We are trying to digest a difficult target with tobacco etch virus protease and was wondering if anybody has experience using this enzyme in organic solvents? Would it be stable in for example 50% ethanol or 50% acetonitrile?
The protein we want to digest is very hydrophobic and surfactants work to some extent to solubilise but not very well.
VTE is a problem that can better inform organic chemists. Affects H bonds of the molecular compounds.
I would try doing the digestion in varying amounts of freshly made and filtered urea, from 0.2 M to 2 M (check pH to make sure it is not altered and use 100 mM of buffer), since some enzymes are stable in high urea concentrations (a N-terminally truncated FIV PR and human topoisomerase I are examples) while the target protein may not be as stable and urea will certainly increase the solubility of a hydrophobic protein.
You could also try different concentrations of dimethylformamide if the TEV PR is active in it, since DMF is a good solvent for hydrophobic peptides.
I suspect that TEV isn't very active in high concentrations of organic solvent, though I'm not sure. David Waugh's TEV FAQ (mcl1.ncifcrf.gov/waugh_tech/faq/tev.pdf) doesn't mention organic solvents or urea, but does say that some detergents interfere with TEV. You might try varying the pH of your reaction; your protein might be more soluble at a pH far from its isoelectric point, and TEV is fairly insensitive to pH between 4 and 9. Remember that TEV is a cysteine protease and needs some reducing agent.
Also, there are some TEV variants with enhanced stability (mostly due to resistance to autolysis). Those might be worth trying in your situation.
Hi James, thanks for the suggestion. How would one go about doing that?
Cobus,
Have you looked at the IMPACT system from NEB? You would have to make a new construct, but then cleavage of the tag would not require a protease.
https://www.neb.com/applications/protein-expression-and-purification/coupled-protein-expression-and-purification/impact-protein-expression
Hi Cobus
I worked many years with enzyme systems in organic solvents and biphase mixtures of water and solvents. Mostly ethanol and methanol are killing completely all kind of Enzyme activities in higher concentrations. DMF and DMSO have very high boiling points so that it might be difficult to get rid of them or recover your products. you can try iso-Propanol and iso-Butanol..acording to the salinity that you offer, you get a biphase mixture that gives good conditions for enzyme and substrat. A 10% buffer or salt concentration should be enough to separate the phases and leave enough aqueos solution for the protease to work
Another option is to make a fine dispersion of your target substrate and use a detergent, a Tween, Brij, Triton or Carbopol ... a non-ionic one migh be the best option, so you can increase your surface, form reactive mycells and decrease particle size to more reactive values.
In both systems also regular enzymes have chances to survive and work.
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