Cleaving GST-fusion proteins

aw at aw at
Fri Oct 6 11:19:23 EST 1995

I have constructed a fusion protein using the pGEX-2T vector 
(Pharamcia).  The protein of interset is expressed (confirmed
by Western blotting) and I am able to purify it to a high degree
using Glutathione-Sepharose 4B.

Now for the problem.........I want to cleave my protein from
GST, using Thrombin.  I have attempted this but the protein
of interest and the fusion protein appear to be
eluted at the same time.  

Therefore, for some reason the cleaved glutathione-S-transferase
is not binding to the column and I am ending up with two bands on 
the SDS-PAGE gel.

Can anyone help me to obtain a purified cleaved protein?

Thanks for your help.

John Lloyd
University of Warwick 

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