> I agree with DK, hydrophobic interaction might be the best solution, it
> might have very high resolution for different folding forms. But it needs
to
> be specifically optimised for every protein....
Indeed. Add to the equation that many proteins do not like high salt. Many
other proteins just do not bind to HIC matrices without unfolding, and yet
more proteins bind and come off together with the partiallyunfolded stuff or
dirt in general.
> 1) what about native IEF ? - but it too can not be used for every
protein -
> many will precipitate.
Chromatoelectrofocussing ? Expensive, may work in some cases, however will
not work in a genera case, I am willing to bet.
> 2) also, if you are working with small proteins stabilised by disulfides,
> may be HPLC will help
These usually either fold OK or fold as a mixture of mis-crosslinked
disulphide adducts. The latter, indeed, are often resolvable by HPLC,
however in many cases HPLC murders the final product.
> may be it would be better just to use the native host, and expressing the
> tagged proteins in it ?
Oftentimes overexpression results in partial misfolding. We just had a case
similar to that where massive overexpression resulted in improper folding
(undetectable by any normal means except for some indications by IEF (even
activity was OK !)).
Hey, look at the bright side - if there were generic methods of protein
purification that worked - who'd need us ?
A.G.E.
