HELP! GST fusion protein cleavage

Dawn Gray gray at oci.utoronto.ca
Mon May 15 13:03:21 EST 1995


Hi,
I am having trouble with premature cleavage of my gst-fusion protein.  I 
am using Pharmacia's pGex2Tk vector.  I can pick up the complete fusion 
protein (the correct size plus the gst) from a crude lysate of an induced 
sample by western blotting.  
However, after purification, I only get GST by western.  When I ran some 
of the supernatant off the GSH-sepharose beads I could pick up the 3'-end 
of my protein (the correct size minus the gst) by western blot.
Therefore, it appears as if my protein is being cleaved sometime during 
lysis.  I have used a different stock of GSH-sepharose, triton X, and 
PBS.  Three of my constructs are in XL1-blue; the fourth is in DH5 - and 
yes, I get cleavage in all of them!!  I have also tried adding a battery 
of protease inhibitors to the PBS in which I resuspend my pellet.
Any suggestions would be greatly appreciated,
Thanks in advance
a very frustrated
Dawn



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