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
