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
