Hey Nick,
Short answer is that there are no magical tricks, as you probably
already expect to hear. Seems there's a lot that you are trying already.
I have some limited experience with some 125kDa, 100kDa proteins.
1. I've had some luck with the E. coli C41, C43 strains. Especially if
your proteins are inherently toxic to E. coli. There are commercial
vendors who sell these strains with the plus or minus pLysS options.
2. Try moving the tags to the other terminus N- to C- or vice versa.
Can't do that for SUMO in any easy manner.
3. Try MBP tag as well
4. Try Studier's autoinduction protocol
5. Try expression with chaperone kit, trigger factor (Takara)
6. You don't mention whether the protein is human etc., but you may
have to move to yeast or insect cells, in the worst case.
DISCLAIMER: I am not paid by Takara to mention their kit.
Good luck!
Raji
-----------
Raji Edayathumangalam
Joint Research Fellow
Brigham and Women's Hospital/
Harvard Medical School
Brandeis University
On Jan 13, 2010, at 4:53 PM, n...@silvaggi.com 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
