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"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