Hi Diana,

I can refer you to the following resources.

http://wolfson.huji.ac.il/purification/PDF/Tag_Protein_Purification/SUMO/LifeSensors_SUMO_Prokar.pdf

http://wolfson.huji.ac.il/purification/PDF/Protease_fusion_cleavage/INVITROGEN_SUMOProtease.pdf

Article A basis for SUMO protease specificity provided by analysis o...

Best,

Dima

The SUMO-tag/SUMO protease system is special because it does not have a particular short recognition sequence (like other proteases) for the protease but the whole SUMO protein is needed to make the protease cut at the Gly-Gly-site at the end of SUMO right before the first amino acid of the target protein (can be any amino acid except for proline). You can contact the EMBL Protein Expression and Purification Core Facility where you can get an expression vector.

This is the strategy that my lab uses:

We perform TA cloning of the gene of interest from an existing plasmid into the pETSumo vector and ligate it, and then you have a your gene in a pETSumo vector containing a Sumo tag and His tag. Both come with the pETSumo vector. After protein purification A histidine tagged Sumo protease digestion will remove both the Sumo tag and His-tag so that your proteins comes of a Ni-NTA column without elution and Sumo protease and both the Sumo-his tag are retained on the resin.

Basically, you have to PCR your gene with forward and reverse primers, and you have to use Taq DNA Polymerase because the technique relies on Taq polymerase adding on extra 3' A to each 3' end of the gene. It just does that automatically. It is the only polymerase that does this, and the pETSumo vector contains T overhangs that pair up with the extra A bases added by Taq polymerase.

This is the kit we use. Read the documentation for details:

https://www.thermofisher.com/order/catalog/product/K30001#/K30001

You just need to carefully design your primers.