Hi Nick, I work with a 120kDa protein and using IPTG 50uM was crucial to get some soluble protein. Try to reduce the amount of IPTG and temperature, increase the induction time (24h,18°C) and volume of culture. It works fine for my protein and may be suitable for yours as well.
Best, Ricardo On Wed, 13 Jan 2010 15:53:22 -0600, nick wrote > Hello All, > > I apologize for the non-CCP4-related query. I have been working for > several weeks now trying, with limited success, to express some very > large proteins (ranging from ~100 to 180 kDa) from pET15b in E. > coli. "Limited success" means I have expressed enough soluble > protein to see on a gel, but not enough to purify. I have tried the > obvious tweaks - changing strains (BL21, BL21-star, Rosetta, pLysS), > screening induction temperature (16 to 37C), [IPTG] (0.3-1.0mM). I > am in the process of subcloning into vectors for (1) SUMO fusion > and (2) periplasmic expression (pET26b). I get the sense from > digging through the literature that high level expression of large > proteins depends mostly on the individual protein and I will > ultimately have to look for homologs. But, this is my first > experience expressing such large proteins and I am curious to know > if anyone out there has some magical trick they wouldn't mind sharing. > > Thanks in advance, > Nick > > ----------------------------------------- > Nicholas R. Silvaggi, Ph.D. > University of Wisconsin-Milwaukee > Department of Chemistry and Biochemistry > 3210 North Cramer Street > Milwaukee, WI 53211 > > Phone: 414-229-2647 > Email: silva...@uwm.edu -- Ricardo Augusto Pereira de Pádua Laboratory of Protein Crystallography of Ribeirão Preto Faculty of Pharmaceutical Sciences of Ribeirão Preto University of São Paulo - Brazil
