bionet.xtallography FAQ

[Mark Israel]

Well, O.K., James...

		The bionet.xtallography FAQ file
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Q1.  Can you crystallize a protein with the His-tag on?
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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 
of Calcium.

--
misrael at scripps.edu			Mark Israel