Removal of GST from digested fusion protein

Phelan, Paul J. via (by Paul.Phelan from
Fri Jan 13 12:29:35 EST 2012

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 GST?  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

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