I am interested in purifying proteins with 1 or 2 disulphide bonds, and have been using enterokinase to cleave off N-terminal tags but we have had issues with poor cleavage and want to try TEV cleavage instead. However TEV protease is usually kept in a high amount of DTT and I am concerned about reducing the disulphide bonds in my purified proteins. I have used TEV protease before with 0.25mM TCEP and it worked well. Is there a way to use TEV with very little or no reducing agent? Perhaps by optimising conditions such as adding glycerol? Also to note our proteins of interest are quite temperature stable. We were thinking of buying in commercial TEV protease so any advice on that is also welcomed.
I don't think it will work without reducing agents.
I have not worked with TEV but I do have experience with the ULP1 SUMO protease, which is a cysteine protease just the same, and what I have seen is that without reducing agents, the reaction does not proceed. I have gone as low as 0.05 mM DTT and there is still considerable activity, but without DTT, there is nothing.
The good news, though, is that at least with ULP1 you can find a sweet spot for DTT concentration where protease activity is still high enough but disulfide bonding is not significantly disturbed (0.1 mM in the two cases I tried), so perhaps you can do the same for you TEV/fusion protein combination.
Note that the paper cited by Hubert Aviolat does not address the situation of having no reducing agents, as the TEV protease was stored in 1 mM DTT and the test reactions were performed using 5 ug of substrate and 0.4 ug of TEV. No indication is given regarding relative volumes, but I am willing to bet that final DTT conc. in the reactions (owing to DTT from the TEV storage buffer) was in the ballpark of 0.05 mM.
Hope it helps!
Hi Nicola Evans TCEP is a potent S-S reducing agent. If you used it before with TEV and had no problems, use it again.
David Waugh's group has an excellent information sheet about TEV proteases. While they do recommend performing the TEV protease cleavage reaction in a buffer with 1 mM DTT, they also recommend that, for proteins containing disulfide bonds, you leave out the DTT and replace with with a redox buffer consisting of 3 mM glutathione and 0.3 mM oxidized glutathione.
See here for more info:
https://mcl1.ncifcrf.gov/waugh_tech/faq/tev.pdf
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