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
