On Sat, 15 Dec 2001, Richard Buick wrote:
> I wonder if anyone could suggest a method for the separation of active from
> inactive (mis-folded) protein. Affinity chromatography is not an option
> since the method must be applied to many different proteins. Ion exchange
> and hydrophobic interaction have been tried with not much success. The
> proteins to be separated are post IMAC.
I could suggest something a bit different. I am more optimistic
than some of the other posters. At the same time, I am not
optimistic about you separating out the different folds of
this protein by gel filtration, mainly because the resolution
of gel filtration is very poor, typically. I would predict
only a widened peak for a mix of different folds, which
might not even be detectable.
I would explore the active site of this protein, and use
of proteases. Many proteases are opportunistic, attacking
active sites of enzymes, etc, when they are exposed. You
could consider adding binding partners, substrates, etc
in the presence of proteases. It obviously helps a great
deal if you have sequence information so you can identify
protease cleavage sites. This protocol can give a pool of
proteins with very different molecular weights that might
separate a lot better by gel filtration. Come to think of
it, you might even be able to combine the protease
treatment with dialysis, depending on the sizes involved.
Consider too, techniques like circular dichroism and
dynamic light scatter. You say you are concerned about
structural differences. I think these should be
possible to identify by these techniques. You should
be able to at least discern between a mixed and a
pure population of folds.
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