Frank O. Fackelmayer
Frank at Fackelmayer.de
Tue Mar 26 04:09:44 EST 2002
It is probably a waste of time to try to clean up the protein in this
way. Instead, it will be better to solve the real problem, proteolysis.
In fact, the "free GST" you observe is the remnant of your fusion
protein being chewed up. This is a very common problem with GST
expression systems: GST is VERY stable against proteolysis, and usually
remains untouched when your fusion partner is already completely
digested by bacterial proteases.
So, avoiding proteolysis is a better way to "remove" the free GST than
trying to get rid of it later. Do all purifications in the cold, work
fast, and include protease inhibitors.
Mark Olson wrote:
> I have a GST fusion protein that I've purified using a Glutathione
> sepharose gel column. Unfortunately, along with my fusion protein, I'm
> getting a large amount of free GST. Since my fusion protein is at 60kd
> and the GST weighs in at 25kd, I was wondering if it was possible to
> simply centrifuge the solution with a membrane that cut off at 40kd so
> the GST would be in the waste.
> Anyone try this or forsee any problems?
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