SENP2 is an enzyme that belongs to the protease family C48. Structurally, SENP2 harbors the C48 catalytic domain which is typically located close to the C terminus and has been reported to engage two SUMO pathways. The first is cleavage processing of small ubiquitin-like modifiers (SUMO1, SUMO2, and SUMO3) propeptides, subsequently leading to protein maturation. The second is the cleavage processing of SUMO1, SUMO2, and SUMO3 from targeted proteins. SENP2 protease has a His-tag for easy removal from a cleavage reaction by using nickel affinity resins.
1. To optimize cleavage conditions, it is recommended to run preliminary cleavage reactions at a small scale.
2. Dilute the target protein sample to 1-2 mg/mL with PBS solution.
3. An effective general range of the SENP2 protease: target protein ratio is 1 µg :50 µg.
4. Reaction can be performed at 4°C-37°C. 4°C is recommended as the starting standard. Incubate the reaction mixture at 4°C for 16 hours.
5. Determine cleavage level of the samples by SDS-PAGE analysis.
6. Once optimize for the cleavage condition, the cleavage reactions can be scaled up to cleave a large amount of the target fusion protein.
- SENP2 protease: target protein ratio of 1 µg :50 µg is used for most fusion protein cleavage. Cleavage efficiency may differ based on structure and properties of each target protein, we recommend testing several enzyme-to-substrate ratios, temperatures, and incubation times.
- We recommend performing longer cleavage time at lower temperatures (4°C) for cleavage efficiency.