GST fusion protein isolation

gc genecutl at mendel.berkeley.edu
Fri Jan 14 15:19:59 EST 1994


In article <QhBhI2_00iV0I4BIJv at andrew.cmu.edu>, "Elaine M. Weidenhammer"
<ew2f+ at andrew.cmu.edu> wrote:
> I'm having a problem with a GST fusion I've made with my favorite gene
> and would appreciate any advice.  I've cloned my gene into the pGEX-3X
> vector.  I see reasonable induction after 4 hours at 37 (not much
> induction at 30 degrees).  The fusion protein appears to be in inclusion
> bodies--after lysis by sonication in a buffer containing 2% Triton
> X-100, my protein is exclusively in the pellet fraction.  I extract the
> pellet in a buffer of (1.5% Sarkosyl, 25mM triethanolamine, 1mM EDTA,
> 10mM DTT) by stirring gently at 4 degrees for 10', followed by
> centrifugation.  I can see my fusion protein present when I run a
> fraction of this supernatant on a protein gel; the problem is that the
> protein  will not stick to the GST-agarose beads.  (I know the beads
> themselves are okay, because I've used beads from the same batch to
> isolate another fusion protein.  However, the latter fusion did NOT
> require extraction from the pellet.)  My hypothesis is that the
> detergent in the buffer I'm using to extract the pellet is inhibiting
> binding to the beads.  Does this sound reasonable, and, if so, does
> anyone have a protocol that will enable me to isolate my protein without
> taking large losses of yield?? Thanks







Do you have any reason to believe that your protein is properly folded?
Considering the fact that it's isolated from inclusion bodies, that
would be the first thing I would think of.  Do you have an activity you
can test?
If you have any of that other protein that does bind to spare, you
can always try putting it in the same buffer and see if that
will stick.










-- 
--gc



More information about the Yeast mailing list