Cleavage of the Fusion Protein
MBP and the target protein are fused by a polylinker containing a TEV protease recognition site for easy removal of the MBP-tag. One unit of TEV Protease will cleave approximately 2 μg of fusion protein. Cleavage should be carried out in 1X TEV Protease Reaction Buffer or in Amylose column elution buffer supplemented with DTT to a final concentration of 1 mM. Depending on the particular fusion protein, the amount of TEV Protease can be adjusted to get an acceptable rate of cleavage.
- If necessary, concentrate the fusion protein to at least 0.5 mg/ml.
- Perform a pilot experiment with a small portion of your protein. For example:
- Combine 15 μg fusion protein and H2O to a volume of 45 μl.
- Add 5 μl of TEV Protease Reaction Buffer (10X) to make a 50 μl total reaction volume.
- Add 1 μl of TEV Protease.
- In a separate tube, combine 5 μg fusion protein, 5 μl TEV Protease Reaction Buffer (10X) and H2O to a volume of 50 μl. Do not add TEV Protease (control sample).
- Incubate reaction and control sample for 1, 3, and 8 hours at 30°C (an additional reaction can be made and incubated for 24 hours at 4°C).
- Take 10 μl of the reaction(s) at the indicated times above and add 5 μl SDS-PAGE Sample Buffer (3X). Take 10 μl of the control sample and add 5 μl SDS-PAGE Sample Buffer (3X) after 8 hours (or longest incubation time).
- Incubate the SDS-PAGE samples for 3-5 minutes at 70-100°C and analyze them by SDS-PAGE.
- Scale up the pilot experiment linearly for the amount of the fusion protein to be cleaved. Save at least a small sample of the uncut fusion as a negative control.
- Check for complete cleavage by SDS-PAGE.
- TEV Protease and the cleaved MBP contain polyhistidine tags at their N-termini. They can be removed from the cleavage reaction by immobilized metal affinity chromatography, such as Nickel or Cobalt resin, thereby isolating the target protein in the flow through.