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gst fusion protein

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Tue Aug 1 00:25:16 EST 1995

In article <Pine.SOL.3.91.950731084921.13640A-100000 at terre>,
alarcoam at IRCM.UMontreal.CA (RAY-Anne-Marie Alarco) wrote:

> 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
> 110 ave. des Pins
> Montreal, Canada

Salut Anne-Marie,

I don't think you have a folding problem with your GST-fusion protein. If
so, you wouldn't get any retained material on the glutathione column.
Maybe, as you suggested, your proteins are in the inclusion bodies. In
this case you are obliged to use urea or guanidinium to solubilize your
protein and then refold the fusion protein. That's not a big deal. I can
give you a protocole.Sometimes you can't get rid of this problem by
playing on IPTG, temperature etc. Sometimes also, some proteins cannot be
expressed at a high yield or at all. I have experienced that my self.
Maybe you have a proteolytic problem with your DH5alpha? I strongly
suggest you to use another E. coli strain. I'm thinking to the BL21 cells.
It can help. Give it a try. If you see no improvement, change the
expression system.

Bon courage



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