I hope others with real world experience can share the pros and cons of their preferred protease.
My criteria...
-Speed of lysis
-Robustness(pH, temperature, and salinity compatibility)
-Storage Life (half life at -80C)
-Price
I have only worked with TEV protease and before I make it a career habit I thought I would ask. In particular I am disappointed with TEV's speed and storage life.
Hi Jacob Bassett . I have lots of experience with proteinases. (please call them proteinases, because they cleave proteins. I'm kinda OCD about this)\s
Using proteinases to cleave off specific sequences on recombinant proteins is a good idea on paper, but like most enzymes, proteinases aren't built for specificity. Proteinases aren't restriction enzymes. They are built to cleave whatever protein comes into their mouth. Specificities are mostly provided by cofactors that they bind to.
Save for TEV and enterokinase, I cant think of other proteinases that can come close to target specificity, but they are frigging expensive and unstable. Forget thrombin and Xa, they aren't even in the same league with respect to specificity.
Personally, I think the next generation of cleavable linkers will forgo proteinases altogether and have chemically sensitive cleavable linker sites, such as proline and aspartate triplets for formic acid cleavage. (Ramos)
Jacob Bassett I'd like to suggest SENP1. I'm not exactly sure what you are attempting to do. If you want to express a protein, capture it on a column, and then cleave off the bit that captures to recover your native protein then the SENP1/Sumo method may work for you. SENP1 isn't extremely efficient at cleaving so I usually leave my samples in the refrigerator overnight. But I've stored it in the -80C for years with no decrease in activity. If you are interested I can give you the details but there are plenty of examples of how this works. And lots of plasmids on Addgene. Just type in SUMO in the search and the first thing that comes up is SUMO conjugated to nsp7. Shoot me an email at [email protected] if you have any questions. We do a lot of E.coli expression and express our own SENP1(Proteinase). I can send you the plasmids for both cloning in your construct and the protease. Both the SUMO tag and the SENP1 are HIS tagged so you just pass them over a nickel column after cleavage and capture the enzyme and tag. Flow through is your purified native protein.