In article <2vgv6a$fdq at mserv1.dl.ac.uk> mbdxb at s-crim1.dl.ac.uk (D.R. Bell)
>We have a GST fusion protein which comes out as insoluble.
>>Has anyone tried solubilising such proteins, and then trying affinity
>purification of the refolded GST-fusion protein?
>>Any comments and protocols welcome!
I'm pretty sure that I've seen this paper referenced here before, but I hope
you'll find it useful anyway:
Solubilization and purification of enzymatically active glutathione
S-transferase (pGEX) fusion proteins, Frangioni and Neel, Analytical
Biochemistry, vol 210, 179-187 (1993).
Authors state that sonicating the cells with sarkosyl produces extract with
higher amounts of soluble, active fusion protein. Sarkosyl worked while
various other ionic or nonionic detergents alone didn't. Best results obtained
with sarkosyl and Triton combined.
I haven't tried using this scheme myself, but a colleague swears by it.
Institute for Molecular Biology and Biophysics
ETH-Honggerberg (Swiss Federal Institute of Technology)
8093 Zurich, Switzerland
email: tan at aeolus.vmsmail.ethz.ch