Can anyone help with protein expression in pGEX vectors?

rpgrant at vax.path.ox.ac.uk rpgrant at vax.path.ox.ac.uk
Tue Oct 6 04:41:34 EST 1992


In article <1992Oct6.003939.10137 at uvm.edu>, lhanson at moose.uvm.edu (lhanson) writes:
> 
> 	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).

One thing you might try is to reduce the temperature of induction (and 
increase the time) with IPTG; try (say) 6h at 30C and/or overnight at 25C.  
This is reputed to help with insoluble fusion products.  Also try varying the 
final concentration of IPTG.

> cannot get affinity purification of the small quantity of fusion protein
> I can get expressed as I believe its in inclusion bodies!

See above.
Also, you might try the pMAL system from New England Biolabs.  This is 
conceptually the same, giving fusions with maltose-binding protein, so you can 
purify the products from an amylose column.  The cleavage from MBP is affected 
with Factor Xa (as with pGEX-3X).  The really neat thing is that they have a 
vector, pMALp2, which has the MAL signal sequence included, so you can purify 
products from the periplasm.  Again, this should help if you get inclusion 
bodies in the cytoplasm.  I did come across a protocol which purported to 
achieve recovery from inclusion bodies, but I can not remember where.

There is another system by Studier, and you can get a catalogue from the 
company which distributes them;

Novagen inc
565 science drive
Madison WI 53711 USA

fax 608-238-1388

phone  800-526-7319 or 608-238-6110.

(Thanks Rod!!!!)  The appropiate bits are the pET vectors.

Best of luck,

Richard Grant,
Sir William Dunn School of Pathology,
Oxford University

Email rpgrant at vax.path.ox.ac.uk



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