Protocol

Preparation of GST Fusion Proteins

This protocol was adapted from "Identification of Protein-Protein Interactions with Glutathione-S-Transferase Fusion Proteins," Chapter 6, in Protein-Protein Interactions, 2nd edition (eds. Golemis and Adams). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2005.

INTRODUCTION

This protocol describes the preparation of glutathione-S-transferase (GST) fusion proteins, which have had a wide range of applications since their introduction as tools for synthesis of recombinant proteins in bacteria. GST was originally selected as a fusion moiety because of several desirable properties. First and foremost, when expressed in bacteria alone, or as a fusion, GST is not sequestered in inclusion bodies (in contrast to previous fusion protein systems). Second, GST can be affinity-purified without denaturation because it binds to immobilized glutathione, which provides the basis for simple purification. Consequently, GST fusion proteins are routinely used for antibody generation and purification, protein-protein interaction studies, and biochemical analysis.

RELATED INFORMATION

An excellent reference for affinity purification of GST fusion proteins is a booklet available from the GE Healthcare Life Sciences Web site (http://www4.gelifesciences.com).

MATERIALS

This protocol also requires equipment and reagents for SDS-Polyacrylamide Gel Electrophoresis of Proteins and Staining Proteins in Gels with Coomassie Blue (see Step 26).

Reagents

Bacterial strain transformed with GST and GST fusion expression plasmids

This protocol is designed for IPTG-inducible bacterial expression vectors. Although variations of GST fusion protein expression vectors are available, the most commonly used versions (available from Amersham Pharmacia) 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 (Amersham, Promega) have been commonly used for expression of recombinant proteins.

Glutathione-Sepharose beads

Isopropyl-ß-D-thio-galactoside (IPTG)

LB broth, containing appropriate antibiotic selection (see Steps 1 and 2)

PBS lysis buffer, freshly prepared

PBS for GST fusion protein preparation, ice cold

PBS with protease inhibitors, freshly prepared

Tris-Cl (50 mM, pH 8.0) containing 20 mM reduced glutathione (Sigma, Amersham)

Equipment

Centrifuge, precooled to 4°C (for centrifuging bacterial cultures; see Steps 6 and beyond)

Chromatography column, disposable (Econo-Column; Bio-Rad)

Concentration buffer exchange unit with low-molecular-weight (MW) cutoff (e.g., Centricon) (optional; see Step 24)

Ice

Incubator (shaking), preset to 37°C

Microcentrifuge and microcentrifuge tubes

Rotator for end-over-end mixing

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 PBS lysis buffer and stored as a 50:50 (v/v) slurry at 4°C.

Sonicator

Spectrophotometer

Tubes and flasks for culturing bacteria (see Steps 1-2)

METHOD

1. 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.

2. Inoculate 1 liter of LB containing the antibiotic selection with the 5-ml overnight culture from Step 1.

3. Grow the cultures at 37°C with shaking to an OD₆₀₀ of 0.5-1.0 (this should take 3-6 hr).

4. Induce expression of the protein by adding IPTG to a final concentration of 0.1 mM.
See Troubleshooting.

5. Incubate the cultures for an additional 3 hours at 37°C with shaking.

6. Centrifuge the bacterial culture at 3500g for 20 minutes at 4°C.

7. Discard the supernatant.
At this point, pellets can be stored frozen at -20°C if necessary.

8. Resuspend the pellet in 20 ml of PBS lysis buffer.

9. Sonicate the bacterial suspension on ice, in short 10-second bursts alternated with 10 seconds of resting on ice. Three cycles of sonication are usually sufficient.
See Troubleshooting.

10. Centrifuge the lysate at 12,000g for 15 minutes at 4°C.

11. Transfer the supernatant to a fresh tube.

12. Add 5 ml of a 50:50 slurry of glutathione-Sepharose beads in PBS lysis buffer.

13. Incubate for 30 minutes at 4°C, rotating the tube end over end to ensure mixing.

14. Centrifuge the samples at 750g for 1 minute at 4°C to pellet the beads. Remove the supernatant.

15. Wash the beads in 5 ml of ice-cold PBS with protease inhibitors.

16. Centrifuge the samples at 750g for 1 minute at 4°C to pellet the beads. Remove the supernatant.

17. Add 5 ml of ice-cold PBS with protease inhibitors. Resuspend the beads by gentle mixing.

18. Centrifuge the samples again at 750g for 1 minute 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.

19. Add 5 ml of ice-cold PBS with protease inhibitors. Resuspend the beads with gentle mixing.

20. Pour the slurry into a disposable chromatography column.

21. Allow the PBS to run out of the column. Wash with 5 ml of ice-cold PBS with protease inhibitors.

22. While the column is flowing, prepare a rack of 10 microcentrifuge tubes labeled 1-10.

23. Elute the fusion protein by adding 5 ml of cold (0°C-4°C) 50 mM Tris-Cl (pH 8.0) containing 20 mM reduced glutathione.

24. Collect ~0.5-ml fractions of the eluate in each microcentrifuge tube.
The eluate may be stored at 4°C. 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 used. Alternatively, a commercially prepared concentration buffer exchange unit (e.g., from Centricon, with a low-MW cutoff) can be used for buffer exchange.

25. 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.

26. Run the samples from the eluates containing protein on an SDS-polyacrylamide gel (see SDS-Polyacrylamide Gel Electrophoresis of Proteins), and stain with Coomassie blue dye (see Staining Proteins in Gels with Coomassie Blue).
The GST moiety is 26 kDa; therefore, add 26 kDa to determine the predicted molecular weight (MW) of the fusion protein.

27. Store the recombinant protein.
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. After prolonged storage, it is important to run an SDS-polyacrylamide gel to check the integrity of the protein.

TROUBLESHOOTING

Problem: Low yield.

[Step 26]

Solution: If low yield is detected 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, analyze them on an SDS-polyacrylamide gel, and 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 24): 1 µl

2% of aliquot of an eluate fraction (Step 24): 10 µl

The GST moiety adds ~26 kDa to the 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 24).
  
  
• Whether the protein was induced (cf. uninduced and induced cultures, aliquots from Step 3 and Step 5).
  
  
• Whether the protein is soluble (cf. aliquots from Step 9 and Step 11).
  
  
• Whether a substantial portion of the fusion protein bound to the beads (cf. aliquots from Step 14 and Step 11).
  
  

Problem: Failure to induce protein expression.

Solution: 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 the following:

1. Titrate the amount of IPTG added. Different proteins are optimally produced using different concentrations of IPTG. If protein expression is problematic, first titrate the amount of IPTG added in Step 4 to determine the optimal conditions for protein induction.

2. Alter the OD₆₀₀ at which the IPTG is added.

3. Lower the temperature of bacterial growth, and induce for longer or shorter times.

Problem: Degradation of the protein.

Solution: 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 and denaturation 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 during Step 9.

Problem: Contamination with bacterial host proteins.

[Step 26]

Solution: Oversonication can 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 this problem occurs in the preparation of the protein of interest, titrate the time of sonication required to release the protein in Step 9.

Problem: Insolubility.

Solution: 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 (e.g., lipid-binding domains). 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).

Problem: Failure to bind the glutathione-Sepharose.

Solution: 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., Smith, D., McDermott, M.J., and van der Slice, R.W. 1999. Refolding and characterization of recombinant human soluble CTLA-4 expressed in Escherichia coli. Protein Expr. Purif 17: 26–32.[Medline]

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.[Medline]

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 Escherichia coli using glutathione-S-transferase-fusion system. Eur. J. Biochem 251: 462–471.[Medline]