HELP! GST fusion protein cleavage
gray at oci.utoronto.ca
Mon May 15 13:03:21 EST 1995
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
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