GST-tagged Yeast Protein Purification   

Abstract

Materials and Reagents

1. Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) (Sigma-Aldrich, catalog number: E0396 )
   
2. Phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626 )
   
3. HEPES (Sigma-Aldrich, catalog number: 54457 )
   
4. Roche protease inhibitor tablets (containing EDTA) (Roche Diagnostics, catalog number: 11697498001 )
   
5. Phosphatase inhibitor (Roche Diagnostics, catalog number: 4906845001 )
   
6. Glutathione (Axxora, catalog number: 157-002-G005 )
   
7. Galactose (Mp Biomedicals, catalog number: 0210174701 )
   
8. Zirconia/Silica beads (Biospec Products, catalog number: 11079105z )
   
9. Glutathione beads (Thermo Fisher Scientific, catalog number: 16100 )
   
10. Tris
    
11. NaCl
    
12. TritonX-100
    
13. Glycerol
    
14. Beta-mercaptoethanol (BME)
    
15. Sc-ura liquid media with raffinose
    
16. Lysis buffer (see Recipes)
    
17. Wash buffer I (see Recipes)
    
18. Wash buffer II (see Recipes)
    
19. Elution buffer (see Recipes)
    

Equipment

1. Table top centrifuge
   
2. Bead beater
   
3. 50 ml Falcon tubes
   

Procedure

1. Strains are grown on sc-ura plates.
   
   1. Start a 5 ml starter culture of sc-ura or sc-ura/raffinose (I prefer to just do everything in autoclaved sc-ura/raffinose) and grow O/N or 16 h at 30 °C.
      
   2. Measure OD₆₀₀ of starter culture and dilute cultures into 50 ml of fresh sc-ura/raffinose to an OD that allows at least 2 cell divisions. I typically inoculate to an OD of 0.1 and allow to grow to an OD 0.6-0.8 before inducing (typically requires roughly 3 h per doubling time for these strains).
      
   3. Induce with 20% galactose when cells at OD 0.6-0.8 to a final galactose concentration of 2%.
      
   4. Induce for 4-5 h.
      
   5. Spin down cells in 50 ml Falcon tubes at 3,000 rpm, wash pellet with water (keep cells on ice), transfer to 2 ml eppendorfs, wash with cold lysis buffer, spin and remove buffer. Freeze cell pellets at -80 °C.
   
   
2. Protein pufication (everything on ice and done in cold room):
   
   1. Add ~ 400 μl of 0.5 mm Zirconia/Silica beads and 450 μl of lysis buffer containing protease and phosphatase inhibitors to cell pellet.
      
   2. Vortex the cells for 45 sec intervals with 2 min on ice for a total of 5 times.
      
   3. Spin tubes at 5,000 rpm for 4 min.
      
   4. Transfer lysate to fresh tubes on ice.
      
   5. Add another 450 μl lysis buffer containing protease and phosphatase inhibitors to cell pellet.
      
   6. Vortex again for another 5 times as above.
      
   7. Spin down the lysate and transfer to the previous lysate.
      
   8. Spin the combined lysate at 14,000 rpm for 10 min to clear lysate.
      
   9. Transfer lysate to fresh tubes being careful not to disturb cell junk at bottom.
      Note: At this point you can either do the binding on the glutathione beads in eppendorfs or in 15 ml Falcon tubes. Some claim that doing the binding step in a larger volume (by adding fresh lysis buffer to the lysate) helps the binding process by diluting inhibitors in the lysate that inhibit the binding process.
      
   10. Add 35 μl of glutathione beads (beads washed 4 times with lysis buffer and then diluted in lysis buffer to aid in transfer of beads uniformly to samples).
       
   11. Rock for 1 h in cold room.
       
   12. Wash beads 3 times with 400 μl of wash buffer I.
       
   13. Wash beads 3 times with 400 μl of wash buffer II.
       
   14. Elute proteins with 50 μl of elution buffer (rock for 30 min). Repeat for total elution of 100 μl.
       
   15. Aliquot elutions and freeze -80 °C.

Recipes

1. Lysis buffer
   

   50 mM	Tris (pH 7.5)
   100 mM	NaCl
   1 mM	EGTA
   0.1%	TritonX-100
   0.1%	BME
   0.5 mM	PMSF

   Roche protease inhibitor tablets (containing EDTA)
   BME, PMSF, and inhibitor tablets are added freshly.
   
2. Wash buffer I
   Exactly the same as lysis buffer except 500 mM NaCl
   
3. Wash buffer II or your preferred kinase buffer
   

   50 mM	HEPES (pH 7.5)
   100 mM	NaCl
   10%	glycerol

4. Elution Buffer or your preferred kinase buffer with glycerol and glutathione:
   

   50 mM	HEPES (pH 7.5)
   100 mM	NaCl
   20%	glycerol
   20 mM	glutathione (reduced form)

   Make sure the pH is around 7.5.
   

References

1. Poon, R. Y. and Hunt, T. (1994). Reversible immunoprecipitation using histidine- or glutathione S-transferase-tagged staphylococcal protein A. Anal Biochem 218(1): 26-33.
   
2. Zhu, H., Bilgin, M., Bangham, R., Hall, D., Casamayor, A., Bertone, P., Lan, N., Jansen, R., Bidlingmaier, S., Houfek, T., Mitchell, T., Miller, P., Dean, R. A., Gerstein, M. and Snyder, M. (2001). Global analysis of protein activities using proteome chips. Science 293(5537): 2101-2105.
   
3. Zhu, H., Klemic, J. F., Chang, S., Bertone, P., Casamayor, A., Klemic, K. G., Smith, D., Gerstein, M., Reed, M. A. and Snyder, M. (2000). Analysis of yeast protein kinases using protein chips. Nat Genet 26(3): 283-289.