, 2009 9:31 PM
*To:* CCP4BB@JISCMAIL.AC.UK
*Subject:* Re: [ccp4bb] Off topic: Mammalian gene expression in E. coli
Hi all,
Once again I seem to have managed to kick up a minor debate on the
bulletin board (Note to self no more posts on SUMO or Apple :-[ ).
With quite a few years of experience
] On Behalf
Of Stephen Weeks
Sent: Thursday, February 26, 2009 9:31 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Off topic: Mammalian gene expression in E. coli
Hi all,
Once again I seem to have managed to kick up a minor debate on
the bulletin board (Note to self no more posts on SUMO
, 2009 9:31 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Off topic: Mammalian gene expression in E. coli
Hi all,
Once again I seem to have managed to kick up a minor debate on
the bulletin board (Note to self no more posts on SUMO or Apple :-
[ ). With quite a few years of experience working
Hello,
The short answer is 'yes'. If you can use both methods :) The issue with
limited proteolysis lies in the questionable state of the full-length
protein - if the stuff is nasty and misfolded, then fagments generated by
proteolytic digest aren't going to be meaningful. On the other hand if
Most of the poorly cleavable fusion proteins (usually MBP-TEV) that
I've seen turned out to be solubly aggregated.
ho
UC Berkeley
--
Date:Fri, 27 Feb 2009 07:23:43 -0500
From:Stephen Weeks stephen.we...@verizon.net
Subject: Re: Off topic: Mammalian gene
to drag such crud along
with it?
Phoebe
Original message
Date: Wed, 25 Feb 2009 14:48:57 -0500
From: Mo Wong mowon...@gmail.com
Subject: Re: [ccp4bb] Off topic: Mammalian gene expression in
E. coli
To: CCP4BB@JISCMAIL.AC.UK
Thanks to all who responded. Actually
nearly impossible to get
rid of completely, thus ruining our ATPase assays.
Is SUMO, being smaller, less likely to drag such crud along
with it?
Phoebe
Original message
Date: Wed, 25 Feb 2009 14:48:57 -0500
From: Mo Wong mowon...@gmail.com
Subject: Re: [ccp4bb] Off topic: Mammalian
impossible to get
rid of completely, thus ruining our ATPase assays.
Is SUMO, being smaller, less likely to drag such crud along
with it?
Phoebe
Original message
Date: Wed, 25 Feb 2009 14:48:57 -0500
From: Mo Wong mowon...@gmail.com
Subject: Re: [ccp4bb] Off topic: Mammalian gene
.
Is SUMO, being smaller, less likely to drag such crud along
with it?
Phoebe
Original message
Date: Wed, 25 Feb 2009 14:48:57 -0500
From: Mo Wong mowon...@gmail.com
Subject: Re: [ccp4bb] Off topic: Mammalian gene expression in
E. coli
To: CCP4BB@JISCMAIL.AC.UK
Hi all,
Once again I seem to have managed to kick up a minor debate on the
bulletin board (Note to self no more posts on SUMO or Apple :-[ ). With quite a few
years of experience working with SUMO I feel I can safely state that it
is a good enhancer of fusion protein production in E. coli. I
Thanks to all who responded. Actually, this bulletin board is better for
help with molecular biology than the molecular biology bulletin board I am
subscribed to!
On Tue, Feb 24, 2009 at 7:47 PM, Stephen Weeks stephen.we...@verizon.netwrote:
Mo,
Just to add my 50 cents, I didn't see any
: 773.608.9185
email: j-kell...@northwestern.edu
***
- Original Message -
From: Raji Edayathumangalam
To: CCP4BB@JISCMAIL.AC.UK
Sent: Wednesday, February 25, 2009 2:01 PM
Subject: Re: [ccp4bb] Off topic: Mammalian gene expression in E
Some useful tips to try can be found at
http://www.embl-hamburg.de/services/protein/production/expression/optimising_exprlevels.html
I've had a recent case where an untagged protein (part of a complex)
was not expressed at all but expressed well when tagged at the
N-terminal with His6 or MBP.
Hi,
The question you have to ask yourself first is - does my gene actually
*have* the rare codons that you're trying to avoid? Experience shows that
usually it's not single codons that are a problem but pairs or triplets of
rare ones. If your gene does not have obviously bad codon combinations
Thanks for the reply.
I've checked my sequence for rare codons; however, what would be useful to a
pseudo-molecular biologist like me is a web server which will look at your
input DNA sequence and guesstimate the success of expression in E. coli
(i.e., consider codon frequency). Does one exist?
Just to add couple of things to what Artem said..
If it is something similar to a mammalian kinase or malaria protein for
example,
1. Recodonizaton can change expression from near zero to substantial
amounts, however,
a) ideally, it needs to be recodonized separately for each target expression
This website is quite useful (via Expasy):
http://gcua.schoedl.de/
Raphael
Mo Wong schrieb:
Thanks for the reply.
I've checked my sequence for rare codons; however, what would be
useful to a pseudo-molecular biologist like me is a web server which
will look at your input DNA sequence and
Hi Mo,
Gene synthesis is definitely something you should try if you can afford
it.
However, I would suggest also trying to change expression plasmid and in
particular induction system and promoter system.
For toxic proteins we had some success using the pBAD (invitrogen)
expression system using
Hi,
Servers to check for rare codons definitely do exist.
http://genomes.urv.es/OPTIMIZER/
http://www.doe-mbi.ucla.edu/~sumchan/caltor.html
and several others
As to comparing Rosetta and Codon+ - your best bet here is just to dig up
product literature from the manufacturers.
Rosetta uses
Mo,
Just to add my 50 cents, I didn't see any mention of the use of
fusion proteins in your original post. GST, MBP or my personal, and
completely biased, favourite SUMO (plus many more proteins) have been
shown to enhance expression when fused to the amino terminus of a target
protein. If
Message-
From: CCP4 bulletin board on behalf of Raphael Gasper
Sent: Tue 2/24/2009 9:56 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Off topic: Mammalian gene expression in E. coli
This website is quite useful (via Expasy):
http://gcua.schoedl.de/
Raphael
Mo Wong schrieb:
Thanks
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