We concentrate the TEV by AmS precipitation. I have attached our protocol. Depending on what your construct is, you might want to add the polyArg tail (improves solubility of TEV dramatically), add the S219V mutation (removes autocatalytic site) and use some sort of codon-enhanced cells (the native TEV gene contains some unfortunate Arg codons that can lead to premature termination). We can store TEV at 400 uM - 11 mg/ml for ages without loss of activity.

I use 20 mM Tris-HCl, pH 8, 300 mM NaCl, 1 mM EDTA and 50% glycerol, and add 10 mM DTT when freezing. We store TEV at -20 C. It doesn't freeze, so I can take out what I need whenever I have to cleave my fusion protein. Having a poly-Arg tail on your TEV construct helps keeping it soluble as well.

Many thanks Klavs for the buffer details:) I will definitely give it a try when I next purify my TEV Protease.

One question: Do you usually concentrate and then dialyse your purified TEV into the described buffer? I am assuming that it is eluted into Immidazole Elution buffer provided your TEV is His tagged as well? 

We concentrate the TEV by AmS precipitation. I have attached our protocol. Depending on what your construct is, you might want to add the polyArg tail (improves solubility of TEV dramatically), add the S219V mutation (removes autocatalytic site) and use some sort of codon-enhanced cells (the native TEV gene contains some unfortunate Arg codons that can lead to premature termination). We can store TEV at 400 uM - 11 mg/ml for ages without loss of activity.

first you have to dialyse your protin against low salt buffer for example 5 mM tris buffer pH 8 to get ride of the imadazole which was in the last purification step then after dialysis you can concentrate using Amicon tubes or lyophilization but I prefere lyphilization as you will not loose any protein and you lyophilize 10 times only to have 100 mM tris at last then you add the 30 - 50 % glycerol and store at - 20  and this is would be enough for your protein

To alter the buffer to something suitable for storage, why not use a size exclusion column? Nickel columns rarely give complete purification (although with TALON sometimes it is very close to complete). You presumably intend to add the TEV to your (hopefully) purified samples - why not add another purification step to minimise contaminants? It's best to add any glycerol to the sample after size exclusion, as the columns run really slowly in glycerol.

I have always stored (S219V) TEV in aliquots in 10-20% glycerol, and generally it thaws fine. I usually keep a working stock at -20, and other stocks at -80 until required. I usually keep it at ~1 mg/mL, as this makes it easy to work out how much to add (~1% of the target protein concentration, unless it's an annoying protein).

Thank you all for the informative answers:) Thanks Klavs for attaching TEV purification protocol.

Nicholas; you are absolutely right- however I find it troublesome to concentrate the TEV to 500ul before purifying it using a size exclusion column (as the loop of our GF Superdex 75 can only take sample volume of 500ul at once). Concentrating to that extent can also cause precipitation; unless I decide to do it in 2 or 3 runs (which is very time consuming).

Klavs, i was reading your protocol, thank you very much for it. so I can see that in the last step you don´t dialyse your protein. So you conserve the protein in the storage buffer and AmS, isn´t it?? 

and you use the protein in another buffer or only in this??