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Expression and Purification of Thioredoxin Fusion Proteins

John McCoy¹, EdwardLa Ville¹

¹Genetics 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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Unit Introduction
Strategic Planning
Basic Protocol: 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
Bibliography
Figures
Tables

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Materials

Basic Protocol: Construction and Expression of a Thioredoxin Fusion Protein

Materials

DNA fragment encoding desired sequence
Thioredoxin expression vectors (Fig. 6.7.1): 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)
IMC plates (see recipe) containing 100 µg/ml ampicillin
CAA/glycerol/ampicillin 100 medium (see recipe)
IMC medium (see recipe) containing 100 µg/ml ampicillin
10 mg/ml tryptophan (see recipe)
SDS-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 Basic Protocol)
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
SDS-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 Basic 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

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 Basic Protocol)
20 mM Tris×Cl (pH 8.0)/2.5 mM EDTA
SDS-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 (Support Protocol 1) and SDS-PAGE (unit 10.1)

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Figures

Figure 6.7.1

Thioredoxin gene fusion expression vectors pTRXFUS, hpTRXFUS, and pALtrxA-781. pALtrxA-781 contains a polylinker sequence at the 3¢ end of the trxA gene. pTRXFUS and hpTRXFUS contain a linker region encoding a peptide that includes the enterokinase cleavage site between the trxA gene and the polylinker. The sequence surrounding the active site loop of thioredoxin has a single RsrII site that can be used to insert peptide coding sequence. The asterisk indicates a translational stop codon. Abbreviations: trxA, E. coli thioredoxin gene; BLA, -lactamase gene; ori, colE1 replication origin; pL, bacteriophage major leftward promoter; aspA terminator, E. coli aspartate amino-transferase transcription terminator.

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Figure 6.7.2

Expression of thioredoxin gene fusions. The gel shows proteins found in the soluble fractions derived from E. coli cells expressing eleven different thioredoxin gene fusions. Lane 1, host E. coli strain GI724 (negative control, 37°C); lane 2, murine interleukin-2 (IL-2; 15°C); lane 3, human IL-3 (15°C); lane 4, murine IL-4 (15°C); lane 5, murine IL-5 (15°C); lane 6, human IL-6 (25°C); lane 7, human MIP-1a (37°C); lane 8, human IL-11 (37°C); lane 9, human macrophage colony-stimulating factor (M-CSF; 37°C); lane 10, murine leukemia inhibitory factor (LIF; 25°C); lane 11, murine steel factor (SF; 37°C); and lane 12, human bone morphogenetic protein-2 (BMP-2; 25°C). Temperatures in parentheses are the production temperature chosen for expressing each fusion. This is a 10% SDS-polyacrylamide gel, stained with Coomassie brilliant blue.

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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.
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	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.
	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 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.
	Shimatake, H. and Rosenberg, M. 1981. Purified 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--lytic protease expression in Escherichia coli reveals that the pro region is required for activity. J. Bacteriol. 171:1320-1325.
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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.