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 guesstimate the success of
expression in E. coli (i.e., consider codon frequency). Does one
exist? Even better, a web server that offers the choice of different
commercial forms of E. coli (i.e., Rosetta and Codon+)? I tried
Googling this a few days ago and didn't find anything too useful?
Cheers!
On Tue, Feb 24, 2009 at 11:00 AM, <ar...@xtals.org
<mailto:ar...@xtals.org>> wrote:
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
still may derive a benefit from codon optimization and especially from
mRNA structure shuffling. There are articles out there that attempt to
present statistically significant evidence and I tend to agree
with them -
the practice of optimizing codon usage AND mRNA structure is a
good and
useful one.
>From what you describe, you're working with a kinase. It is not
at all
uncommon to have toxicity of kinases for E. coli. Additional issues
include heterogenous phosphorylation if the kinase is normally
active or
can auto-activate in E.coli (classical example is PKA which can
phosphorylate itself in up to 30 places given the right conditions).
Toxicity isn't difficult to spot - classical signs include
microcolonies,
heterogenous colonies, slow growth, plasmid instability and so on.
Even
with a native gene you would likely notice these symptoms to some
extent.
Incidentally - toxicity usually equals folding, i.e. for a kinase
to be
toxic at least some of it has to be folded enough to work. This is
actually *good news* because toxicity can be combated on a
different level
than lack of folded expression. For example, co-expression with
phosphatases tends to work miracles for kinase-based toxicity.
Finally, to answer your question directly - yes, i've seen several
cases
of proteins not expressing even with rare codon tRNA supplemented in
trans, but expressing well from optimized DNA. Again - optimized for
codons AND structure, as I've never separated the two processes.
Synthetic DNA is cheap these days. If you can afford it - it's
useful to
try before taking the next step. In this case the obvious next step is
attempt at expression in insect cells - kinases usually work out
really
well in IC.
Artem
--
Raphael Gasper-Schönenbrücher
Max-Planck-Institut für molekulare Physiologie
Abt. für strukturelle Biologie
Otto-Hahn-Straße 11
44227 Dortmund, Germany
Tel: +49/231/133-2121
Fax: +49/231/133-2199
