Dear Andreas, maybe the problem is rather rooted in your expression set up. If the protein was soluble in the small scale test, it is not necessarily the case in a fermenter run. The parameters may have changed so strongly that your protein is now expressed mostly insoluble. To my experience, such things can even happen scaling up from small flasks to bigger ones.
For some proteins it might be better to use fresh pellets. In my hands so far, I have not observed that freezing of unbroken cells matters, yet as I'm working on membrane proteins, I have noticed that as soon as the cells are broken and membranes are prepared, it can matter indeed if and for how long they are stored frozen. I guess, it needs to be tested for individual cases. Best wishes, Sabine On Mon, September 29, 2014 5:02 pm, Andreas Förster wrote: > Dear all, > > I've encountered people who refuse to freeze cells and always lyse fresh > pellets. Better protein, they say. I've never had reason to do so > myself, or even to believe in their voodoo. Up until now, maybe. > > My protein expresses well and is almost all in the soluble fraction in > an expression test from a fresh pellet. The large-scale expression from > the same pellet, now frozen and thawed, yielded 90% insoluble protein. > > If it's the freezing that dooms the protein, I'm happy to redo the > fermentor run. Are there other examples out there of this? > > Thanks. > > > Andreas > > > > > -- > Andreas Förster > Crystallization and X-ray Facility Manager > Centre for Structural Biology > Imperial College London >
