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 <[log in to unmask]> 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: [log in to unmask]
> 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 <[log in to unmask]>
> 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 <[log in to unmask]> 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: [log in to unmask]
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