Abstract
The rare AGG codon in Escherichia coli has been reassigned to code non-canonical amino acids (ncAAs) by using the PylRS-tRNA(Pyl)(CCU) pair. When N(ε) -alloc-lysine was used as a PylRS substrate, almost quantitative occupancy of N(ε) -alloc-lysine at an AGG codon site was achieved in minimal medium. ncAAs can be potentially incorporated at the AGG codon with varying efficiencies, depending on their activities towards corresponding enzymes. As AGG is a sense codon, the approach reported here resolves the typical low ncAA incorporation issue that has been associated with ncAA mutagenesis and therefore allows bulk preparation of proteins with site-selectively incorporated ncAAs for applications such as therapeutic protein production.