Protein purification

Artem Evdokimov AEVDOKIMOZ at cinci.rr.com
Sat Dec 15 12:19:40 EST 2001


Dear Richard,

What you are asking for is somewhat impossible, as a general case :) My
meager 5-6 years of purifying proteins have taught me that it is almost
impossible to really predict protein behavior during purification process.
In other words, one cannot have the cake and eat it, at least so far.

Here's some reasons why:

1) all proteins are different, thus it is impossible to devise ONE
non-affinity substrate which will just bind them all, and the problem
becomes even worse when you have partially folded forms in solution. Generic
affinity tags are the best solution to mass purification to-date.
2) denatured or partially denatured protein will most likely stick to the
native protein and to everything else.
3) some forms of denatured proteins are nearly indistinguisheable (by their
physico-chemical properties) from fully folded forms, yet they're inactive
and 'bad'.
4) some proteins are so difficult to work with that purification of *any
material at all* becomes a problem, not to mention ennrichment of the
active, folded material.
5) IMAC can kill proteins - and can't be used for everything, since many
things won't bind to it even when they are coupled to an affinity tag.

I can only suggest that you try to optimize your expression (by
experimenting with and carefully selecting groth conditions, strains,
constructs, fusion partners and tags) for the highest yield of folded
protein. This will be individual for each individual case - again, there's
no golden solution for all. (There may be a semi-generic 'optimum' for
groups of proteins, e.g. many small acidic bacterial proteins will fold well
in a certain range of conditions & expression vectors, but this does not
mean that mammalian small acidic proteins will fold well in those). Use more
than one tag for purification - this improves your chances for separation.
Don't scoff at HIC - it often allows partially denatured protein to unfold
even further which causes it to stick really tightly to the matrix. The
inherent danger of HIC is, of course, that it may unfold your protein of
interest.

And so on.

Generally speaking, hire an expert and bite the bullet - purificatin is
difficult and quite unpredictable :)

A.G.E.


"Richard Buick" <Richard.Buick at btinternet.com> wrote in message
news:9vfusg$ni8$2 at knossos.btinternet.com...
> Hi
> 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.
> Thanks
> Rick
>
> --
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>
> ***************************************************
> Richard J. Buick
>
> E-mail: mail at richardbuick.com
> URL: http://www.richardbuick.com
> Tel(GSM): +44 7801 416792
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> ***************************************************
> Richard J. Buick
>
> E-mail: mail at richardbuick.com
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