sometimes His-tags lead to aggregation mediated by metals, we solved the problem by adding
EDTA after purification on the Ni column, it was very clear on gel filtration. Aggregation was reversible.
It worked on other proteins expressed in our lab which had the same problem.
We published this on Acta F, and I found more papers reporting the same finding :
<x-tad-bigger>Renzi F, Panetta G, Vallone B, Brunori M, Arceci M, Bozzoni I, Laneve P, Caffarelli E.</x-tad-bigger>
<x-tad-bigger> </x-tad-bigger><x-tad-bigger>Large-scale purification and crystallization of the endoribonuclease XendoU: troubleshooting with His-tagged proteins.
Acta Crystallograph Sect F Struct Biol Cryst Commun. 2006 Mar 1;62(Pt 3):298-301. Epub 2006 Feb 28.
</x-tad-bigger>
Yoshida H, Hensgens CM, van der Laan JM, Sutherland JD, Hart DJ, Dijkstra BW.
An approach to prevent aggregation during the purification and crystallization of wild type acyl coenzyme A: isopenicillin N acyltransferase from Penicillium chrysogenum.
Protein Expr Purif. 2005 May;41(1):61-7.
Good luck,
Beatrice
Il giorno 22/set/06, alle 21:50, Jenny ha scritto:
Hi All,Beatrice Vallone
I'm trying to purify a protein which is expressed in soluble form.It's a His-tag protein, so when I run His-column, it's really easy to clog.In the next step size exclusion purification, the protein come off at the void volume, which suggests that the protein is severely aggregated.Does any one have any suggestions that I can modify the purification condition so as to avoid aggregation problem?Or can I add GuHCl during His column purification so that it won't clog so easily?Thanks a lot.
Jenny
Dept. of Biochemical Sciences
University of Rome "La Sapienza"
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