GST-fusion protein ?????

[Bernard P. Murray, PhD]

In article <3692E849.9D7687F6 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 there any ways to recover my protein ?
> Thanks for help.

Assuming you can elute the undigested protein by some means
(GSH or other conditions) can you cleave the protein in
solution?  If so you could do this and then purify your
protein away from the tag using the GSH-sepharose.
     If the problem is poor digestion then it is worth
exploring a range of conditions.  The suppliers of the
thrombin give fairly conservative initial conditions to
stop you shredding your protein but if there is little or
no cutting then try warmer, longer or a different buffer
(eg. add a little Ca2+).
     I hope that this helps,
          Bernard
-- 
Bernard P. Murray, PhD
Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA