Refolding urea-solubilized protein
un691cs at genius.embnet.dkfz-heidelberg.de
Thu Nov 3 16:40:30 EST 1994
> I have overproduced a recombinant plant protein in E. coli, and it is
> insoluble. I can solubilize it in urea and isolate it by its Histidine tag
> on a nickel column, but I need to get rid of the urea and hopefully refold
> it correctly to reconstitute its activity.
> When I try to dialyze to remove the urea, the protein precipitates. What
> are some techniques used to promote folding? Should I add anything that
> will help stabilize the protein while it folds? What concentration is best
> for the protein in the dialysis tubing? Are there other techniques that
> might be better than dialysis? (I tried gradual removal by dialysis into
> lesser concentrations of urea, and it looked OK at 4 M, but precipitated at
> 2 M).
> If anybody can recommend any articles or books that might also help, these
> would be greatly appreciated.
I have the same problem. Currently, I dialyze first against NaPo4 (5 mM)
, 0.01% Triton X-100 and 6 M urea. Then I remove the Urea by very slow
dialysis against the same without Urea, this takes appr. 3 days. The
protein precipitates, so I centrifuhe and use the supernatant. The
concentration is about 20 micrograms / ml... and is biologically active.
Since I am working with a member of the TGF-beta family, I am thinking of
trying a very low pH (currently it is at 6.8), which may help.
hope this helps a little.
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