Incorporating, Quantifying, and Leveraging Noncanonical Amino Acids in Yeast.

Genetic code expansion has allowed for extraordinary advances in enhancing protein chemical diversity and functionality, but there remains a critical need for understanding and engineering genetic code expansion systems for improved efficiency. Incorporation of noncanonical amino acids (ncAAs) at stop codons provides a site-specific method for introducing unique chemistry into proteins, though often at reduced yields compared to wild-type proteins. A powerful platform for ncAA incorporation supports both the expression and evaluation of chemically diverse proteins for a broad range of applications. In yeast, ncAAs have been used to study dynamic cellular processes such as protein-protein interactions and also allow for exploration of eukaryotic-specific biology such as epigenetics. Furthermore, yeast display is an advantageous technology for engineering and screening the properties of proteins in high throughput. The protocols presented in this chapter describe detailed methods for the yeast-based genetic encoding of ncAAs in proteins intracellularly or on the yeast surface. In addition, methods are presented for modifying proteins on the yeast surface using bioorthogonal chemical reactions and evaluating reaction efficiency. Finally, protocols are included for the preparation of libraries that involve genetic code expansion. Libraries of proteins that contain ncAAs or libraries of the cellular machinery required to encode ncAAs can be constructed and screened in high throughput for many biological and chemical applications. Efficient incorporation of ncAAs facilitates elucidation of fundamental eukaryotic biology and advances tools for enzyme and genome engineering to evolve host cells that are better able to accommodate alternative genetic codes.