Purification and Cleavage
The protocol below is an example optimized for 8 mg of target protein. It is worth estimating how much target protein is applied to the column, as this allows one to minimize the amount of protease added.
- Fill the syringe or pump tubing with distilled water. Remove the stopper and connect the column to the syringe (use the connector supplied), laboratory pump, or chromatography system “drop to drop” to avoid introducing air into the system.
- Remove the snap-off end at the column outlet.
- Wash out the ethanol with 3 to 5 column volumes of distilled water.
- Equilibrate the column with at least 5 column volumes of binding buffer. Recommended flow rates are 1 ml/min (1 ml column) and 5 ml/min (5 ml column).
- Apply the pretreated sample using a syringe fitted to the Luer connector or by pumping it onto the column. For best results, use a flow rate of 0.2 to 1 ml/min (1 ml column) and 0.5 to 5 ml/min (5 ml column) during sample application.
- Wash with binding buffer (generally at least 5 to 10 column volumes) until the absorbance reaches a steady baseline or no material remains in the effluent. Maintain a flow rate of 1 to 2 ml/min (1 ml column) and 5 to 10 ml/min (5 ml column) for washing.
- Elute the GST-tagged protein with 5 to 10 column volumes of elution buffer. Maintain flow rates of 1 to 2 ml/min (1 ml column) or 1 to 5 ml/min (5 ml column). Collect the eluate (0.5 to 1 ml/tube for 1 ml column, 1 to 2 ml/tube for 5 ml column). Pool fractions containing the GST-tagged protein (monitored by UV absorption at A280).
- Remove the free reduced glutathione from the eluate using a quick buffer exchange on a desalting column, depending on the sample volume.
- (a) For PreScission Protease, add 1 µl (2 units) of PreScission Protease for each 100 µg of tagged protein in the buffer-exchanged eluate.
(b) For thrombin and Factor Xa, add 10 µl (10 units) of thrombin or Factor Xa solution for each mg of tagged protein in the buffer-exchanged eluate.
- (a) For PreScission Protease, incubate at 5 °C for 4 h.
(b) For thrombin and Factor Xa, incubate at room temperature (22 °C to 25 °C) for 2 to 16 h.
The incubation times are starting points and may need to be changed for an optimal yield of cleaved target protein.
- Once digestion is complete, apply the sample to an equilibrated GSTrap column as described above (steps 1 to 6) to remove the GST moiety of the tagged protein.
For PreScission Protease: The flowthrough 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 column. 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 flowthrough 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 column. The thrombin or Factor Xa can be removed from the protein of interest in one step using a HiTrap Benzamidine FF (high sub) column in series after a GSTrap column. In this process, the cleaved, tagged protein and thrombin or Factor Xa is washed from the GSTrap column onto the HiTrap Benzamidine FF (high sub) column. This second column captures the thrombin or Factor Xa, thus enabling the collection of pure protease-free protein in the eluent. If GST GraviTrap is used, the eluted fraction is loaded with a syringe onto the HiTrap Benzamidine FF (high sub) column.