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Codon Bias and Heterologous Protein Expression


Contributed by Steve Anderson, Ph.D..


Expression of proteins in a host other than the organism from which the protein is derived, or heterologous protein expression, is an important tool in biotechnology. However, sometimes your protein of interest is poorly expressed in your host of choice. Why might this happen?


One reason could be codon bias.  A look at the genetic code (see Table) reveals that there are multiple codons for several amino acids. For example Arg, Leu, and Ser have six codons each. Thus many different coding sequences are possible for the same protein. As a result of evolution different organisms prefer different codons for the same amino acid. Typically this occurs at the third or “wobble” position of a codon. In fact, codon bias exists not only across species, but even between tissues of the same species/organism, or for genes expressed at different levels in the same organism. Potential mechanisms for this include the total GC content of the organism’s genome, mRNA folding, the presence of rare or non-preferred codons near the translation initiation site, tRNA availability, or a combination of these and other mechanisms.


Codon Reverse Table


If your gene sequence uses a different set of codons than those commonly used by your host, expression is likely to be disappointingly low. This suboptimal codon usage can result in significantly reduced heterologous protein expression in a selected host organism, such as expressing a human gene in E.coli.


Expression can sometimes be greatly increased by re-engineering a gene or cDNA to use the codons preferred by the host. Gustafsson et al.  present a table with many examples of gene sequence optimization and its effect on expression. For example, expression of human IL-2 in E. coli was increased 16 fold in one report by re-engineering the coding sequence of IL-2. Likewise, expression of mouse Ig kappa in yeast was increased more than 50 fold, and expression of mouse c-Fos in E. coli was increased from undetectable levels to approximately 20% of the total soluble protein. In addition, expression of many viral proteins can be increased by hundreds to more than 1000 fold in their typical host cells.


If you’re having trouble expressing your protein of interest you might want to consider investigating codon bias. Re-engineering your cDNA to use codons that better match your host’s preferences could result in significant increases in expression of your protein in your desired host.

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1. Gustafsson C, Govindarajan S, Minshull J. 2004. Codon bias and heterologous protein expression. TRENDS in Biotechnol. Jul;22(7):346-353.

2. Plotkin JB, Kudla G. 2011. Synonymous but not the same: the causes and consequences of codon bias. Nat Rev Genet. Jan;12(1):32–42.