The incorporation of non-canonical and unnatural amino acids into proteins is a powerful way to selectively modify proteins and alter their functions. This can be achieved by a growing number of genetic code expansion and protein ligation methods. Stop codon suppression and quadruplet codons allow for site-specific incorporation of amino acids, while stochastic replacement of natural amino acids leads to proteome wide modifications. On the other hand, chemical or enzyme-mediated ligation methods enable the combination of synthetically modified peptides and native proteins. Non-canonical amino acids can carry a variety of functional groups, including, but not limited to, handles for click chemistry, crosslinkers, and artificial post-translational modifications. Among the major applications are the fluorescent labeling of proteins for microscopy, interaction studies of proteins and other biomolecules, and biosensors and nano-machines. Despite its great potential, the incorporation of non-canonical amino acids into proteins is also a technically challenging task and sometimes suffers from poor reproducibility. While plasmids and reagents are often readily shared within the community, experimental details may not always include all the necessary information due to their limited format.

The aim of this Methods Collection is to provide detailed videos that showcase all the critical steps of various genetic code expansion techniques, as well as their applications.