GST Fusion Proteins in Yeast

Dan Zabetakis dan at cubsps.bio.columbia.edu
Sat Jul 8 10:02:26 EST 1995


In article <3tkgd9$pt at news.service.uci.edu>,
Bill Brickey  <wwbricke at uci.edu> wrote:
>      1. Is there something in yeast extracts which interferes with
>         binding?  Are there alternate protocols ?


   I don't think there is any overall problem in yeast. People in my lab have
expressed GST fusions in yeast, but purification was limited by the lower
expression levels.
  There may be other explanations. A protein I have a GST fusion with seems
to stick very tightly to my column (Pharmacia pre-packed). Our procedure
called for elution with 1mM glutathione but I found that it wasn't coming
off. I thought it was not sticking, but when I used more glutatione (I now
elute with 20mM in 7 column volumes) a ton came tumbling off the column.
In earlier experiments I didn't see the protein in the high salt washes we
do at the end either, which is strange.

DanZ


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