DK wrote:
> In article <mailman.899.1239820815.13724.methods from net.bio.net>, "Paul J. Phelan" <Paul.Phelan from tufts.edu> wrote:
>> 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?
>> Because GST forms dimers (which is surprisingly little known and
> responsible for probably countless incorrect conclusions in published
> papers), you can only do it two ways: 1) cleave off GST if your
> expression construct allows it, 2) run chromatography that
> separates GSG from your GST fusion in the presence of high urea
> concentration (at least 1M and as high as 2M may be necessary).
> This is only an option of your fusion partner survives such
> conditions.
>> DK
Getting rid of trace GST after cleavage is not trivial IMO. After
expressing a GST fusion protein we cleaved away the GST using a thrombin
cleavage site and ran an affinity column to get rid of uncleaved fusion
protein and free GST. The resulting flowthrough has a single band on
Coomassie blue stained gel but still has GST present as measured by ELISA.
We've switched to plan B - a different construct.
Allison
