Saccharomyces cerevisiae-derived Small ubiquitin-like modifier (SUMO, Smt3) is commonly used as a protein fusion domain to facilitate expression and purification of recombinant proteins, and a Saccharomyces cerevisiae-derived SUMO-specific protease(Ulp1) is then used to remove SUMO tag from these proteins in a ‘scarless’ manner. SUMO Protease cleaves in a highly specific manner, recognizing the tertiary structure of the SUMO tag, rather than an amino acid sequence, and hydrolyzes the peptide bond in the x-Gly-Gly-x sequence after the Gly-Gly bond at the C-terminus of the SUMO tag. The SUMO Protease cleavage proteins over wide ranges of temperature (4℃-30℃), ionic strengths(0-400 mM NaCl) and pH(7.0-9.0), and easily removed from the cleavage reaction by Immobilized Metal Affinity chromatography (IMAC).
1. SUMO Protease;
2. 10X SUMO Protease Buffer + Salt: 500 mM PB, pH 7.4,
2% Igepal (NP-40), 1.5 M NaCl, 10 mM DTT;
3. 10X SUMO Protease Buffer – Salt: 500 mM PB, pH 7.4,
2% Igepal (NP-40), 10 mM DTT;
1.Add the following to a microcentrifuge tube:
Fusion Protein | 20μg |
10X SUMO Protease Buffer +/– Salt
| 5μl |
SUMO
Protease
| 10U |
ddH2O
| To 50μl |
2. Mix and incubate at 30°C, Remove 5μl aliquots at
1, 2, 4, and 6 hours.
3. Analyze by SDS-PAGE.
- Keep the concentration of Imidazole less then 150mM, or the activity of
the SUMO Protease can be adversely affected.
- For most fusion proteins, SUMO Protease functions
optimally in a reaction mixture containing 150 mM NaCl; however, conditions may
be optimized by varying the NaCl concentration from 100 mM to 300 mM. Remember
to take into account the contribution of salt from the enzyme and from your
substrate. When setting up your cleavage reaction, use the appropriate 10X SUMO
Protease Buffer +/- Salt.
- Researchers need to optimize their specific
reaction conditions. As an initial suggestion, 20 units of SUMO protease can be
used per 40μg of target protein for 1 hour at 30 °C, or overnight at 2–8 °C.
The cleavage efficiency can then be estimated by SDS-PAGE.
One unit of SUMO Protease cleaves ≥85% of 2 μg control substrate in 1 h at 30°C.
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