GST-fusion expression problem

Chris Houchens houchens at med.uvm.edu
Sun Aug 18 11:49:26 EST 1996


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




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