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 board....
Given the breadth of knowledge of the CCP4BBers, molecular biology is
still down the CCP4BBer's alley! Doesn't shock me that some molecular
biologists responded :)
The questions usually boils down to: To respond or not to respond?
Cheers,
Raji
On Feb 25, 2009, at 2:48 PM, Mo Wong wrote:
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.net> 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!
