Separating GST from GST-fusion protein

Pow Joshi via methods%40net.bio.net (by pow.joshi from gmail.com)
Thu Apr 16 12:37:22 EST 2009


2009/4/15 Paul J. Phelan <Paul.Phelan from tufts.edu>:
> I think I asked this question before on this forum, but I thought I'd try it
> again just in case anyone has any new "tricks" or ideas, because I'm having
> the same old problem again.  I am trying to purify a GST-fusion protein, and
> it's contaminated with GST.  The GST-fusion has a mol. wt. of 40,000 and GST
> is 26,000, but on a Sephacryl S100 gel filtration column, both proteins are
> co-eluting in a single, beautiful peak.  Anyone would think that such a
> textbook peak would only contain pure protein, but no - half of it is GST!
>  I even re-ran the gel filtration column in a buffer containing 0.4 M NaCl
> to try to separate the two proteins, but I got the same single peak, so the
> GST must be still there.
> Now I am not talking about digesting a GST-fusion and then removing GST; I
> have no problem doing that, but I am trying to purify some intact
> GST-fusion, without taking GST with it: does anyone have a magic trick to
> get rid of GST?


ah, only if it is absolutely imperative that you have a pure protein,
and have some time at hand: perhaps you could generate polyclonals
against the whole mixture of gst+ gst-protein; then drain out the gst
antibodies against a gst column.... once that is done, and you are
left with the antibodies against your protein, which you can then use
to affinity purify your fusion .... Gee, but I guess it's far too
involved anyways .... perhaps Dima's and Wo's ideas are better to try
first....

best,
Pow
>
> Thank you
>
> Paul Phelan
>
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