Refolding urea-solubilized protein

mmp at mmp at
Wed Nov 2 12:44:54 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.


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