In our case, incubation in the presence of imidazole can knock the
protein out of solution- this was gleaned from crystallization
conditions containing imidazole. The best we could come up with is that
the imidazole groups from the His-tag were interacting with neighboring
protein in solution, causing them to precipitate. Another theory is
that trace amounts of Ni may leech off the column during purification
and coordinate with multiple His-tags on the pure protein, causing them
to aggregate.
Hope that helps,
-Chris
Jacob Keller wrote:
Chris,
Can you or others speculate, perhaps in light of the structure, why a
his-tag would cause precipitation?
Jacob Keller
*******************************************
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
Dallos Laboratory
F. Searle 1-240
2240 Campus Drive
Evanston IL 60208
lab: 847.491.2438
cel: 773.608.9185
email: [EMAIL PROTECTED]
*******************************************
----- Original Message ----- From: "Christopher Bahl"
<[EMAIL PROTECTED]>
To: <CCP4BB@JISCMAIL.AC.UK>
Sent: Tuesday, November 11, 2008 10:06 AM
Subject: Re: [ccp4bb] crystallization of proteins with His-tag and/or
c-myc tags
Hi Jorge,
We have had cases both for and against leaving His-tags on proteins.
In one case, the presence of the His-tag was causing the protein to
precipitate, blocking our attempts at crystallization until we
removed it. However, we also had a different protein that
crystallized beautifully with the His-tag left on. I can't speak to
the impact the tag had on crystallization since we never bothered to
remove it and it was not visible in the structure. My (rather
unhelpful) input is that it greatly depends on the individual protein
as to whether or not it is beneficial to remove the His-tag prior to
crystallization. -Chris
Tim Gruene wrote:
Hi Jorge,
using a system where you can cleave off the His-tag (with TEV,
FactorX, etc) adds a purification step which is complementary to the
first purification step (Ni-column etc). In my experience this
results in very pure protein which makes it more likely to crystallise.
Therefore I would always choose such a system and not spend much
time on trying to crystallise the protein with the His-tag attached.
Tim
--
Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen
GPG Key ID = A46BEE1A
On Tue, 11 Nov 2008, [EMAIL PROTECTED] wrote:
Dear all,
Concerning the crystallization of proteins with a His-tag, based
upon discussions on this bulletin board and on the article
Mike Carson, David H. Johnson, Heather McDonald, Christie Brouillette
and Lawrence J. DeLucas, His-tag impact on structure, Acta Cryst.
D63, 295-301, (2007).
I understand that as a general approach one should try to
crystallize the protein with the His-tag (yet it might crystallize, so
no need to care the work of taking out the tag); then, if this is not
successful, go to the procedure to either express (and purify) it
without the His tag or take it out later. Any observations/advices?
But one other question to add is what if the protein is expressed
with both a c-myc tag and a His-tag (you might consider also the case
in which only the c-myc tag is present). Any experience on the
effect of
the c-myc tag on crystallization? References are welcome yet I
could not
find much googling around...
Thanks,
Jorge
