Expression and Purification of Thioredoxin Fusion Proteins

John McCoy1, Edward La Ville1

1 Genetics Institute, Cambridge, Massachusetts
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 6.7
DOI:  10.1002/0471140864.ps0607s10
Online Posting Date:  May, 2001
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Abstract

This unit describes a gene fusion expression system that uses thioredoxin, the product of the Escherichia coli trxA gene, as the fusion partner. The inherent thermal stability of thioredoxin and its susceptibility to quantitative release from the E. coli cytoplasm by osmotic shock can also be exploited as useful tools for thioredoxin fusion protein purification. In this protocol, a fusion of trxA to any desired gene is constructed and the resulting fusion protein is expressed in an appropriate host strain. Additional protocols describe E. coli cell lysis and fractionation, osmotic release of thioredoxin fusion proteins from the E. coli cytoplasm, and heat treatment to purify some thioredoxin fusion proteins.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Construction and Expression of a Thioredoxin Fusion Protein
  • Support Protocol 1: E. coli Lysis Using a French Pressure Cell
  • Support Protocol 2: Osmotic Release of Thioredoxin Fusion Proteins
  • Support Protocol 3: Purification of Thioredoxin Fusion Proteins by Heat Treatment
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Construction and Expression of a Thioredoxin Fusion Protein

  Materials
  • DNA fragment encoding desired sequence
  • Thioredoxin expression vectors (Fig. ): pTRXFUS or pALtrxA‐781 (Genetics Institute or Invitrogen) or hpTRXFUS (Genetics Institute)
  • E. coli strain GI724 (Genetics Institute or Invitrogen), grown in LB medium and made competent
  • LB medium (unit 5.2)
  • recipeIMC plates (see recipe) containing 100 µg/ml ampicillin
  • recipeCAA/glycerol/ampicillin 100 medium (see recipe)
  • recipeIMC medium (see recipe) containing 100 µg/ml ampicillin
  • recipe10 mg/ml tryptophan (see recipe)
  • recipeSDS‐PAGE sample buffer (see recipe)
  • 30°C convection incubator
  • 18 × 50–mm culture tubes
  • Roller drum (New Brunswick Scientific)
  • 250‐ml culture flask
  • 70°C water bath
  • Microcentrifuge, 4°C
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1), and Coomassie brilliant blue staining (unit 10.5)

Support Protocol 1: E. coli Lysis Using a French Pressure Cell

  Materials
  • Cell pellet from 4‐hr post‐induction culture (see protocol 1)
  • 20 mM Tris⋅Cl, pH 8.0 ( appendix 2E), 4°C
  • Lysis buffer: 20 mM Tris⋅Cl (pH 8.0) with protease inhibitors (optional)—0.5 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM p‐aminobenzamidine (PABA), and 5 mM EDTA
  • recipeSDS‐PAGE sample buffer (see recipe)
  • French press and 3.5‐ml mini‐cell (SLM Instruments), 4°C
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1)

Support Protocol 2: Osmotic Release of Thioredoxin Fusion Proteins

  Materials
  • Cell pellet from 4‐hr post‐induction cultures (see protocol 1)
  • 20 mM Tris⋅Cl (pH 8.0)/2.5 mM EDTA/20% (w/v) sucrose, ice‐cold
  • 20 mM Tris⋅Cl (pH 8.0)/2.5 mM EDTA, ice‐cold
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1)

Support Protocol 3: Purification of Thioredoxin Fusion Proteins by Heat Treatment

  Materials
  • Cell pellet from 4‐hr post‐induction cultures (See protocol 1)
  • 20 mM Tris⋅Cl (pH 8.0)/2.5 mM EDTA
  • recipeSDS‐PAGE sample buffer (see recipe)
  • 80°C water bath
  • 10‐ml glass‐walled tube
  • Additional reagents and equipment for lysis using a French pressure cell ( protocol 2) and SDS‐PAGE (unit 10.1)
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Figures

Videos

Literature Cited

Literature Cited
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   LaVallie, E.R., Rehemtulla, A., Racie, L.A., DiBlasio, E.A., Ferenz, C., Grant, K.L., Light, A., and McCoy, J.M. 1993a. Cloning and functional expression of a cDNA encoding the catalytic subunit of bovine enterokinase. J. Biol. Chem. 268:23311‐23317.
   LaVallie, E.R., DiBlasio, E.A., Kovacic, S., Grant, K.L., Schendel, P.F., and McCoy, J.M. 1993b. A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm. Bio/Technology 11:187‐193.
   Lu, Z., DiBlasio‐Smith, E.A., Grant, K.L., Warne, N.W., LaVallie, E.R., Collins‐Racie, L.A., Follettie, M.T., Williamson, M.J., and McCoy, J.M. 1996. Histidine patch thioredoxins. Mutant forms of thioredoxin with metal chelating affinity that provide for convenient purifications of thioredoxin fusion proteins. J. Biol. Chem. 271:5059‐5065.
   Lunn, C.A. and Pigiet, V.P. 1982. Localization of thioredoxin from Escherichia coli in an osmotically sensitive compartment. J. Biol.Chem. 257:11424‐11430.
   Mazzarella, R.A., Srinivasan, M., Haugejorden, S.M., and Green, M. 1990. ERp72, an abundant luminal endoplasmic reticulum protein, contains three copies of the active site sequences of protein disulfide isomerase. J. Biol. Chem. 265:1094‐1101.
   McCoy, J.M. 1992. Heat‐shock proteins and their potential uses for pharmaceutical protein production in microorganisms. In Stability of Protein Pharmaceuticals, Part B. (T. Ahern and M. Manning, eds.) pp 287‐316. Plenum Press, New York.
   Mieschendahl, M., Petri, T., and Hanggi, U. 1986. A novel prophage independent trp regulated lambda pL expression system. Bio/Technology 4:802‐808.
   Mitraki, A. and King, J. 1989. Protein folding intermediates and inclusion body formation. Bio/Technology 7:690‐697.
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   Papouchado, M.L., Valdez, S.N., Ghiringhelli, D., Poskus, E., and Ermacora, M.R. 1997. Expression of properly folded human glutamate decarboxylase 65 as a fusion protein in Escherichia coli. Eur. J. Biochem. 246:350‐359.
   Riggs, P. 1994. Expression and purification of maltose‐binding protein fusions. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.F. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 16.6.1‐16.6.14. John Wiley & Sons, New York.
   Shatzman, A.R., Gross, M.S., and Rosenberg, M. 1990. Expression using vectors with phage λ regulatory sequences. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.F. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 16.3.1‐16.3.11. John Wiley & Sons, New York.
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Key Reference
   Ausubel, F.M., Brent, R., Kingston, R.F., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. (eds.) 1997. Current Protocols in Molecular Biology. John Wiley & Sons, New York.
  A source of protocols for cloning and analyzing DNA.
Internet Resources
   http://www.invitrogen.com/manuals.html
  Source for protocols on affinity‐based purification.
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