Thrombin cleavage problem

Jim Woodgett jwoodget at oci.utoronto.ca
Thu Aug 18 10:33:16 EST 1994


In article <32v7dl$33m at mserv1.dl.ac.uk>, grggta at picr.cr.man.ac.uk (Graham 
Atherton) writes:
  
> We are attempting to purify a protein expressed by the Pharmacia 
> GEX system. The protein expresses well and binds to the beads well 
> when purifying but once treated with thrombin to cleave the 
> fused polypeptide from our protein we get three or four bands of 
> smaller MW than our protein. It is thus possible that the thrombin 
> is cleaving our protein in several places? 
> Has this happened to anyone else and if so, what can be done? 

Thrombin cleavage of GST fusion proteins can be a real pain and is highly 
dependent on the protein of interest (which is true of any protease cleavage 
strategies).  There are two things you can do to increase your chances of 
getting it to work more specifically.

1. Use the most highly purified thrombin you can buy (highest specific 
activity and price..).

2.  Use pGexKG which was developed in Jack Dixons lab and is based on pGex2T 
but has a glycine polylinker to increase accessibility of the thrombin to its 
cleavage site.  See Anal. Biochem. 192, 262-267 (1991).

This won't guarantee success as your protein may be susceptible to protease 
cleavage.  

Jim








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