Hi! I am cloning and purifying proteins using the pET SUMO Expression system from ThermoFischer. I have found that the supplied SUMO protease is not especially efficient and have read that increasing the concentration of the protease can be helpful. However, SUMO protease is expensive and I have heard that many researchers produce their own SUMO protease. Does anyone have a protocol for how to clone and purify their own SUMO protease? Preferably with a His-tag so that it can be separated from the protein of interest? I found the article, "Preparation of SUMO proteases and kinetic analysis using endogenous substrates" but found it pretty confusing.
Thank you!!!
why don't you just put a thrombin or TEV cleavage site between SUMO and your protein. TEV in particular is highly selective and easy to prepare.
I used pET-15b (Amp.) & pET-28a (Kan.) for my proteins and cut his-tag using thrombin. You can try...
Hi Lauren,
We use an in-house expressed and purified SUMO protease. It works so much better than the commercially available stuff.
Try addgene:
https://www.addgene.org/64697/
If you find the a SUMOStar sequence I would be very interested.
Good luck
Thank you all for the helpful information! I am fairly new to research and had not heard of Addgene. Thanks!
Hi Lauren, just to add to some of the other posts, and clarify. Sumo protease, or Ulp1 protease expresses robustly and you'll get milligrams of it from 1L E.coli culture using any pET vectors. One batch should last you a long time.
The advantages of SUMO protease, unlike some of the other proteases like 3C, TEV or Thrombin, is that you'll get no residual amino acids after cleavage, which is great for many purposes.
Good luck.
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