pGEX-fusions

k_garson at icrf.icnet.uk k_garson at icrf.icnet.uk
Wed Jul 7 04:27:52 EST 1993


In article <munro.741763893 at sbsu1>, munro at sbsu1.aukuni.ac.nz (Gordon Munro) 
writes:
> My fusion protein is 70kDa,  but if I run a sample of th glutathione purified
> fusion on a SDS PAGE gel,  I get 2 bands,  one at 70 kDa as expected and one at 26kDa (about the right size for GST).   Unfortunatley the smaller band
> is by far the more prominent one sugesting that most of the bacteria in the 
> culture are only producing GST, not the fusion.
> I start with a single colony known to contain the fusion and have not treated the fusion with thrombin prior to sds-page,  so theoretically there should be no GST produced at all.  Has anyone had any expersience with  spontaneous loss of th
> their insert in pGEX-KG?  Or does anyone know if the fusion protein is unstable?
> 
> Any comments/ideas appreciated
> 
> Gordon Munro
> School of Biological Sciences
> Auckland University
> New Zealand

I have had similar problems and it appears that the linker region between GST
and your inserted amino acids is sensitive to contaminating proteases. Although
most of my protein preps are stable, on occasion I see a GST size band as well.
In one or two cases, storage of this sample eventually resulted in complete
cleavage of the fusion protein to the GST  band. Unfortunately, the rest of 
the fusion degraded completely such that I was left only with GST! Try more
stringent washes or different host strains.

Good Luck!

Ken Garson
ICRF, London, UK
k_garson at icrf.icnet.uk



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