Expression and Purification of GST-tagged Proteins from E. coli   

Abstract

Materials and Reagents

1. Escherichia coli (E. coli) strain of choice
   
2. IPTG (Sigma-Aldrich)
   
3. Imidazaole (Sigma-Aldrich)
   
4. Protease inhibitor (Roche Diagnostics)
   
5. Lysozyme (Sigma-Aldrich)
   
6. Glutothion sepharose 4B beads (Thermo Fisher Scientific/Pierce Antibodies, catalog number: 15160 )
   
7. Glutathione (Thermo Fisher Scientific/Pierce Antibodies, catalog number: 78259 )
   
8. NP-40 (Sigma-Aldrich)
   
9. Bacto trypton
   
10. Yeast extract
    
11. Liquid nitrogen
    
12. NaCl
    
13. KCl
    
14. Na₂HPO₄
    
15. KH₂PO₄
    
16. Protease inhibitor
    
17. Lysis buffer (see Recipes)
    
18. 1x PBS buffer (see Recipes)
    
19. Elution buffer (see Recipes)
    
20. 2x YT medium (see Recipes)
    

Equipment

1. Sonicator
   
2. Centrifuges
   
3. Pellete
   
4. Water bath
   

Procedure

1. Small scale expression and purification test
   1. Grow 5 ml E. coli culture harboring the expression construct in 2x YT medium overnight (12-16 h) at 37 °C.
      
   2. The next day, 1: 20 dilute the overnight E. coli culture into fresh 2x YT medium. Grow at 37 °C to OD₆₀₀ of 0.6-0.8. Depending on E.coli strain and status, the time should be around 2-3 h.
      
   3. Induce the culture with 0.1 mM IPTG. Take 10 ml culture out at 0 h, 1 h, 2 h, 4 h and overnight, spin down and flash freeze in liquid nitrogen, then stored in -80 °C.
      
   4. Once all the samples are collected, take the pellete, resuspend in 1.5 ml lysis buffer. Incubate on ice for 30 min.
      
   5. Sonicate each sample on ice for 15 sec for twice.
      
   6. Centrifuge the lysate at top speed at 4 °C for 30 min. Separate the supernatant and pelletes. Resuspend the pelletes in 1.5 ml lysis buffer. Take 10 μl from supernatant and pellete suspension for sampling.
      
   7. Add the 10 L glutothion sepharose 4B beads, incubate at 4 °C for 1 h and wash with 200 μl 1x PBS three times. Collect the supernatant after spinning down the beads for sampling.
      
   8. Elute the beads with 10 μl elution buffer.
      
   9. Run the samples from each step in appropriate SDS-PAGE gel to determine the expression and purification condition, and protein expression amount.
      Note: If there is solubility issue, try decrease the IPTG concentration and expression temperature.
      
   
   
2. Large scale expression and purification
   1. Grow 50 ml E.coli overnight culture at 37 °C.
      
   2. Dilute the over night culture to 1 L culture and grow at 37 °C till OD₆₀₀=0.4-0.8.
      
   3. Induce with 0.1 mM IPTG and grow at 37 °C for appropriate amount of time.
      
   4. Harvest the cells at 4,000 x g for 10 min at 4 °C. Flash freeze the pellete in liquid N₂ and store at -80 °C.
      
   5. Thaw the cells in room temperature (RT) water bath. Put in ice and resuspend in 5-7x pellete volume of lysis buffer supplemented with protease inhibitor and lysozyme (See small scale expression and purification test). Incubate on ice for 30 min.
      
   6. Sonicate on ice for 15 sec for 3 times, until the viscosity decrease. Be careful not let the lysate temperature goes beyond 10 °C. Save 10 μl for sample.
      
   7. Centrifuge the lysate at 40,000 x g for 30 min at 4 °C. Discard the pellete. Save samples from supernatant and pelletes.
      
   8. According to the estimated GST protein amount from small scale expression and purification test and binding capacity of glutothione beads in the manual, add appropriate amount of beads. To ensure purity, the amount of express proteins should be in excess of beads binding capacity.
      
   9. Add 1% NP-40 to reduce the unspecific binding. Incubate at 4 °C for 30 min.
      
   10. Transfer the slurry into a disposable column and collect the flow through.
       
   11. Wash the beads with 20x beads volume PBS. Collect the flow through.
       
   12. Add 1x beads volume of elution buffer. Collect the elution.
       
   13. Repeat step 12 for 5 times.
       
   14. After elution, take small amount of beads, add SDS loading buffer for sample.
       
   15. Run the samples collected from lysate, pellete, supernatant, flow through, wash, elute and beads in SDS-PAGE gel.
       
   16. Collect the elution containing your GST-proteins and dialysis against PBS at 4 °C for at least 1 h for three times.
       
   17. Flash freeze at liquid nitrogen and store at -80 °C.
       

Recipes

1. 2x YT medium

   Bacto trypton	16 g
   Yeast extract	5 g
   NaCl	5 g
   H2O	800 ml

   Adjust pH to 7.2
   Add H₂O to 1 L
   Autoclaved
   
2. PBS buffer (pH 7.4) (1 L)

   NaCl	8 g
   KCl	0.2 g
   Na2HPO4	1.44 g
   KH2PO4	0.24 g
   H2O	800 ml

   Adjust pH to 7.4.
   Adjust volume to 1 L with additional distilled H₂O
   Sterilize by autoclaving
   
3. Lysis buffer

   50 mM	NaH2PO4 (pH 8.0)
   300 mM	NaCl
   10 mM	Imidzaole,

   1x Protease inhibitor
   1 mg ml^-1 lysozyme
   
4. Elution buffer

   20 mM	glutathione
   75 mM	Tris (pH 8.0)
   150 mM	NaCl

Acknowledgments

This protocol was adapted from Reference 1. Funding from the NIH supported this work.

References

1. Pierce Glutathione Agarose Instructions booklet: http://www.piercenet.com/instructions/2162248.pdf.