I have worked with the optimization of several viral proteins, from different viruses/families, with different results.
When Menzella, 2011 was published I tried that method. I found quite the contrary result, in that randomization did far poorer than straight maximization of codon usage. I chucked this up to working within the context of viral infection (though it was also true in transient expression from plasmid transfection under CMV promotion).
I was quite excited by Pechmann and Frydman's Nature article which highlighted the importance of the location of the codon within the protein when regarding optimal vs suboptimal codon selection. Oddly enough we were able to deploy this information most successfully with the purposeful deoptimization of a protein.
Pairing a locational awareness, screening for secondary structure, and then maximizing codon optimization has had the best results for more than a year. By maximization, I mean choosing the most optimal codon in every instance that doesn't violate:
- Needed restriction sites
- Predicated Secondary Structure
- Location gradient to a lesser extent (ibid.)
This is all well and good, but we've been having problems with my institutions preferred vendor for gene synthesis. A post-doc that just joined my lab recommended a company I had never heard of before, and they posed an optimization strategy I had never considered before. Instead of maximizing codon optimization, they propose a "balanced" codon optimization that matches the normal distribution of codons in the target organism.
For example, the codon bias for alanine in humans breaks down (roughly) as follows:
- GCC 65%
- GCT 20%
- GCA 11%
- GCG 4%
As opposed to a maximization strategy which would seek to use "GCC" when ever possible and only when there was an issue switch to "GCT," the balanced stratgy would seek to replicate the above distribution as closely as possible.
Has anyone tried a balanced codon approach, especially when compared to a maximization technique? My overall goal is to maximize expression of naturally poorly expressing protein. I know that straight maximization can occasionally lead to poor expression for a variety of reasons (often fatal secondary structure), but this seems like it would hardly increase expression at all.
I would be further interested if anyone else had experience within a viral context.
I know that there have been a few related questions, mostly in the context of E. Coli. I don't think this is a duplicate, but would be willing to remove if it is seen as such. If I run into some extra funds I may test this empirically, but often when I'm trying such a test it's only in relation to 2-3 proteins, which might not be representative.