Hi Nick, A couple of other suggestions:
(1) Try an E. coli K strain (e.g. JM109). They're different in glucose metabolism (JN Phue et al, 2005, Biotechnol Bioeng 90:805820) and in my experience tend to grow more slowly - which seems to help improve yields of large constructs.
(2) Optimal oxygenation also seems to be very important in producing large constructs. Not sure what vessels you're using but Fernback flasks work well for me. Or a fermenter might solve all you problems.
Good luck, Will.
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
