HELP! GST fusion protein cleavage

Dawn Gray gray at oci.utoronto.ca
Tue May 23 11:45:36 EST 1995


Thanks to everyone for their help.  It seems like I am the only one who 
doesn't know about the Bl21 (ompT-) strain. There is an ompT site in the 
vector adjacent to the thrombin site which I now believe was causing all my  
sorrow.  I am moving my constructs now.  Thanks again.  Dawn.

On 18 May 1995, William P. Prichett wrote:

> 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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