On Nov 11, 2008, at 16:12, 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.
3C protease seems to work really well and would recommend it over TEV
or FX.
One could look at the His-SUMO tag ideas for alternative idea for
cleavage and some aid in solubilization.
Therefore I would always choose such a system and not spend much
time on trying to crystallise the protein with the His-tag attached.
If you have the His-protein always try to crystalize it. We got one
very nice structure with a His-zipper from the Tag helping to form the
lattice.
Consider the no-tag as yet another alternative for crystallization
trials - but if that does not work cleave it !
A.
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
