Inducing GST fusion proteins - help needed.

Aron B. Jaffe jaffe at a1.mscf.upenn.edu
Fri Apr 28 17:12:30 EST 1995


grieger at ksu.ksu.edu (David M Grieger) wrote:
>
> Dear recombinant protein enthusiasts:
> My student is trying to produce a fusion protein of 40kD with the GST-pGEX 
> expression system.  After purifying through a glutathione columun, we end up with a few band of larger molecular weight than wanted, and the fusion product is very low in yield.  We have tried altering the IPTG concentration from  0.1mM to 1.0mM and also decreased the growing temp. from 37 to 30 C.  Not sure if we'
> re just not inducing the plasmid efficiently or if the protein is being packaged into inclusion bodies.  ANY suggestions to increase our yield would be greatly appreciated.  If you come thru, the "check's in the mail".  Thankyou
>   

To see if the protein is in inclusion bodies, add Laemmli buffer to the
bacterial pellet and run that out on the gel... if it's in there, you 
should see a band that is obviously induced.  If induction is the 
problem, try inducing at different points (when OD is .5, .6, etc.)
and for different times... 

As far as the larger bands you are seeing... your student may not
be washing the beads well enough (you can never wash too much),
or you may have an inefficient stop codon and get some read-through...

good luck

aron



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