As Ladislav said, TEV has been bioengineered to increase solubility/stability. If you know your sequence you can check for the 3 most commonly used mutants. S219V eliminates autolysis. L56V and S135G increase the solubility greatly (up to 40mg/mL as a double mutant). However, without these mutations it will begin aggregating at 1mg/mL. Also, you have 300mM NaCl in your original conditions which will make it precipitate out even faster. So check your sequence and remove the salt. If you only have the S219V mutation, you should keep your TEV stocks at 1mg/mL or less with ~50% glycerol. 

See http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2211701/pdf/2360.pdf for details.

Hi Valerie, I purified some different tagged protein and then I removed the tags with TEV protease. One of these proteins seemed cut resistant in conditions similar to your. Also my protein tended to precipitate. I tried some different condition, then I solved the problem by omitting the DTT in the reaction buffer (no more precipitation and 80% tag removal). So, you can try to lowering the reducing agents DTT and glutathione.

Thank Raffaele,

I am growing up 4L of cells for more pilot experiments, and that is one thing I will definitely try!

Val

You can also try on bead digestion instead of digestion after elution

Hi Valery! Did you try to cut you protein without elution when it is still on the column? You can wash GST -sepharose with extensive amount of PBS buffer, equilibrate with buffer recommended for TEV digestion ( I recommend to use 1mM DTT or 0.5mM TCEP), add protease and incubate over night at 4C. Next day your protein should be in a flow throw( you can also wash GST sepharose by PBS) and GST and uncut protein should stay on a column. This method is good If your expression is OK and you have a lot of protein because you will not cut 100% of you protein on column.

Good luck, Tetyana

Dear Valery!

TEV performance can somtimes be quite unpredictable.

We have not experienced precipitation of TEV so far, but lots of other issues.

It is quite useful to monitor the ratio of TEV concentration to the concentration of cut protein for aliquots from the same batch of TEV. (e.g. write it in a book accessible to all you use the same batches of TEV). When you have a clue about the different activities of your TEV preps, you have the benefit to get the amounts of TEV required for cutting a certain concentration of protein right in the first place.

Have you checked the TEV construct that you are using? There are stabilized versions of TEV around, which could help you with your problem. However, you might be using such a stabilized version already.

We have made good experiences to combine the TEV cleavage with the dialysis of the protein, as the high amounts of imidazole or other components in your elution buffer might affect the TEV stability or activity. So usually, we dialyse while cutting overnight. Just be aware of this: If the tag which you want to remove contains solubilizing proteins, then your cut protein might start precipitating during this dialysis step.

So, I'd check first the construct and modify the buffer conditions (antioxidants) as suggested above. Maybe you can combine it with the required dialysis? In case this does not help, try another batch of TEV prep and monitor these events with all colleagues who use the same batches of TEV.

Good luck,

Frank

Thanks everyone! I am going to try to optimize the cleavage conditions first. I will try on column cleavage, as well as cleaving during dialysis. Hopefully one of these things will work :)

Hi Valerie,

If you can share with us whether the suggestions worked or not it would be fantastic! As I'm going through problems of my own with TEV digestion of MBP fusion proteins

Thanks :)

We always had glycerol in our buffer with DTT and the TEV never precipitated. This TEV was used for hundreds of proteins at NIEHs. You may want to use less of your protein and/ or make it less concentrated. You may have a solubility problem with your protein. It may not be the TEV. The TEV may precipitate with your protein, because it is binding to it in order to cleave it. Or maybe the cleaved material is what is insoluble? Did you run a gel on the ppt to see what is there?

It could depend on the TEV solubility mutations. Try this one: http://www.protean.cz/en/recombinant-protein/28/tev-protease

As Ladislav said, TEV has been bioengineered to increase solubility/stability. If you know your sequence you can check for the 3 most commonly used mutants. S219V eliminates autolysis. L56V and S135G increase the solubility greatly (up to 40mg/mL as a double mutant). However, without these mutations it will begin aggregating at 1mg/mL. Also, you have 300mM NaCl in your original conditions which will make it precipitate out even faster. So check your sequence and remove the salt. If you only have the S219V mutation, you should keep your TEV stocks at 1mg/mL or less with ~50% glycerol. 

