| ABSTRACT |
| Glutathione-S-Transferase (GST) fusion proteins have a wide range of applications. This protocol is designed for IPTG-inducible bacterial expression vectors. |
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| MATERIALS |
| Buffers, Solutions, and Reagents |
- Reduced glutathione, 20 mM prepared in 50 mM Tris-HCl (pH 8.0)
- Isopropyl-β-D-thio-galactoside (IPTG)
- LB broth, containing appropriate antibiotic selection
- Lysis buffer: PBS + 1% Triton X-100, supplemented just before use with protease inhibitors (e.g, final concentrations of 2 µg/ml aprotinin , 1 µg/ml leupeptin , and 25 µg/ml phenylmethylsulfonyl fluoride [PMSF])
- Phosphate-buffered saline (PBS), ice cold
- 150 mM NaCl
- 20 mM sodium phosphate (pH 7.4)
- PBS supplemented just prior to use with protease inhibitors (e.g., final concentrations of 2 µg/ml aprotinin, 1 µg/ml leupeptin , and 25 µg/ml PMSF)
- Appropriate antibiotics for plasmid selection (see step 1)
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| Bacterial Strains |
- Bacterial strain transformed with GST and GST fusion expression plasmids (see note to step 1)
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| Special Equipment |
- Centrifuge, precooled to 4°C
- Shaking incubator, preset to 37°C
- Rotator for end-over-end mixing
- Disposable chromatography column
- Glutathione-Sepharose beads
- Sonicator
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| Additional Reagents |
- This protocol also requires equipment and reagents for SDS-PAGE, including Coomassie blue (see Sambrook and Russell 2001)
- SDS-PAGE sample buffer, 2x (Sambrook and Russell 2001)
- 100 mM Tris-HCl (pH 6.8)
- 4%(w/v) SDS (electrophoresis grade)
- 0.2% (w/v) bromophenol blue
- 20% (v/v) glycerol
- 200 mM dithiothreitol (DTT) or β-mercaptoethanol
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| METHOD |
| Inoculate one colony of each bacterial strain expressing each construct (GST alone, GST fusion proteins) into individual 5-ml aliquots of LB broth containing appropriate antibiotic selection. Grow overnight at 37°C with shaking. Although variations of GST fusion protein expression vectors are available, the most commonly used versions include the sequence encoding the GST moiety followed by a multiple cloning site; an IPTG-inducible promoter; the ampicillin-resistance gene; the lacI gene for expression control; and a bacterial origin of replication. Many bacterial strains can be used, including those commonly used for cloning. Alternatively, protease-deficient strains such as BL21 have been commonly used for expression of recombinant proteins. Inoculate 1 liter of LB containing antibiotic selection with the 5-ml overnight culture from step 1. Grow at 37°C with shaking to an OD600 of 0.5-1.0 (should take 3-6 hr). Induce expression of the protein by adding IPTG to a final concentration of 0.1 mM. Different proteins are optimally produced using different concentrations of IPTG. If protein expression is problematic, first titrate the amount of IPTG added to determine the optimal conditions for protein induction. Incubate for an additional 3 hr at 37°C with shaking. Centrifuge the bacterial culture at 3500g for 20 min at 4°C. Discard the supernatant. At this point, pellets can be stored frozen at -20°C if necessary. Resuspend the pellet in 20 ml of lysis buffer supplemented with protease inhibitors. Sonicate the bacterial suspension on ice, in short 10-sec bursts alternated with 10 sec resting on ice. Three cycles of sonication are usually sufficient. Oversonication can result in degradation and denaturation of the fusion protein. It can also result in contamination with bacterial host proteins (this can be detected after elution of the preparation on an SDS-PAGE gel, and staining with Coomassie). If these problems occur in the preparation of the protein of interest, titration of the time of sonication required to release the protein is useful. Centrifuge the lysate at 12,000g for 15 min at 4°C at this stage. Transfer the supernatant to a fresh tube. Add 5 ml of a 50:50 slurry of glutathione-Sepharose beads in lysis buffer. Commercially available glutathione-Sepharose beads are often provided in a solution containing alcohols or other ingredients. Prior to use, these resins should be washed with lysis buffer and stored as a 50:50 (v/v) slurry at 4°C. Incubate for 30 min at 4°C, rotating the tube end over end to ensure mixing. Centrifuge at 750g for 1 min at 4°C to pellet the beads. Remove the supernatant. Wash the beads in 5 ml of ice-cold PBS supplemented with protease inhibitors. Centrifuge at 750g for 1 min at 4°C to pellet the beads. Remove the supernatant. Add 5 ml of ice-cold PBS supplemented with protease inhibitors. Resuspend the beads by gentle mixing. Centrifuge again at 750g for 1 min at 4°C to pellet the beads. Remove the supernatant. The fusion protein can be stored on the beads at 4°C at this stage. This is appropriate if the protein is to be labeled or used in a GST pull-down experiment. Add 5 ml of ice-cold PBS supplemented with protease inhibitors. Resuspend the beads with gentle mixing. Pour the slurry into a commercially available disposable chromatography column. Allow the PBS to run out of the column. Wash with 5 ml of ice-cold PBS supplemented with protease inhibitors. While the column is flowing, prepare a rack of 10 microcentrifuge tubes labeled 1-10. Elute the fusion protein by adding 5 ml of cold (0-4°C) 20 mM reduced glutathione in 50 mM Tris (pH 8.0). Collect ~0.5-ml fractions of the eluate in each microcentrifuge tube. Store the eluate at 4°C prior to use. The column can be stored in PBS at 4°C properly sealed to prevent desiccation and contamination. The eluted proteins are in a solution containing 20 mM glutathione. In most instances, it is optimal to remove the glutathione. This can be accomplished by dialysis against the buffer that is most compatible with the assay in which the protein will be utilized. Alternatively, a commercially prepared concentration buffer exchange unit (with a low MW cutoff) can be used for buffer exchange. Perform