Can anyone help with protein expression in pGEX vectors?

lhanson lhanson at moose.uvm.edu
Mon Oct 5 19:39:39 EST 1992


	I've been trying to express 2 cDNAs in pGEX-2T.  Both cDNAs are
from a fungal library and encode proteins involved in mating reactions.
The predicted fusion protein should be about 67 kdal; glutathione S-transferase
(26 kdal) + cDNA encoded peptide (40 kdal).  I have followed the protocol
given in "Current Protocols in Molecular Biology" and haven't been having
much luck.  I can often see what appears to be an intensification of a
protein band around 70 kdal in induced cultures (as compared to an uninduced
control).  The fusion protein is expressed at significantly lower levels than
the parental vector alone expresses GST (gluathione S-transferase).

	Does anyone know of any "tricks" I might use to improve expression?
I've tried inducing in early log phase (1.5 hrs after inoculation) as well as
later (4-5 hrs post inoculation).  I've even tried growing plates of ~600-1000
colonies overnight and then suspending them in LB (as a slurry) and then adding
inducer (IPTG to 1-3.5 mM final).  This last technique works great for the
parental vector alone but not for fusions.  I think I've tried the other
obvious experiments such as upping the ampicillin to 200 ug/ml, checking
my plasmids to ensure they are the constructs I believe them to be, using
Terrific broth as opposed to LB.

	I'm after enough protein to use in rasing antibodies.  Affinity
purification of large cultures isn't the answer at this point either.  I
cannot get affinity purification of the small quantity of fusion protein
I can get expressed as I believe its in inclusion bodies!

	Lee Hanson
	lhanson at moose.uvm.edu

	Department of Botany
	University of Vermont



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