See http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2211701/pdf/2360.pdf for details.

The same thing happened to me yesterday. Upon incubation O/N with TEV protease, in the morning i saw a white thready precipitated formed. After running the SDS gels, following coomassie staining, i do not see my actual protein (either uncleaved or cleaved), or even the TEV protease. I believe the proteins (my protein with TEV) might have formed a conglomerate and precipitated. IS this  a possibility and how can i dissolve this and incubate again. Will Glycerol help? Suggestions are really appreciated. 

Definitely a very late answer, but for those viewing later I thought I'd chip in.

Based on the initial description I would not assume that the TEV protease itself precipitated, but it is more likely that your GST-tagged protein precipitated after TEV removed the GST tag. I had this occur more times than I care to remember, but I found that the addition of 5-10% glycerol tended to dramatically improve the solubility of my protein (often human protein kinases). Like Frank described, I usually cleaved off the GST tag during an overnight dialysis step, making sure to include 5-10% glycerol in the dialysis buffer. I also observed on some occasions that particular protein constructs were quite resistant to TEV proteolysis, such that I had to add as much as 1:5 TEV:protein to get efficient cleavage.

Hope this helps someone!

Hey Justin

Appreciate your update. I currently tried to do an on column cleavage O/N with TEV protease. The cleaved protein seemed to precipitate. The TEV however was present in the elute. I did use 10% glycerol in the cleaving buffer, however that did not seem to help though. The reason i am trying an on column cleavage is to minimize the number of steps to obtain the pure protein. Please let me know if you have more ideas on this? Also can the TEV protease be reused?

I never had much luck with on column cleavage, and I would expect that since the protein is so highly concentrated onto the resin that it may actually be more prone to aggregation after the cleavage of the tag. My tried and true method was to first affinity purify my protein (usually 6xHIS w/ a Nickel column), then dialyze overnight at 4C with TEV protease (1:20 w/w in 25 mM Tris/ 300 mM NaCl/ 5% glycerol/ 2mM DTT), then the next day I would run the cleaved protein back over the Nickel column and since the TEV protease was itself His-tagged it would stick to the resin while your cleaved protein flows through (so make sure to collect the flow through!). Note if you are using a nickel column, you should spike a small amount of imidazole (20 mM or so) into your sample before running this second column cleanup, since Ni-NTA is extraordinarily sticky in the absence of any imidazole. You could stop at this point, but I usually then ran a gel filtration column to remove aggregated (but not precipitated) protein, since kinases love to aggregate.

I have a question about this topic. What is the most optimal buffer to maintain this pretentious TEV enzyme? To store at -80°C or -20°C? My protein is a tetramer and I can not add too many "sweets" to improve the digestion.

Filip,

Typically people store TEV protease in a Tris buffer (50-100mM), pH 8.0, 300mM NaCl, glycerol (usually 5-20%), and DTT between 0.5-2.0mM. Some also have a little bit of imidazole (5-10mM) in their buffers.

I would highly recommend flash-freezing and storing at -80 °C over -20 °C.

If DTT is problematic for your system, which is fairly common, TCEP or another more gentle reducing agent could be substituted.

Good luck!

Thank you for answer! This information helps me a lot, tomorrow I will purify the TEV enzyme.

Thank dr. Stiers!

Hi Filip Alina

I am working on a tetramer protein too and it forms inclusion bodies when only tagged with His. I’m considering using an MBP-fused version. I was wondering if you used the same tag and were you able to successfully obtain the cleaved form of the protein? Thank you!

I apologize for the late response, Kiall Suazo.

I can not say they form especially inclusion bodies, but a slight aggregation occurs. To my tetrameric protein I want to remove His-Tag sequence with a protease.

Protease TEV has an MBP at the N-terminus. Digestion attempted in different solutions does not exceed 50-55%. Indeed protein aggregation after His -tag removal seems to disappear (I checked on the SEC).

But on the chromatogram, immediately after the tetrameric enzyme, a ”hidden „ sign appears. On the gel, this new fragment has a molecular weight close to the tetrameric enzyme. It looks like the enzyme is truncated after this digestion.

I don't know if my answer is helpful.

I still investigate! :)