You could also try upping the tev:prot ratio, such that the protein is ~100% cleaved, then do IMAC or simply some other, non-IMAC chromatography step, such as ion exchange or SEC, depending on the size and charge of your protein relative to TEV.
JPK On Fri, Apr 8, 2011 at 8:17 AM, Zhijie Li <zhijie...@utoronto.ca> wrote: > Hi Anita, > > Admittedly, there are proteins that naturally bind to Ni-NTA so tightly that > they co-elute with our His6-tagged proteins even on an imidazole > gradient. However, we do need some luck to come across a protein with such > property. For most proteins, they would just flow through the Ni-NTA in the > presence of 10-20mM imidazole. Are you sure that what you saw as a lower > molecular weight band on SDS gel was not really a clipped form of your > protein that failed to be cleaved by TEV and still carried a His tag when > undenatured? > > I guess that your TEV is also His-tagged, so seeing it in the Ni-NTA > fraction is reasonable. But I would expect some sort of separation from your > protein on the imidazole gradient even if the peaks are overlapping. Also, > on Q column, TEV, being an extremely basic protein, simply won't bind. If > you saw the "TEV" band in the Q fractions, then that suggests that you may > have incorrectly identified your protein bands on the SDS gel. > > It would be interesting to see your SDS gel. Also providing more specific > details of your chromatographies may help a lot. For example, what was the > approximate concentration of imidazole when your peak came out from the > Ni-NTA when eluted with a gradient? What was the condition you used for Q > column and what is your protein's PI? There are just too many factors that > could effect the performance of the ion-exchange chromatography. > > On the other hand, if it is true that your protein binds to Ni-NTA so well > even without a His tag, then why not try expressing it alone without a His > tag? Shouldn't you be able to purify it easily with Ni-NTA? > > Finally, the difficulty in TEV cleavage could indicate a construction > problem. I assume that your protein is N-terminally His-tagged. To my > experience, TEV wants one or two more amino acids between the G/S in > "ENLYFQ^G/S" and the folded protein domain, i.e., it wants some space on the > right hand side of the cleavage site. Adding one or two amino acids after > the current cleavage site may help. > > Zhijie > > From: anita p > Sent: Friday, April 08, 2011 5:10 AM > To: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] Tev Cleavage issue !! > Thanks everyone for your suggestions ! > Artem has pointed out that low diffraction of the crystal might be because > of other problems .. If you could highlight a bit more on this issue it > would be helpful for me. > I have tried to seperate the cleaved, uncleaved and TEV over MonoQ column > but there was a single peak and all of them came out together, there was no > seperation. > I even tried to cleave the protein at 30 degress and it starts > precipitating. > I have also tried binding it to the IMAC as Martin has suggested but then I > get a single peak while running the imidazole gradient and its tev, cleaved > and uncleaved together. > And I also get the flowthrough while loading unto the column which should be > theoritically the cleaved one but it is a combination of cleaved uncleaved > and tev. > > awaiting for bit more suggestions > Anita > > On Fri, Apr 8, 2011 at 10:48 AM, Artem Evdokimov <artem.evdoki...@gmail.com> > wrote: >> >> 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 >> > > -- ******************************************* Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu *******************************************
