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 Bioe
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) Opt
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
O
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
Hi Nick,
Some success has been reported for large soluble proteins using the C41(DE3)
and C43(DE3) *E. coli* strains (see the paper by Miroux & Walker). Also you
can try another promoter/expression system , the pBAD expression system
based on arabinose induction.
Hope this helps.
Best
--
Pasca
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
