I would guess that your GST tag is still bound to glutathione, as you suggest. Could you use a nickel column in your first purification step, instead of the glutathione sepharose? You might even be able to add your His-TEV onto the column for the proteolysis, then elute off the nickel column and put everything onto your glutathione sepharose.
Irit On Jan 13, 2012, at 9:29 AM, Phelan, Paul J. wrote: > I am trying to purify a protein, starting with a fusion that is double-tagged > with GST and poly-His, and my protein of interest also has an n-terminal > poly-His tag of its own. So the fusion protein is > polyHis-GST-polyHis(Protein). An initial step with glutathione Sepharose > works just fine, the fusion protein binds and is eluted with 10 mM > glutathione. Digestion of the fusion protein (with His-tagged TEV protease) > also works, and gives me the polyHis-GST tag, plus polyHis(Protein). BUT - > when I re-run the digested protein over glutathione Sepharose, the > polyHis-GST is not removed, and just passes through the column, along with > the polyHis(Protein). Has anyone else seen a problem with GST from a > digested fusion protein not binding to glutathione Sepharose? Could it be > that the GST still has glutathione tightly bound to it? (The fusion protein > is digested by extensive dialysis with TEV protease). Could it be that the > polyHis-GST is not behaving the same as non-tagged ! GS! > T? This seems doubtful, since the fusion protein binds to glutathione > Sepharose in the first step. Before anyone suggests using a Nickel resin to > remove the polyHis-GST, that's also a problem, since my protein of interest > (and also the TEV protease) is His-tagged too. > > Any suggestions/insights would be welcome! > > Paul Phelan > Tufts University > Boston > > _______________________________________________ > Methods mailing list > [email protected] > http://www.bio.net/biomail/listinfo/methods _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
