Nick,
We have recently got much improved expression (both total amount and in
soluble form) of 72 kDa protein by using the EnBase Flo cell cultivation kit
from Biosilta (www.biosilta.com). The method is based on enzymatic slow
release of glucose from a starch(?)-like polymer - thus mimicking a
fed-batch principle of bioreactor cultivations, but in shake flasks.
Induction is done at a much higher cell density than conventionally, and
long induction periods (24 h) can be used. Consequently the yields become
higher and the folding/refolding machinery has time to make more correctly
folded protein. Smaller cultivation volumes can be used, yet results in more
cells and product. We also tried with a 100 kDa protein without great
improvement yet. But the kit is certainly worth a go.
Tuomo Glumoff
==========================================
Tuomo Glumoff, FT Tuomo Glumoff, Ph.D.
lehtori, dosentti lecturer and docent
Oulun yliopisto University of Oulu
Biokemian laitos Dept. Biochemistry
PL 3000 BOX 3000
90014 OULUN YLIOPISTO FI-90014
UNIVERSITY OF OULU
Finland
==========================================
Kontaktitiedot - Contact information:
www.biochem.oulu.fi/henkilokunta/glumoff/
==========================================
matkapuh. / mobile: +358-(0)50-5226136
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-----Original Message-----
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Will
Stanley
Sent: 14. tammikuuta 2010 3:18
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Expression of large proteins in E. coli
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 90:805-820)
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
