resin with bound fusion protein (see protocol 4, step 7)
  • PBS ( appendix 2E)

Alternate Protocol 4: Batch Protease Cleavage and Separation of Fusion Protein on Glutathione Resin

  Materials
  • Solution of fusion protein that has been cleaved with thrombin or factor Xa and dialyzed into PBS/EDTA/PMSF buffer (see protocol 5, step 4)
  • Glutathione buffer (see recipe)
  • PBS/EDTA/PMSF buffer (see recipe)
  • 2.5 × 8–cm glutathione‐Sepharose 4B column (e.g., Bio‐Rad Econo)
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1)

Support Protocol 1: Affinity Chromatography Purification of Polypeptides after Enzymatic Cleavage

  Materials
  • Cleaved fusion protein (see protocol 8, step 6, or protocol 6, step 5)
  • PBS ( appendix 2E)
  • Centriprep concentrator with appropriate MWCO (Amicon)
  • Low‐speed refrigerated centrifuge (e.g., Beckman J6‐B and JS‐4.2 rotor or equivalent, 4°C), or 0.22‐um low‐protein‐binding filter (Costar)
  • HPLC system (see unit 8.7)
  • Gel‐filtration columns (see unit 8.3)
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1)
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Figures

Videos

Literature Cited

   Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. (eds.). 2008. Current Protocols in Molecular Biology. John Wiley & Sons, Hoboken, N.J.
   Brymora, A., Valova, V.A., and Robinson, P.J. 2004. Protein‐protein interactions identified by pull‐down experiments and mass spectrometry. Curr. Protoc. Cell Biol. 22: 17.5.1‐17.5.51.
   Davies, A.H., Jowett, J.B.M., and Jones, I.M. 1993. Recombinant baculovirus vectors expressing glutathione‐S‐transferase fusion proteins. Biotechnology 11: 933‐936.
   Frangioni, J.V. 1992. Solubilization and purification of enzymatically active glutathione‐S‐transferase (pGEX) fusion proteins. Anal. Biochem. 210: 179‐187.
   Gearing, D.P., Nicola, N.A., Metcalf, D., Foote, S., Willson, T.A., Gough, N.M., and Williams, R.L. 1989. Production of leukemia inhibitory factor in Escherichia coli by a novel procedure and its use in maintaining embryonic stem cells in culture. Biotechnology 7: 1157‐1161.
   Grieco, F., Hull, J., and Hull, R. 1992. An improved procedure for the purification of protein fused with glutathione‐S‐transferase. Biotechniques 13: 856‐857.
   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. 1991. New vectors for high level expression of recombinant proteins in bacteria. Anal. Biochem. 202: 293‐298.
   Jung, J.W., Jung, S.H., Kim, H.S., Yuk, J.S., Park, J.B., Kim, Y.M., Han, J.A., Kim, P.H., and Ha, K.S. 2006. High‐throughput analysis of GST‐fusion protein expression and activity‐dependent protein interactions on GST‐fusion protein arrays with a spectral surface plasmon resonance biosensor. Proteomics 6: 1110‐1120.
   Mitchell, D.A., Marshall, T.K., and Deschenes, R.J. 1993. Vectors for the overexpression of glutathione‐S‐transferase fusion proteins in yeast. Yeast 9: 715‐722.
   Saaem, I., Papasotiropoulos, V., Wang, T., Soteropoulos, P., and Libera, M., 2007. Hydrogel‐based protein nanoarrays. J. Nanosci Nanotechnol. 7: 2623‐2632.
   Sambrook, J., and Russell, D. 2001. Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
   Singh, C.R. and Asano, K. 2007. Localization and characterization of protein‐protein interaction sites. Methods Enzymol. 429: 139‐161.
   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.
   Smyth, D.R., Mrozkiewicz, M.K., McGrath, W.J., Listwan, P., and Kobe, B. 2003. Crystal structures of fusion proteins with large‐affinity tags. Protein Sci. 12: 1313‐1322.
   Vikis, H.G. and Guan, K.L. 2004. Glutathione‐S‐transferase‐fusion based assays for studying protein‐protein interactions. Methods Mol. Bio. 261: 175‐186.
   Yip, Y.L., Smith, G., and Ward, R.L. 2001. Comparison of phage pIII, pViii and GST as carrier proteins for peptide immunization in Balb/c mice. Immun Lett. 79: 197‐202.
   Zhan, Y., Song, X., and Zhou, G.W. 2001. Structural analysis of regulatory protein domains using GST‐fusion proteins. Gene 281: 1‐9.
Key Reference
   Smith and Johnson, 1988. See above.
  Original description of the pGEX system.
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