How to Remove the SUMO Tag After Purification
The Small Ubiquitin-like Modifier (SUMO) tag is a widely utilized fusion tag that facilitates protein solubility and stability. It can be specifically cleaved by SUMO-specific proteases, such as SENP1 or SENP2, to yield the target protein in its untagged form. The standard protocol for SUMO tag removal consists of the following steps
Expression and Purification of SUMO-Tagged Protein
The target protein is expressed using a SUMO-fusion expression system, commonly in Escherichia coli, and subsequently purified via affinity chromatography.
Selection of the Appropriate SENP Enzyme
A SUMO-specific protease, typically SENP1 or SENP2, is selected to cleave the SUMO tag efficiently.
Enzymatic Cleavage of the SUMO Tag
The purified SUMO-tagged protein is incubated with the selected SENP protease at an optimized enzyme-to-substrate ratio, typically ranging from 1:50 to 1:200, depending on experimental conditions.
Incubation Under Optimal Conditions
The reaction is conducted at an appropriate temperature (4°C–25°C) and pH, allowing sufficient time (several hours or overnight) for complete cleavage.
Removal of SENP Protease and SUMO Tag Fragments
Given that both the SUMO tag and SENP protease typically contain His-tags, nickel-NTA affinity chromatography can be employed to selectively retain these components. The cleaved target protein is collected in the flow-through fraction.
Verification of Cleavage Efficiency
The extent of SUMO tag removal is assessed using SDS-PAGE and Western blot analysis to confirm the purity and integrity of the target protein.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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