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.

  1. If necessary, concentrate the fusion protein to at least 0.5 mg/ml.

  2. Perform a pilot experiment with a small portion of your protein. For example:

    1. Combine 15 μg fusion protein and H2O to a volume of 45 μl.

    2. Add 5 μl of TEV Protease Reaction Buffer (10X) to make a 50 μl total reaction volume.

    3. Add 1 μl of TEV Protease.

    4. 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).

    5. 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).

    6. 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).

    7. Incubate the SDS-PAGE samples for 3-5 minutes at 70-100°C and analyze them by SDS-PAGE.

  3. 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.

  4. Check for complete cleavage by SDS-PAGE.

  5. 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.