Protein refolding - (Jul/21/2011 )

Dear all,

I ordered the production of FGF-B in E. coli and I obtained the purified protein in PBS plus 8M urea. I was told the protein went to inclusion bodies and thus, they had to used urea to dissolve the protein. The point is that I need the native protein. Does anyone know of a good protocol to refold this protein or a related one? If not that specific, any more general protocol?
I was thinking of dialysis... is it possible to progressively reduce the urea concentration? Should I use glycerol in the dialysis buffer as well?
Thanks a lot in advance!

I'm not sure of a dialysis protocol, but if you are going to do it, I just found out about a product from Merck called D-Tube dialyser. I don't know too much about it but I will probably be purchasing to do some dialysis. I know this doesn't help your problem directly, but just an FYI anyway.

http://www.merck-chemicals.com/australia/life-science-research/d-tube-dialyzer-mini-mwco-12-14-kda/EMD_BIO-71505/p_TVib.s1ODP0AAAEj8Rl9.zLX

Hola of course you could establish your own refolding protocol, diluting urea and dyalizing or ultrafiltrting. But in my experience always is better purify a soluble expressed protein unless it is an smaall amount, than refold a huge amount, because you could obtain pure protein with low activity.I have seen that commercial protein is obtained from E.coli, so is soluble produced or is easy refolding. I have seen that some people produced fusion proteins with GST, thioredoxin or SUMO. As I see that you are in a big intitution with expression service, think in use your construction for light soluble expression if there is, or starting from soluble fusion protein.
Returning to refolding, if it has S-S intramolecular you have to add reducer to your urea solution, and having present that intermolecular S-S (reaggregation) could be formed if the concentration is too high or the pH is near the isoelectric point . Buena suerte