Incubation with MgCl2/ATP/KCl facilitates the release of the Cpn60. See: R. E. Joseph; A. H. Andreotti, Protein Expr. Purif. 2008, 60, 194–197.
Best, Cynthia __________________________________ Cynthia Kinsland, Ph. D. Director, Protein Production Facility Cornell University B78 ST Olin Lab Ithaca, NY 14853-2703 Tel: (607) 255-8844 E-mail: [email protected] On Sep 24, 2012, at 10:38 AM, Katherine Sippel 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
