Expression and Purification of Thioredoxin Fusion Proteins

John McCoy1, Edward LaVallie1

1 Genetics Institute, Cambridge, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 16.8
DOI:  10.1002/0471142727.mb1608s28
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 colitrxA gene, as the fusion partner. The system is particularly useful for high‐level production of soluble fusion proteins in the E. coli cytoplasm; in many cases heterologous proteins produced as thioredoxin fusion proteins are correctly folded and display full biological activity. Protein fusions to His‐patch Trx can usually be purified in a single step from cell lysates. Additional protocols describe E. coli cell lysis using a French pressure cell 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
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

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

  MaterialsFor recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 22; for suppliers, see appendix 44.
  • 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 (unit 1.8)
  • LB medium (unit 1.1)
  • recipeIMC plates containing 100 µg/ml ampicillin (see recipe)
  • recipeCAA/glycerol/ampicillin 100 medium (see recipe)
  • recipeIMC medium containing 100 µg/ml ampicillin (see recipe)
  • 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 subcloning of DNA fragments (unit 3.16), transforming competent E. coli cells (unit 1.8), preparing miniprep DNA (unit 1.6), restriction mapping (unit 3.2), direct sequencing of plasmid DNA (units 7.3 & 7.47.4), SDS‐PAGE (unit 10.210.2), and Coomassie brilliant blue staining (unit 10.6)

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

  Additional MaterialsFor recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 22; for suppliers, see appendix 44.
  • Cell pellet from 4‐hr post‐induction culture ( protocol 1basic protocol)
  • 20 mM Tris⋅Cl, pH 8.0 ( appendix 22), 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
  • French press and 3.5‐ml mini‐cell (Fig. ; SLM Instruments), 4°C

Support Protocol 2: Osmotic Release of Thioredoxin Fusion Proteins

  Additional MaterialsFor recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 22; for suppliers, see appendix 44.
  • Cell pellet from 4‐hr post‐induction cultures ( protocol 1basic protocol)
  • 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

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

  Additional MaterialsFor recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 44; for suppliers, see appendix 44.
  • Cell pellet from 4‐hr post‐induction cultures ( protocol 1basic protocol)
  • 20 mM Tris⋅Cl (pH 8.0)/2.5 mM EDTA
  • 80°C water bath
  • 10‐ml glass‐walled tube
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Figures

Videos

Literature Cited

Literature Cited
   Bardwell, J.C.A., McGovern, K., and Beckwith, J. 1991. Identification of a protein required for disulfide bond formation in vivo. Cell 61:581‐589.
   Edman, J.C., Ellis, L., Blacher, R.W., Roth, R.A., and Rutter, W.J. 1985. Sequence of protein disulphide isomerase and implications of its relationship to thioredoxin. Nature 317:267‐270.
   Holmgren, A. 1985. Thioredoxin. Ann. Rev. Biochem. 54:237‐271.
   Katti, S.K., LeMaster, D.M., and Eklund, H. 1990. Crystal structure of thioredoxin from Escherichia coli at 1.68 angstroms resolution. J. Mol. Biol. 212:167‐184.
   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., 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.
   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.
   Norrander, J., Kempe, T., and Messing, J. 1983. Construction of improved M13 vectors using oligonucleotide‐directed mutagenesis. Gene 26:101‐106.
   Shimatake, H. and Rosenberg, M. 1981. Purified λ regulatory protein cII positively activates promoters for lysogenic development. Nature 292:128‐132.
   Shinde, U., Chatterjee, S., and Inouye, M. 1993. Folding pathway mediated by an intramolecular chaperone. Proc. Natl. Acad. Sci.U.S.A. 90:6924‐6928.
   Silen, J.L., Frank, D., Fujishige, A., Bone, R., and Agard, D.A. 1989. Analysis of prepro‐α‐lytic protease expression in Escherichia coli reveals that the pro region is required for activity. J. Bacteriol. 171:1320‐1325.
   Stormo, G.D., Schneider, T.D., and Gold, L. 1982. Characterization of translation initiation sites in E. coli. Nucl. Acids Res. 10:2971‐2996.
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