HELP! GST fusion protein cleavage

William P. Prichett William_P_Prichett%notes at SB.COM
Thu May 18 16:42:32 EST 1995


Dawn,
     I have recently been purifing GST fusion proteins that a colleague
constructed and he had used BL21 as the host strain.  I have almost
undetectable 29k GST coming down with the GSH sepharose(by coomassie), with
strong bands of our fusion protein of interest(I can sonicate my cells in
only PBS without any difference in the band pattern).  You may want to try
that strain.  Also, since you are have apparent problems with the thrombin
cleavage site, you could sub clone into one of the later pGEX series
plasmids that contains the Factor Xa cleavage site.  Also, try checking
that you are not over sonicating your E. Coli, as this would tend to help
liberate a lot of free proteases.  Hope it works out.

Bill

In article <Pine.SUN.3.91.950515134811.5804A-100000 at pip>, Dawn Gray
<gray at oci.utoronto.ca> wrote:

> 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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