Is it possible that your ligand is covalently attached? Any idea of the Kd?
On Thu, Oct 7, 2010 at 10:13 PM, Daniel Bonsor <[email protected]> wrote: > "It refolds properly according to the CD spectra but it some how manages to > hold on to trace amounts of ligand despite serial dialysis (500ml to 5ml of > sample) in 8M, 6M, 4M, 2M 1M urea followed by 50mM Tris. I also have a > homolog that abjectly refuses to refold in either urea or guanidine, though > it does turn the dialysis tubing into a lovely snow globe." > > I am assuming that the protein is tagged in someway and that you add your > purified protein to Urea/Guanidine and refold by dialysis. > > If you protein is His-tagged, I would unfold it on the column and flush a > large excess of Binding buffer (containing denaturant) to remove the ligand > and elute the protein in elution buffer (containing denaturant) and then > refold, to ensure that no ligand is present during refolding. If this is what > you have done, ignore this. > > For the protein that cannot be refolding, have you investigated thermal > denaturation. I have removed a 1kDa ligand by placing a nickel column in a > water bath set near to the Tm of the protein. After flushing an excess of > prewarmed buffer through, the water bath was switched off, allowed to cool to > RT and then protein eluted off. Around ~95% was refolded. The unfolded > protein was then separated by size-exclusion. > > > Though I have no evidence for this and I am just thinking out aloud (please > could someone correct me if I am wrong, have evidence to the contrary or > both), if the ligand is not present in the periplasm, and the protein is > targeted through the sec pathway (which recognizes unfolded proteins), > purification from the periplasm could circumvent refolding if you are lucky > and you do not get cytoplasmic contamination. > > All the best > > > Dan >
