Did you try different Temperatures, such as 20, 25, 30, 37 degrees? Does your buffer contains L-arginine or mixtures of L-arginine and L-glutamic acid? These additives inhibit the activity of proteases at a concentration greater than 50 mM in a buffer.

I tried but i had the problem of protein precipitation so had to carry out the cleavage at 4 deg C. I dont have any of the additives that you mentioned.

How long did you let the reaction go? Sometimes we have to let the reaction go a week or longer to get full cleavage. One of my colleagues believes that increasing the salt concentration (to 500 mM NaCl) may help as it may disrupt intermolecular interactions that occlude the TEV cleavage site from the protease. You may also want to check David Waugh's recommendations for other ideas: https://mcl1.ncifcrf.gov/waugh_tech/faq/tev.pdf

Good luck.

In my experience TEV tolerates quite well 0.5 M NaCl and 0.5 M imidazole in cleavage reaction. The protease itself is quite soluble and express and purify to a really high level (S219V mutant). The wild type self-cleave so I use the mutant one ( 4 degree C and ON incubation is enough). I have been using TEV with many different kind of proteins to get rid of affinity and solubilization tag with considerable success. I have worked with both ENLYFQ/G or S (PMID: 12074568). I haven't tried a recognition site with C at the P1´ part of the scissile bond. Do you have a rationale why you wanted to use C at the P1´ part of the cleavage site? For example: C may come from the protein you want to study and you do not want any extra aa insertion at the N terminus. My first argument would be having C at P1´ part may result in an sub-optimal cleavage site assuming that you get rid of the 6M GuHCl from the cleavage buffer. You may also want to check if the protease is wild type or stable (S219V) one and it is active over the period of incubation. Steric hindrance is also a concern but from your information it appears that you are getting quite bit of cleavage but it is not pushing it to the product side enough. I would suspect that TEV protease, even it is a horribly stable protein, perhaps is getting inactivated unless you have evidence it is not. Suboptimal steric hindrance or rather breathing of the N terminus may result in some kind of cleavage but it doesn’t explain why it is not pushing it enough after 50%. So my optimization of cleavage would consider i) suboptimal cleavage site: C at P1´ site ii) keeping the TEV active over the period of incubation. If the situation continues and it affects downstream usage of the protein, I would perhaps switch to the sumo protease which recognizes the native structure of its substrate rather a small peptide like in case of TEV protease. Hope this helps. Good luck !

The observation that your reaction only results in 50% cleavage no matter how long it is left for or how much enzyme you add (I am assuming here) suggests to me you have two populations of protein present. It could be that these represent e.g. correctly folded and incorrectly folded (and potentially aggregated) protein. Perhaps the TEV is only able to cleave the correctly folded fraction of protein. If this is the case, you may be best to simply use the fraction of protein that is cleaved and ignore the remainder. You could easily remove the uncleaved sample with another IMAC step.

Dear Pratyush,

Thanks for your detailed suggestions. The step preceding my TEV cut is an IMAC using Gua.HCl, hence I do a ON dialysis of the combined fractions from this step to get rid of the Gua and then check the conc of the protein the next morning. I need an N-term Cys for the downstream processes so cant really help there. the TEV that we are using is the one you mentioned which is resistant to self-cleavage. Also my buffer for the ON TEV cut is just 1x TBS (50 mM Tris, 150 mM NaCl, pH 8) with no other additive. In the reaction mix however, I add 1mM DTT (thats the final conc.).

Dear Roger,

Thats exactly my thought on this issue and as a matter of fact today I will be trying a 2nd IMAC so that we can expect the cleaved fragment lacking the His tag to come in the flowthrough or early fractions. Thanks and I hope for the best.

Cysteine at P1' shouldn't be an issue. See this paper: Article The P1 ' specificity of tobacco etch virus protease

If aggregation is an issue, maybe you should do a size exclusion step prior to setting up the TEV digest to get rid of the aggregate. There's still some potential that his tags on the aggregate may be surface exposed and copurify with your unaggregated TEV-cleaved sample.

Does the final cleave reaction contain imidazole? In my experience, Imidazole severely inhibits TEV protease cleavage.

No It doesn't. I dialyse my protein ON against sufficient volume of the cleavage buffer and the next morning I start the TEV cleavage.

I have always included the TEV protease in the dialysis bag with the newly purified substrate - as the imidazole gets dialysed out, TEV activity subsequently increases and over the duration of 16-18 hours and 2-3 buffer changes, TEV activity is ~100%. I would assume my information is redundant in case you have already tried TEV digestion successfully with some other substrate and that this particular case is unique in its manifestation

Certainly, that's the usual practice.The problem here is that I have 6M Gua.HCl in the sample (after I perform an IMAC with Gua and Gua plus Imidazole system), now in case the denatured TEV precipitates out inside the dialysis bag and never gets refolded during dialysis then what I do ? Thats why I always try the cleavage 'after' I have gotten rid of Gua etc by ON dialysis.

TEV sequence signal is ENLYFQG/S, so you need a Glycin or Serin after the Q. Then, you can add your cisteine. Having a Glycin will let the Sulfur of the cystein ready to use.

Alternatively, you can use pET His-SUMO GFP vector, where HisSUMO is cleaved with SENP2 protease, and it did not leave any residue after cutting

https://www.embl.de/pepcore/pepcore_services/cloning/sumo/

Good luck

Thanks but as you can see my question is about an year old and I have already optimized my cleavage conditions and a lot more over this one year.

haha glad your problem is solved. it would probably help if you could share how you optimized your conditions for anyone else visiting this thread with a similar issue in the future.

Somnath Mukherjee

Hi somnath, will you please share that, how did you finally cleave your protein and what condition you have used? Thanks

Imidazole can inhibit TEV protease activity to 50%.

It is recommended that you do buffer exchange before you start the cleavage of your protein.

Somnath Mukherjee Hi, can you please share how did you solve this problem?

Sorry this isn't an answer - Does anyone know what the maximum amount of Imidazole you can get away with in a TEV digest is and still have ok cleavage?

I've seen Dr Das' answer but I want to see if anyone else had success with some amount of Imidazole.

Heidar Koning Generally, you should have less than 50 mM imidazole in your TEV cleavage reaction, but I personally like to remove all imidazole in my reaction.