Prepare Your Sample

Express Your GST-tagged Protein: Ensure the GST-tagged protein is expressed and purified using a standard GST purification method (such as using glutathione-affinity chromatography).

Concentrate Your Sample: After purification, concentrate the protein solution if necessary, and dialyze to remove excess salt and other contaminants that may inhibit thrombin activity.

Optimize Thrombin Cleavage Conditions

Thrombin Addition: Add thrombin to your protein solution. A typical thrombin concentration is around 1–2 units per mg of protein. You can use more thrombin if necessary, but the quantity will depend on the size of your protein and the desired cleavage efficiency.

Buffer Conditions: The thrombin cleavage is usually done in a buffer with a composition like:

50 mM Tris-HCl (pH 7.5–8.0) (optimal pH for thrombin activity)

150mM NaCl

1–2 mM CaCl₂ (necessary for thrombin activity, but avoid excessive calcium that might interfere with downstream steps)

Optional: A small percentage of glycerol (~10%) may be used to help stabilize the protein.

Incubation: Incubate the mixture at room temperature or 4°C for several hours, typically 4-16 hours. You may need to test cleavage efficiency by sampling the mixture over time.

Cleavage Monitoring

SDS-PAGE: Run a small aliquot on an SDS-PAGE gel to monitor the cleavage progress. You should observe the cleavage of the GST tag (approximately 26 kDa) from your target protein. After cleavage, the GST tag should appear as a separate band, and your protein will be in its native form.

Post-Cleavage Purification

Removal of Thrombin and GST tag: To separate the cleaved GST tag and thrombin from your target protein, use a second round of affinity chromatography.

GST-Removal: Use glutathione-agarose beads to bind the cleaved GST-tag and remove it from the target protein.

Thrombin Removal: You can remove thrombin by using an additional affinity column or heparin columns, as thrombin binds heparin.

Alternatively, if thrombin has high specificity and doesn't interact significantly with your protein, size-exclusion chromatography (SEC) or ion exchange chromatography can be used to separate the thrombin and any residual GST tag from your protein.

Final Purification and Activity Check

Check Protein Activity: After cleavage and purification, assess the activity of your target protein. Depending on the protein’s properties, you may need to optimize the storage and handling conditions to avoid denaturation.

Concentration: If necessary, concentrate your protein using an appropriate concentration method (e.g., centrifugal filter units) without compromising the protein’s activity.

Thrombin inhibitors: If you need to stop thrombin cleavage at a specific time point, you can add a thrombin inhibitor (e.g., PPACK) to halt the cleavage reaction.

Thrombin specificity: Ensure that thrombin recognition sites (e.g., Leu-Val-Pro-Arg-Gly-Ser) are properly exposed in your fusion protein. If not, thrombin may not cleave efficiently.