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
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
--
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
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
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)
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
