GST fusion protein solubility

Arnoud van Vliet arnoud at vanvliet.u-net.com
Tue Jan 13 20:11:26 EST 1998


Hi Attanasio,

when I was doing my PhD I had the same problem; nice expressions with
the GST system, but insoluble. A collegue got me to try the 6His
system from Qiagen (pQE9) and I was very happy with the results. The
protein was still insoluble, but I was able to purify it denaturing
and use it in an ELISA (and that was my goal).

Bottomline: what you wanna use the protein for? If it's for ELISA's or
immunisations, insoluble 6His proteins are great, If you want to do
function assays (gelshifts, enzyme assays) then it might not be good
as the denaturing conditions are exactly what they say they are:
denaturing thus linearising and removing possible functions.

just my two cents worth

good luck & hope this helps

Arnoud

Arnoud van Vliet, PhD
Dept of Genetics
Univ. of Leicester, UK

avv2 at le.ac.uk / arnoud at vanvliet.u-net.com

On 13 Jan 1998 04:41:46 -0800, atanasio at GUGU.USAL.ES (atanasio) wrote:

>Hi,
>
>I am experiencing troubles with GST fusion proteins (total Mr 65000). 
>Most of the protein stays in the inclusion body pellet. It can be 
>solubilized by 8M urea, but thereafter the fusion protein will not 
>efficiently bind to GSH-agarose. We have tried to grow bacteria at RT, 
>but with limited success. We have also tried to make a MBP fusion but we 
>are experiencing the same troubles.
>
>Does anybody now to how to make the fusion proteins soluble without 
>interfering with their ability to interact with the affinity column?. 
>Do you think it is worth to shift to poly-His tagging?.
>
>Thanks for all, and best of luck to the readers.
>
>
>Atanasio Pandiella
>Instituto de Microbiología Bioquímica
>Edificio Departamental
>Avda. del Campo Charro s/n
>37007-Salamanca-Spain
>
>Phone: +34-23-120561
>fax: +34-23-224876
>e-mail: atanasio at gugu.usal.es




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