GST-fusion expression problem

Didier Thomas dmthomas at orion.oac.uci.edu
Wed Aug 21 02:26:48 EST 1996


In article <4v7him$rc9 at swen.emba.uvm.edu>, houchens at med.uvm.edu (Chris
Houchens) wrote:

> I have made several GST-fusion expression clones (in pGEX-2T) designed to 
> express either a full-length protein of a gene or various N- and 
> C-terminal truncations of the same gene. The inserts were generated by 
> PCR and, therefore, the C-terminal truncations contain the same 
> N-terminal sequence (confirmed by sequencing the fusion junction). 
> 
> THE PROBLEM IS: while all the truncations induce to relatively high 
> levels, I am unable to detect ANY induction of my full-length protein 
> (even though, again, the full length and C-terminal truncations have the 
> same 5" sequence). I would at least expect to see induction of some 
> protein due to protein degradation of introduction of a stop codon in the 
> insert due to PCR. But I see nothing at all!!! The -IPTG and +IPTG sample 
> of boiled cells are identical. Can anyone provide some insight as to what 
> might be happening? The bacteria carrying the full-length fusion seem to 
> grow as well as all my other constructions, so I do not think that 
> toxicity is a problem (but I could be wrong). I have also tried 
> expressing the full-length in a number of bacterial cells including DH5's 
> and TOPP1's (next is BL21's). Any help would be greatly appreciated, If 
> you could e-mail directly, that would be great. Thanks in advance for 
> your help.
> 
> Chris Houchens
> houchens at salus.med.uvm.edu

Hi Chris,

Your problem maybe that your full length protein is too large. In the
original paper describing the pGEX expression system, it was already clear
that there is an inverse relationship between the length of the fusion
protein and the 
yield of expression. In brief: the shorter the better. I experienced
myself this problem with my constructs in a bacterial expression system.
This problem disappears when GST fusion proteins are expressed in an
eucaryotic system.

Hope it helps

Didier

-- 
Didier Thomas
University of California, Irvine
College of Medicine
Dept. of Biological Chemistry
Med. Sci. I, Room D214
Irvine, CA 92717
USA



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