For starters, you could re-clone the protein with e.g. just a His tag or move the tag to another end, or put some distance between the end of TEV site and the protein; or perhaps use no tag at all -- or a different one?
Is it possible that the tag is messing you up - yes. Is it 'probable' - I can't say that I know because I've crystallized literally dozens of proteins with His-tags attached, and more than a few with His-tag and cleavage site. I prefer a plain His-tag or a cleaved one, to be sure - but I am not quick to blame the tag (since there are so many other possible things to blame). Based on the behavior of your protein after cleavage, it may be that you have oligomer(s) forming in solution such that cleaved and uncleaved proteins do not segregate. You may wish to explore other kinds of chromatographic separation e.g. ion exchange of HIC - they may or may not work out. You can also consider cleaving your protein at lower concentration, in the presence of detergents or polyols, etc. Cheers, Artem On Thu, Apr 7, 2011 at 9:37 PM, anita p <crystals...@gmail.com> wrote: > Hi Crystallographers, > I am working of 23 Kda protein with a Nterminal His tag and a TEV > cleavage site. > I am getting crystals with the his tag and tev site intact, but they dont > diffract. > *Is it probable that they dont diffract because of the extra his tag and > the tev site?* > > I am trying to get rid of this tag but the reaction is optimum at 10:1 > protein to TEV ratio in micrograms overnight incubation without shaking. > I tried to run it on histrap column after this reaction but I am not able > to purify cleaved protein from TEV and uncleaved. > I have tried several times but I get 3 bands ie., the TEV, uncleaved and > Cleaved. > I have also tried to use the Nibeads instead of the histrap column, but no > difference is seen. > * Is there a possible way to approach this problem?* > > Suggestions awaited > Anita >
