gst fusion protein

RAY-Anne-Marie Alarco alarcoam at IRCM.UMontreal.CA
Mon Jul 31 08:11:59 EST 1995


Hi everyone,
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    

Anne-Marie Alarco
IRCM
110 ave. des Pins
Montreal, Canada




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