Apologies for revisiting this topic but I wanted to post a summary of the responses for the compulsive archive searchers like me.
Several people suggested the addition of ATP (5 mM) to the buffers either at lysis or once the protein is bound to the column (see ref: R. E. Joseph; A. H. Andreotti, Protein Expr. Purif. 2008, 60, 194–197.). I've attempted both options and both are pretty effective (~80% removal). Due to protein quirks I've have to drop the IMAC and go old school using straight ion exchange which actually cleans up all the residual Cpn60-bound protein (Cpn60 has a theoretical pI of 4.8) so downstream polishing is pretty easy. Additional suggestions included: 1) I once had a protein like this. From SDS-PAGE it looked like the protein was completely insoluble, but there was 1-2% which folded properly and bound to Ni-NTA with 6xHIS. This was enough to purify and crystallize. Take the supernatant and bind it to your capture resin. See what sticks. 2) Have you considered moving to a eukaryotic expression host like sf9/Tni, S2, HEK, or Pichia Pastoris? Can you co-express the protein with another protein you know it interacts with? 3) Try bumping up the salt and glycerol in your wash buffer to get rid of it. 4) Maybe you can try some useful ideas for your case listed in this study: Protein Expr Purif. 2007 April; 52(2): 280–285. *really cool paper BTW* 5) I used a panel of chaperonin plasmids from Takara and found one that folded my enzyme without co-purification. Thanks for all your help, Katherine On Mon, Sep 24, 2012 at 9:38 AM, Katherine Sippel < [email protected]> wrote: > Hi All, > > I've recently swapped over to expression in the ArcticExpress(DE3) cells > for a particularly rock-like protein. I've got soluble expression but I'm > having the issue of Cpn60 (the overexpressed chaperonin) piggybacking along > with the tagged protein. Google-fu and the CCP4bb archives indicate that > this is a known issue but I haven't seen a solution thus far. Does anyone > out there have any tricks up their sleeve? > > Also in case you were wondering in the various BL21 lines, even at > extremely low temperatures and 10 uM IPTG, it expresses well but produces > inclusion bodies that are completely insoluble in 8M urea, 6M guanidine, > high temperature, high pH, high reducing agent, and a number of detergents > so swapping back to another cell line isn't in the cards. > > Any help would be appreciated. > > Cheers, > Katherine >
