Balanced vs Maximized Codon Optimization

Question

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:

1. Needed restriction sites
2. Predicated Secondary Structure
3. 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:

1. GCC 65%
2. GCT 20%
3. GCA 11%
4. 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.

Answer 1

Codon usage optimization is still pretty much heurestic. The most common factor we think are important in E. Coli are:

• optimization to highly expressed genes, i.e. maximization of codon adaptation index http://www.ncbi.nlm.nih.gov/pubmed/19359587/
• keeping the optimal GC content http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0107319
• not introducing motifs that are somehow detrimental, e.g. restriction enzymes, or some dinucleotides http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0056642
• adapting to available tRNA http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007002
• minimizing folding energy (in the first ca. 30 codons) and minimizing the amount of hairpins at the 5' end http://www.sciencedirect.com/science/article/pii/S0014579305010719
• optimization of 'codon context' e.g. dicodons but also using nonoptimal codons between secondary structure for more accurate folding http://www.ncbi.nlm.nih.gov/pubmed/15772378
• optimization of codon balance and also maybe the introduction of a 'ramp of slow codons' in the beginning of a gene to avoid 'traffic jams' of ribosomes later on. http://www.ncbi.nlm.nih.gov/pubmed/20403329

Most probably it's a mixture of all of those.

Disclaimer: We are working on a codon optimization strategy for e.coli/yeast/hela and it works in e. coli but is yet untested in human cells. If you want to take a long shot just shoot me a message and we could try it. Apart from this, I would also offer you to have a look at your sequence and see if there is anything 'strange' from the codon usage point of view and what I think what be the best optimization strategy.

Answer 2

In a side-by-side comparison of 3 viral proteins (2 from paramyxoviridae, 1 from caliciviridae) each with both a "maximizing" and "matched" codon bias, we found the "maximized" version produced more protein the "matched" version in each instance. Protein levels were quantified via Western and flow, cell lines tested were 293T, HEp2 (HeLa contaminate), and Vero.

This is certainly not wide enough testing for any kind of general statement, it's just what we found. I think this will take a cell bio or pure micro-bio lab to fully study. We're in infectious diseases, and this question would be off grant and off topic for the paper. If we can find a way to squeeze it into a publication we will.

I'm going to accept Jan's answer because it contains more information on optimization, but with this answer in place to:

1. Address what we observed.
2. Note that Jan's doesn't offer any information on "matching" the codon bias of the target host (other than perhaps noting optimization is heuristic).