GST fusion prot isolation

Elaine M. Weidenhammer ew2f+ at andrew.cmu.edu
Fri Jan 14 12:20:36 EST 1994


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
elaine




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