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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, [log in to unmask] 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