Incorporation of Non-Canonical Amino Acids 121
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Fig. 1 Approaches for genetic code expansion with non-canonical amino acids. a In vivo nonsense
suppression method: a non-canonical amino acid (ncAA, green star) is synthetically acylated to a
suppressor tRNA (ncAA-tRNASUP) and microinjected into Xenopus laevis oocytes together with RNA
coding for the protein of interest. To enable site-specific incorporation of the ncAA a codon is repur-
posed, most commonly it is the amber stop codon TAG (SUP, site of suppression). b Alternatively,
tRNA can be misacylated inside the cell by aminoacyl-tRNA synthetases (aa-RSs). Orthogonal aa-RS
and tRNA pairs can be co-evolved to be specific for certain ncAAs. For incorporation of ncAAs ( green
stars), the DNA encoding these pairs is transfected into the expression system of choice together with
the gene of interest carrying a repurposed codon at the site of mutagenesis (SUP). The ncAA is taken
up by the cell directly from the extracellular growth media. c Protein ligation through the use of bac-
terial intein sequences allows for chemical ligation of recombinant and synthetic peptide fragments