In article <3692E895.F5E48209 at leonis.nus.edu.sg>,
Dongxu Liu <medp7060 at leonis.nus.edu.sg> wrote:
>Does anyone have the following problem as I ?
>>I expressed my protein as GST-fusion protein which bound to gluthione
>sepharose 4B. Unfortunately, my proteins still remains in the Sepharose
>after thrombin digestion on the matrix.
Are you having trouble removing the fusion protein from the matrix
with free glutathione? In other words, how certain are you that
the thrombin digestion worked? If it didn't, then the protein may be
stuck for the reason it was supposed to stick in the first place; by
binding to the glutathione on the column.
In the future, if you don't have experience purifying your fusion
protein to a column, it might be worth running a pilot experiment using
a few microliters of beads in a microfuge tube with small amounts of
your extract to make certain that you can both cleave and recover your
protein. This beats losing mass quantities of your protein in one shot
before figuring out if it can even be done. I've made several GST fusion
proteins, one very large and difficult to deal with. It couldn't be
cleaved with thrombin, which could be due to any number of problems (e.g.
steric masking of the thrombin cleavage site due to the structure of your
cloned domain).. I had no problem recovering my protein (with the GST
domain still attached) with glutathione, however.
Stacy Ferguson
