Cleavage and Purification of GST-Tagged Protein Bound to Glutathione Sepharose in Batch Mode

Glutathione Sepharose High Performance, Glutathione Sepharose 4 Fast Flow, and Glutathione Sepharose 4B can all be used for cleavage and purification of GST-tagged proteins in batch.

Recommended Buffers

Binding buffer:	PBS: 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3
For PreScission Protease cleavage:
Elution buffer:  Cleavage buffer: PreScission Protease	50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, pH 7.0
For thrombin cleavage:
Elution buffer:  Cleavage buffer: Thrombin solution:	50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0 PBS: 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4,pH 7.3 Dissolve 500 units in 0.5 ml of PBS prechilled to 4 °C. Swirl gently. Store solution in small aliquots at -80 °C to preserve activity.
For Factor Xa cleavage:
Elution buffer:  Cleavage buffer: Factor Xa solution:	50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0 50 mM Tris-HCl, 150 mM NaCl, 1 mM CaCl2, pH 7.5 Dissolve 400 units of Factor Xa in 4 °C water to give a final solution of 1 unit/µL. Swirl gently. Store solution in small aliquots at -80 °C to preserve activity.

Preparation of Glutathione Sepharose media and binding of protein

Glutathione Sepharose media are supplied in 20% ethanol. The media are used at a final slurry concentration of 50%.

1. Determine the bed volume of Glutathione Sepharose required for your purification.
   
   
2. Gently shake the bottle to resuspend the slurry.
   
   
3. Use a pipette or measuring cylinder to remove sufficient slurry for use and transfer to an appropriate container/tube.
   
   
4. Sediment the chromatography medium by centrifugation at 500 × g for 5 min. Carefully decant the supernatant.
   
   
5. Wash the Glutathione Sepharose by adding 5 ml of PBS per 1 mL of 50% slurry.

Glutathione Sepharose must be thoroughly washed with PBS to remove the ethanol storage solution because residual ethanol may interfere with subsequent procedures.

6. Sediment the chromatography medium by centrifugation at 500 × g for 5 min. Carefully decant the supernatant.

7. Repeat steps 5 and 6 once for a total of two washes. Add the cell lysate to the prepared Glutathione Sepharose and incubate for at least 30 min at room temperature, using gentle agitation such as end-over-end rotation.

Purification and Cleavage

Assume 8 Mg of GST-tagged protein bound per mL of chromatography medium.

1. Wash the tagged-protein-bound Glutathione Sepharose with 10 bed volumes of cleavage buffer. Bed volume is equal to 0.5× the volume of the 50% Glutathione Sepharose slurry used.
   
   
2. (a) Prepare the PreScission Protease mix:
   For each mL of Glutathione Sepharose bed volume, prepare a mixture of 80 µL (160 units) of PreScission Protease and 920 µL of cleavage buffer at 5 °C.
   (b) Prepare the thrombin mix:
   For each mL of Glutathione Sepharose bed volume, prepare a mixture of 80 µL (80 units) of thrombin and 920 µL of cleavage buffer.
   (c) Prepare the Factor Xa mix:
   For each mL of Glutathione Sepharose bed volume, prepare a mixture of 80 µL (80 units) of Factor Xa and 920 µL of cleavage buffer.
   
   
3. Add the mixture to the Glutathione Sepharose. Gently shake or rotate the suspension end-over-end.
   
   
4. (a) For PreScission Protease, incubate at 5 °C for 4 h.
   (b) For thrombin or Factor Xa, incubate at room temperature (22 °C to 25 °C) for 2 to 16 h.

The incubation times in steps 4a and 4b are starting points and may need to be changed for an optimal yield of cleaved target protein.

5. Following incubation, wash out the untagged protein with approximately three bed volumes of cleavage buffer. Centrifuge the suspension at 500 × g for 5 min to pellet the Glutathione Sepharose. Carefully transfer the eluate to a tube.

For PreScission Protease: The eluate will contain the protein of interest, while the GST moiety of the tagged protein and the PreScission Protease will remain bound to the Glutathione Sepharose. This means that the protein of interest will not be contaminated with protease and thus no additional purification will be required to purify the target protein from the protease.

For thrombin and Factor Xa: The eluate will contain the protein of interest and thrombin or Factor Xa, respectively, while the GST moiety of the tagged protein will remain bound to the Glutathione Sepharose. The thrombin or Factor Xa can be removed from the protein of interest using HiTrap Benzamidine FF (high sub). This column captures the thrombin or Factor Xa, thus enabling the collection of pure protease-free protein in the eluent.