Bernard,
Thank you for your reply.
I did check the digestion of my GST fusion protein by thrombin, at least 80
% was cut measured by the SDS-PAGE. Now I can get my protein off the beads
together with the GST itself and the GST-fusion protein by using higher
concentration of reduced glutathione. The problem now is how to separate
them.
pI MW
GST 6.19 27K
my protein 6.24 46K
Any suggestions will be highly appreciated.
Dongxu, Singapore
Bernard P. Murray, PhD wrote:
> 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
