gst fusion protein
alarcoam at IRCM.UMontreal.CA
Mon Jul 31 08:11:59 EST 1995
I am trying to raise antibodies against part of a yeast membrane protein.
In order to do so, I have generated two constructs designed to express two
different cytosolic portions of my protein (24
and 38 kDa each) in frame with the GST (PGEX system, Pharmacia).
On a small scale level, the bacterias (E. coli DH5alpha) do not express a
lot of the fusion protein. I have tried different induction conditions
(temperature, amount of IPTG, time of induction) but nothing seems to
significantly improve the amount of expressed fusion protein. In large
scale culture, the elution of the glutathione sepharose beads shows even
less protein than the small scale. Does anyone have any suggestion ? Does
anyone ever used proteins isolated from inclusion bodies to raise
antibodies in rabbits ? I can't really assay for the refolding of the
fusion protein since I don't have any enzymatic assay for my protein.
Thank you in advance for all your tips
110 ave. des Pins
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