Protein refolding - (Jul/21/2011 )
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.
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