Separating GST from GST-fusion protein

Paul J. Phelan via methods%40net.bio.net (by Paul.Phelan from tufts.edu)
Wed Apr 15 12:47:56 EST 2009


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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