GST fusion proteins HELP

John Philo jphilo at amgen.com
Fri Sep 27 10:02:45 EST 1996


berez at WARREN.MED.HARVARD.EDU wrote:
> 
> I am trying to express a leech homeobox protein in bacteria to make an
> antibody.  The protein is insoluble.  I followed the Frangioni and
> Neel paper for solubilizing using sarkosyl.  I get a good amount of
> the protein into the supernatant, then I add Triton to restore binding
> to glutathione sepharose but I don't get a good yeild.  I get back the
> tiniest fraction of what is in the supernatant.  I also seem to get
> poor elution from the beads.  Any ideas?
> 
> Ashley Bruce

The experience of my colleagues here with GST-fusions is that often when
the GST fails to keep the fusion soluble (i.e. the fusion ends up in
inclusion bodies) then it is extremely difficult to get the GST portion
to refold into a conformation capable of recognizing the resin.  One is
really playing a kinetic competition game---if the GST portion folds
faster than its partner can cause aggregation/precipitation, then the
fusion stays soluble, you have some chance that the fusion partner will
fold too, and the resin will work for purification.  But once the
partner drags the GST into an aggregate, the GST portion seems to be
essentially irreversibly unfolded.

You may have to try to get by with the tiny fraction that remains
soluble (if there is any), or try another purification tag.

John Philo, Protein Chemistry
Amgen Inc., Thousand Oaks, CA
jphilo at amgen.com
*** Disclaimer: These are the opinions of the poster not Amgen Inc.***



More information about the Proteins mailing list