I am purifying the SUMO-specific cysteine protease Ulp1, which I am recombinantly expressing in E. coli. My goal is to inhibit host cell proteases from cleaving my recombinant Ulp1 protease during lysis, prior to when I load my cell lysate on the column. I know that Ulp1, being a cysteine protease, will likely be inhibited by the protease inhibitors during lysis. However, as long as Ulp1 does not get irreversibly modified, I assume that a sufficient amount of washing the column and buffer exchanges after elution should dilute out the protease inhibitors. Is this a safe assumption? Or will the protease inhibitors irreversibly inhibit Ulp1, leaving me with highly-purified protein with zero activity? Importantly, my wash, elution, and buffer exchange buffers will be devoid of protease inhibitors.
http://iti.stanford.edu/research/documents/ProteaseInhibitionGuide.pdf
Those cocktails are proprietary, but most likely have at least leupeptin and some sort of chloromethylketone, both of which are irreversible inhibitors. Roche tech support should at least be able to tell you that much. Alternatively, you could make your own cocktail of reversible inhibitors... Good luck!
I would make my own cocktail with AEBSF, pepstatin, EDTA and maybe some others. And if you want to initially keep the activity of your proteinase low, don't include DTT or other reducing agents in the buffer. But then when you want to assay or activate it, add the reducing agents. Sometimes if the proteinase is really active, it will chew on itself. PS, I think E-64 is in the cocktail.
Small correction to Bradford's comment: Leupeptin *is* reversible.
https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-purification-isolation/cell-lysis-fractionation/protease-phosphatase-inhibition.html
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