making disulphide bonds to aid refolding

Michael Witty mw132 at
Wed Jan 9 02:17:33 EST 2002

Dear All,
        my particular protein at the moment, a phage coat protein,
contains disulphides which cause a ladder of monomers: one-mers right up
to sizes that precipitate.  I can break them with 10mM DTT, but when I try
to refold the protein by removing DTT and 8M urea (protein bound to
Ni-NTA, to prevent agregation) I get back my ladder of multimers.  I
suspect that the "good" disulphides are slow to form and the "bad"
disulphides are fast to form.
     My solution would be to speed up disulphide formation while protein
is bound to Ni-NTA: is there a convenient reagent for this?  I have read
discussion of DMSO on this newsgroup before.  Is this the only commonly
used reagent?  What concentration do people use?
     Also, should I expect a protein to snap back into shape upon removing
8M urea or is it a time consuming process?
     TIA for all comments.  Regards, Mike.

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