Hello, how to remove the SUMO tag after purification?
The SUMO tag is a commonly used fusion protein tag that can be specifically removed by specific enzymes (such as SENP1 or SENP2), resulting in the target protein without the tag. The common procedure for removing the SUMO tag is as follows:
1. Expression and purification of SUMO-tagged protein:
First, use an expression system with the SUMO tag, such as E. coli, to express and purify your target protein.
2. Choose the appropriate SENP enzyme:
Choose the appropriate SENP enzyme to remove the SUMO tag. SENP1 and SENP2 are the most commonly used.
3. Add SENP enzyme to the fusion protein sample:
Add the SENP enzyme to the purified SUMO fusion protein solution according to the recommended enzyme:substrate ratio. The ratio can vary from 1:50 to 1:200, and the specific ratio needs to be optimized based on experimental conditions.
4. Incubation:
Incubate at an appropriate temperature (between 4°C and 25°C) and pH for several hours or overnight.
5.Remove SENP enzyme and SUMO tag:
Since the SUMO tag and SENP enzyme likely both carry His tags, you can use nickel-NTA affinity chromatography again to separate uncut SUMO fusion protein, SENP enzyme, SUMO tag, and target protein. The eluate should contain only the target protein, as the SENP enzyme and SUMO tag will be absorbed by the nickel beads.
6.Verify the effectiveness of tag removal:
Finally, techniques such as SDS-PAGE and Western Blot can be used to check the purity of the protein to ensure that the SUMO tag has been completely removed.
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