The old tricks could also be useful:
lower the induction temperature and IPTG concentration, and shorten the
induction time....
Does the degradation occur during induction or purification?
Pete
On Wed, 3 May 2000, Rick Thorne wrote:
> Dear Eva
>> All GST-fusions are different but I can offer you some guidelines which
> might help you.
>> First step might be to alter the strain of bacteria you are using for a
> more specialized strain eg: BL21-DE3 from Stratagene (look at the web
> site to learn more about these). Alternatives are available from other
> companies (but I never used them).
>> If this does not help, if at all practicable, make new construct/s with
> smaller bits of the protein you want to use. This sometimes removes
> parts of the molecule that encourage its wholesale destruction.
>> regards,
>> Rick
>> Eva Chen wrote:
>> > Is there anybody having experience on the purification of GST fusion
> > proteins?
> > Our lab doesn't have much experience and now there has been a serious
> > problem of degradation of the GST fusion protein. We have tried the
> > protease inhibitor from Sigma and Roche, and the whole purification
> > process was accomplished in a 4 C cold room. But the proteolysed
> > products are still there.
> >
> > Any suggestions are highly appreciated. Thank you all for your
> > attention!
> >
> > Eva Y. Chen
> > yi-hui.chen at mcmail.vanderbilt.edu> > Jim Sutcliffe Lab
> > 615-936-3627
> > Department of Molecular Physiology and Biophysics
> > Vanderbilt University School of Medicine
> > Nashville, TN 37232
> > USA
>>
