Concerning the contamination of recombinantly expressed proteins with
bacterial chaperones, one might wish to try a complete removal using ATP
and denatured protein as a "bait" as described here:
Protein Expr Purif. 2000 Oct;20(1):45-7.
Separation of copurifying GroEL from glutathione-S-transferase fusion
proteins.
Rohman M, Harrison-Lavoie KJ.
Might work or not but could save a lot of time consuming search for the
right fusion-tag.
Florian
Phoebe Rice wrote:
We haven't tried SUMO, but had some frustrating results with
GST fusions. They did improve expression and solubility - BUT
in one case the target protein precipitated immediately when
the tag was cleaved off, and resisted all attempts to bring it
back to life. In another case, the fusion protein dragged
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 <[email protected]>
Subject: Re: [ccp4bb] Off topic: Mammalian gene expression in
E. coli
To: [email protected]
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
<[email protected]> wrote:
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 you fear you have toxicity, simply
tracking the OD600 pre and post induction normally
tell you if this is happening. I've worked with
proteins that basically baselined the cell growth
upon induction and, as Artem stated, at least I
knew my protein was being made albeit at very low
levels.
Stephen
--
Stephen Weeks, Ph. D.
Drexel University College of Medicine
Department of Biochemistry and Molecular Biology
Room 10102 New College Building
245 N. 15th St.
Philadelphia, PA 19102
Phone: (+) 215-762-7316
Fax: (+) 215-762-4452
Mo Wong wrote:
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 am trying to
express a human protein in bacteria where the
only modified amino acids are 3 phosphorylated
serines. I’ve gone through the usual hoopla of
trying to get it expressed in E. coli
(Rosetta/Codon+ cells, varying IPTG, low
temperature, etc). Sequencing confirms my insert
is correct, but from coomassie gel inspection, I
appear to get near zero induction (I need to do
a Western to get a clearer assessment). I’ve
heard about custom gene synthesis, and it
appears Mr. Gene (https://www.mrgene.com/) would
be a good avenue to look into as they optimize
the ORF taking into account codon usage in E.
coli (though I’m not sure they examine
putative mRNA substructure formation like some
companies do). It’s only 49c per base pair, so
doesn’t seem too cost prohibitive. My only
concern is that if this protein is toxic, I
could be wasting money.
So I was wondering, has anyone seen the
expression for a particular protein change from
zero in Rosetta/Codon+ cells using "native"
sequeneces to being largely overexpressed in
BL21(DE3) cells using codon optimized sequences?
For folks who have had a similar problem to the
one I've described, would you recommend that I
first try using a codon optimized sequence in E.
coli over testing protein expression in
yeast/insect cells, or the other way round?
Thanks!
Phoebe A. Rice
Assoc. Prof., Dept. of Biochemistry & Molecular Biology
The University of Chicago
phone 773 834 1723
http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123
RNA is really nifty
DNA is over fifty
We have put them
both in one book
Please do take a
really good look
http://www.rsc.org/shop/books/2008/9780854042722.asp
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