GST fusion protein

rdudley at aherf.edu rdudley at aherf.edu
Wed Jul 8 07:49:41 EST 1998


In article <6nsj13$p6c at mserv1.dl.ac.uk>,
  Luc CAMOIN <camoin at cochin.inserm.fr> wrote:
> Dear netters,
>
> I expressed a fusion protein produced in E Coli. The fusion protein is a
> GST tagged protein and the predicted molecular weight is around 45 kDa. My
> protein of interest is a very hydrophobic protein of 19 kDa.
> Purification on Glutathione sepharose 4B and western blot with anti GST
> revealed only a band around 25 kDa. This molecular weight and reactivity
> with anti GST could suggest that this protein is GST alone.
> Can we conclude that the contact between GST and the protein of interest is
> a protease sensitive region? Is this a known problem? Did you also find
> this problem in your experiments? Can you give me references about this
> problem?
<snip>

Make sure there isn't a frame shift between the GST and your protein.  Also,
is there the chance that you have a mixed population in your cultures? 
(i.e., some bacteria contain a GST-only plasmid, and some contain a
GST+insert plasmid)  I only aske because I was working with a protein that
had a very hydrophibic region, and eventually found it would stick to the
glutathione sepharose in a non-specific manner.  We coul dnever elute it with
GST alone.  You may want to do a trial experiment where you run out a sample
of every step along the way--including crude lysae, unbound fraction,
purified sample, and some of the sepharose--and do a western blot.

rich
rdudley at aherf.edu

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