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
