misrael at scripps.edu
Thu Nov 7 20:47:57 EST 1996
Well, O.K., James...
The bionet.xtallography FAQ file
Q1. Can you crystallize a protein with the His-tag on?
xtalrox at sba.cerf.net (Bob Cudney) writes:
In a show of hands at a recent meeting (Recent Advances in
Macromolecular Crystallization - RAMC) the consensus among those who
had tried to crystallize His-tagged proteins was in favor to remove
the tag in order to obtain crystals.
There were a few who had obtained crystals with the His-tag in
place, but the vast majority had the best success with the His-tag
removed. The literature is light in this area as you indicated.
Of the crystallization reports in 1995 and 1996 there are several
involving His-tags. Again, the majority removed the His-tag.
If sample permits, perhaps you can screen the sample with the
His-tag intact, and then if no crystals are obtained remove the tag
and repeat the screen.
Let us know of your results. This is a rapidly growing area in
crystal growth and we could all benefit from your experience.
leemor at cshl.org (Leemor Joshua-Tor) writes:
Yes, we have successfully crystallized a protein and a couple of
mutants of that same protein with a his-tag on the N-terminus. They
also diffract really well, so there's no interference.
pk11 at cornell.edu (Palangpon Kongsaeree) writes:
In our lab, the native and hig-tagged proteins are crystallized
under very similar conditions. And the his-tagged protein seems to
crystallize a bit more easily, at least in several other forms and
conditions. Well, this depends on the nature of the protein and the
packing in the crystal lattice. Another protein in the lab, without
the tagged-histidines, never crystallizes.
gregers at palmer.u-strasbg.fr (Gregers) writes:
I have also worked with a tagged protein, and my experience is
that I had to cleave the tag (which is very easy, leave overnight
with thrombin in the coldroom, no unwanted degradation). Before tag
cleavage, we had no crystals. After cleavage the crystals appeared
immediately. So my advice is: Do not be afraid to try, it is very
easy and can change things. Furthermore, you can use relatively
cheap thrombin from SIGMA instead of some molecular biology company.
If you use His tag, and elute with imidazole, make sure to remove
the imidazole before cleavage. Thrombin requires a few millimolar
misrael at scripps.edu Mark Israel
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