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Separating GST from GST-fusion protein

Separating GST from GST-fusion protein - Protocols and Methods Forum

Separating GST from GST-fusion protein - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 04-15-2009, 05:47 PM
Paul J. Phelan
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Default Separating GST from GST-fusion protein



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?

Thank you

Paul Phelan

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  #2  
Old 04-16-2009, 12:17 AM
DK
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Default Separating GST from GST-fusion protein

In article <[Only registered users see links. ].net> , "Paul J. Phelan" <[Only registered users see links. ]> wrote:

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
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  #3  
Old 04-16-2009, 06:59 AM
WS
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Default Separating GST from GST-fusion protein

Hi Paul,

you might want to consider isoelectric focusing. For a nice
inexpensive colection of protocols, see Shen 2008. Methods in
Molecular Biology, vol. 424, Sample Preparation and Fractionation,
Volume 1, 187-203.

Best regards,

Wo
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  #4  
Old 04-20-2009, 02:44 PM
Allison
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Default Separating GST from GST-fusion protein

DK wrote:

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
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  #5  
Old 04-20-2009, 05:56 PM
DK
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Default Separating GST from GST-fusion protein

In article <49ec8633$0$9446$[Only registered users see links. ]>, Allison <[Only registered users see links. ]> wrote:

Running eluate over second GI column would get rid of the
residual GST.


That's right. GST is usually more trouble than it is worth.
Given the abundance of good fusion partners, all of them better
than GST in one way or another, it's a wonder why people
still use GST at all.

DK
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