Expression of toxic proteins:

ijiwaru at wheel.dcn.davis.ca.us ijiwaru at wheel.dcn.davis.ca.us
Wed Jul 10 14:31:15 EST 1996


In article <4s161g$aqe at delphi.bc.edu>, (KP) wrote:


> Let me try again.  I am using a pGex expression from pharmacia.  I induced
> it with IPTG and repressed the endogenous proteins with 2% glucose. 
> Basically, I followed the protocols provided by Pharmacia.   Also, I grew
> the bug in 2XYT media.  I did checked for my proteins in the insoluble
> fraction but nothing is detected.
> Thanks.

Its not endogenous proteins you will repress with the addition of 2%
glucose, its catabolite repression of your Plac.  Try doing a fresh
transformation of E.coli (strain of choice, preferably lacIq - especially
if its been over a month since you've done so) with your expression
plasmid.  Grow in liquid culture in the presence of glucose (I've found
that about 0.25% to 0.5% is sufficient for Plac in the context of
Pharmacia's pCANTAB5E).  When the culture has reached late log phase,
pellet the cells and rinse with medium minus glucose.  Pellet the cells
again and resuspend in medium minus glucose plus IPTG and grow for 2-4
hours.  Harvest and check for protein expression by your favorite assay. 
I grew mine in plain old LB.  I did find that you cannot simply add IPTG
to a glucose containing culture, you won't induce.

Hope this helps.

Lyle Najita
Plant Pathology
University of California - Davis



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