a protein assay on the eluted fractions. The results of the protein assay will indicate which of the eluate tubes contains the fusion protein. Run the samples from the eluates containing protein on an SDS-polyacrylamide gel and stain with Coomassie blue dye. The GST moiety is 26 kD; therefore, add 26 kD to the predicted molecular weight of your fusion protein to determine the anticipated molecular weight. At this point, the recombinant protein can be stored. The method of storage must be determined empirically. For example, a protein to be used subsequently in an enzymatic assay may require specific handling as compared to a protein to be used in a protein-protein interaction study. Most proteins can be stored for short periods of time at 4°C. In general, freeze-thaw cycles should be avoided. For the assays described below, it is important to run a SDS-polyacrylamide gel to check the integrity of the protein after prolonged storage. |
| Troubleshooting |
| Low Yield. If the yield is low at the end of the purification, repeat protein purification and compare the levels of the fusion protein present at the different stages of preparation. Remove aliquots from the preparation at the steps detailed below. Combine the samples with SDS sample buffer and analyze them on a SDS-polyacrylamide gel (stain with Coomassie blue). Volumes are based on a starting volume of a liter of culture; adjust accordingly. - 15 µl of uninduced culture from step 3 (prior to addition of IPTG) - 15 µl of induced culture from step 5 - 0.2% of supernatant at step 9 (total cell lysate): 40 µl - 0.2% of supernatant at step 11 (soluble lysate): 40 µl - 0.2% of supernatant at step 14 (lysate after incubation with beads): 40 µl - 0.2% of aliquot of an eluate fraction (step 26): 1 µl - 2% of aliquot of an eluate fraction (step 26): 10 µl The GST moiety adds ~26 kD molecular mass. If protein degradation is occurring, the MW of the majority recovered species may be significantly less than the predicted MW; run the gel accordingly. Results from this gel will show: The yield, and the integrity of the protein (aliquots from step 26). Whether the protein was induced (compare uninduced and induced cultures, aliquots from step 3 and step 5). Whether the protein is soluble (compare aliquots from step 9 and step 11). Whether a substantial portion of the fusion protein bound to the beads (compare aliquots from step 14 and step 11). Failure to Induce Protein Expression. To determine whether the induction conditions are working, prepare a culture of GST in parallel with the GST fusion protein. If GST is produced, but the fusion protein is not, optimization of induction of the fusion protein is necessary. Growth conditions that can be varied to address this problem include titrating the amount of IPTG added, altering the OD600 at which the IPTG is added, lowering the temperature of bacterial growth, and inducing for longer or shorter times. Degradation of the Protein. The problem of protein degradation can be addressed by using protease-deficient (lon-) bacterial strains. There are many commercially available bacterial strains designed for protein expression with appropriate genetic backgrounds to minimize protein degradation (e.g., BL21). Degradation can also result from oversonication. Determine the optimal sonication time for the protein of interest by performing small-batch preparations and testing different sonication times. Insolubility. Insolubility can be ameliorated by inducing expression at lower temperatures (30°C or less) and for longer times. The use of different detergents during lysis can also help (an excellent reference for this problem is Frangoni and Neel 1993). Another way to avoid this difficulty is to isolate the insoluble protein and subsequently refold it (Volkel et al. 1998; Cox et al. 1999), but this is not always possible. It may be that the fusion protein contains domains that promote insolubility or aggregation (lipid-binding domains, for example). Altering the fusion protein, where possible, to exclude these domains can improve solubility. However, if the protein cannot be modified, the solution may be to transfer the protein of interest to a His-tagged vector (which allows affinity purification of denatured proteins). Failure to Bind the Glutathione-Sepharose. Excessive sonication can often interfere with glutathione-Sepharose binding. To address this possibility, decrease the time and intensity of sonication. Addition of DTT, to a final concentration of 5 mM, prior to lysis can also increase the binding of some fusion proteins. |
| REFERENCES |
| Cox, G.N., Pratt D., McDermott M.J. and van der Slice R.W. 1999. Refolding and characterization of recombinant human soluble CTLA-4 expressed in E. coli. Protein Expr. Purif . 17 : 26-32. Frangoni J.V. and Neel B.G. 1993. Solubilization and purification of enzymatically active glutathione-S- transferase (GEX) fusion proteins. Anal. Biochem . 210 : 179-187. Sambrook J. and Russell D.W. 2001. Molecular Cloning: A Laboratory Manual Third Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York Volkel D., Blankenfeldt W., and Schomburg D. 1998. Large-scale production, purification and refolding of the full-length cellular prion protein from Syrian golden hamster in E. coli using glutathione S- transferase-fusion system. Eur. J. Biochem . 251 : 462-471. |
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| Anyone using the procedures in this protocol does so at their own risk. Cold Spring Harbor Laboratory makes no representations or warranties with respect to the material set forth in this protocol and has no liability in connection with the use of these materials. Materials used in this protocol may be considered hazardous and should be used with caution. For a full listing of cautions regarding these material, please consult: Protein: Protein Interactions, Second Edition: A Molecular Cloning Manual , edited by Erica A. Golemis and Peter D. Adams, © 2005 by Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, p. 89. |
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