Why rebalance codon usage?
The genetic code is composed of 64 codons with only 21 amino acid and “stop” assignments. Therefore, degeneracy is inherently designed into translation. Preferential usage of particular codons varies by organism. For example, leucine is specified by 6 distinct codons, some of which are rarely used. By rebalancing codon usage within a reading frame, preferred leucine codons are selected over rarely used codons. This is thought to increase the yield of heterologous expression [1]. The free, online IDT Codon Optimization Tool can help you rebalance codon usage for a sequence from one species to that for the organism chosen for expression.
How the Codon Optimization Tool works
The Codon Optimization Tool was written using a codon sampling strategy [2] in which the reading frame is recoded based on the frequencies of each codon’s usage in the new organism.
As an example, codon optimizations of sequences that will be expressed in human cell lines assign the phenylalanine codon UUU 46% and UUC 54% of the time (see Table 1, amino acid F). In addition, codons with <10% frequency are eliminated, with remaining codons renormalized to 100%. For example, codons CUA and UUA designate leucine (amino acid L), but are rarely used. Thus, they would not be assigned, and the remaining codons for leucine (UUG, CUU, CUC, and CUG) would be renormalized to 100%.
Table 1. Human codon table [8]. Stars (*) denote stop codons.
Will codon optimization affect protein expression?
There is currently no known method that is predictive of protein expression, although the concept of the codon adaptation index has proven most predictive for expression by E. coli [3,4]. While codon optimization can improve expression, it does not provide a guarantee. The amount of increase in protein expression through codon optimization will vary, depending on the particular protein and organism. Additionally, expression levels can be influenced by many other factors, including tRNA copies [5], mRNA stability [6], protein folding kinetics [7], protein stability, protein transport, toxicity of the protein within the expression cell environment, and a host of other factors that vary for each protein and organism. As a result, any particular optimization demands experimental verification.
Access IDT’s free, online Codon Optimization Tool and get started.
We also provide a step-by-step tutorial for using the Codon Optimization Tool. View it here.