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
Subject: Re: Off topic: Mammalian gene expression in E. coli
Just i
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 you
ehalf
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 or Apple :-
ckly, not waste time trying to
>> optimize a hopeless case, and decide whether to proceed with more
>> trials or to go in a different direction.
>>
>> Artem
>>
>> ---
>> When the Weasel comes to give New Year's greetings to the Chickens
>> no good int
hursday, 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 or Apple :-[ ).
With quite a few
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
r 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
Subject: Re: [ccp4bb] Off topic: Mammalian gene expression in
E. coli
To: CCP4BB@JISCMAIL.AC.UK
Th
were 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 20
chaperones into the prep that were 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
Subject: Re: [ccp4bb] Off to
, 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
> >Subject: Re: [ccp4bb] Off topic: Mam
gt;Subject: Re: [ccp4bb] Off topic: Mammalian gene expression in
E. coli
>To: CCP4BB@JISCMAIL.AC.UK
>
> 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!
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. Also
7.491.2438
cel: 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 expre
Did you not know that the CCP4BB-- even when all else appears to be
melting down-- is Nature's sustained gift to humankind? Did you also
not know that astronauts, astronomers, astrologers, artists,
musicians, sportsmen, scientists, politicians, writers and everyone
else subscribes to the bo
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 wrote:
> Mo,
> Just to add my 50 cents, I didn't see any mention of the use of fusion
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 for
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
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 pRARE
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
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
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
s
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? Ev
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 you
I thought I'd post this to the CCP4bb, as judging by previous posts, it
seems I could get some useful insight into my problem...
This is question has probably been asked by people for a long as molecular
biology has been around, but hopefully my question isn't a complete rehash
of other peoples: I
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