making disulphide bonds to aid refolding

[Emir Khatipov]

"Michael Witty" <mw132 at mole.bio.cam.ac.uk> wrote in message
news:Pine.SGI.4.33.0201090647330.5850536-100000 at mole.bio.cam.ac.uk...
> 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.
>
Michael,
You can try braking disulfides with TCEP (e.g., Pierce) which is a more
powerful reductant than DTT (plus it does not smell :) and available as
neutral pH solution.
As for re-formation of disulfides, I believe the best would be air
oxidation, which is slow and thus would be in accordance with you remark
about different speeds of formation of good and bad disulfides. I would try
the following: stir 0.1-0.5 mg/ml solution of your protein in a 10-20mM
buffer pH 7.4-7.8 for a 2-4 days at +4C. Take samples occasionally to
monitor bond formation using DTNB assay. Concentrate protein on Ni column.
Alternative methods include air oxidation in presence of granulated
activated charcoal (same amount as of a protein) - charcoal adsorbs small
air bubbled on its surface and this facilitates oxidation of the protein on
its surface. There is also a method of oxidation with K3Fe(CN)6, as well as
with DMSO that you mentioned. But those methods are more rigid and fast, whi
ch might be a disadvantage in your case. Please email me if you need
detailed protocols. I have them somewhere on a Current Protocols CD. But
generally it is as I said - low concentration solution of the protein to
minimize intermolecular cross-bridging, pH over 7 but below 8, air or
sparging with pure oxygen (rarely required though).

Emir