Thrombin cleavage problem
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
> 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
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