Questions and Answers

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Why we need to optimize the codon usage for yeast

The short answer is: Because each codon has multiple possible tRNAs that recognize that codon, and the abundance of tRNAs differs between species.

As an example, there are several different codons that code for Serine (UCU, UCC, UCA, UCG) but in one species it may be that there are lots of tRNAs that recognize UCU, but very few for UCU, UCA and UCG. Thus, if you want high expression, you'll want to use UCU as your codon for serine.

Here's the mapping of codons to amino acids:

and here's the codon usage table for S. cerevisiae:

Now, getting high expression isn't quite as simple as just choosing the most-used codon for each amino acid. There are other factors. For example, it may actually give higher expression to use some less-used codons at the beginning of a gene, giving the ribosome a slow start, and some proteins require that the ribosome slows down in certain regions in order for the protein to fold correctly (presumably one part needs time to fold before the next part is added).

Another important factor is mRNA secondary structure. If the mRNA sequence is such that there are sequences complimentary bases on the same mRNA strand, then it can fold in on itself and prevent or limit ribosome binding. So, codon optimization tools try to take a set of factors into account at the same time and create the optimal compromise codon usage that is expected to get the highest expression.

Even though codon usage optimization can be important it's often not necessary at all. For many proteins you will get some level of expression if you simply copy the gene from its host organism with PCR and transform it into your cell with an appropriate plasmid vector. It's likely however that we'll get much lower expression levels, but that's not always a bad thing (an expression that's too high may put a high burden on the cell and reduce survivability or for secreted proteins it may saturate the secretory pathway).

We're going to optimize codon usage for high expression and use an inducible promoter to limit how much is expressed. That allows us to experiment with expression levels without changing the DNA again. It also allows us to wait until a culture is in its exponential growth phase before inducing expression, which is one way of getting a higher over-all protein yield from a batch culture.