[log in to unmask]" type="cite">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, <[log in to unmask]> 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