Approaches to genetically modify proteins for enhanced interaction, NMR probing and bioconjugation

The genetic incorporation of non-canonical amino acids (ncAAs) site-specifically into proteins, also known as "genetic code expansion (GCE)", is a powerful method to introduce new chemistry into native polypeptide chains. This alteration in protein chemistry allows us to selectively attach reporter groups, or to precisely enhance protein properties, and has broad applications in biotechnology and medical research. In this thesis, new selection methods based on fluorescence-activated cell sorting (FACS) are established and improved to develop specific aminoacyl-tRNA synthetases (aaRSs) for the incorporation of p-pentafluorosulfanyl-L-phenylalanine (SF5Phe), N6-(trifluoroacetyl)-L-lysine (TFA-Lys), N6-(((trimethylsilyl)-methoxy)carbonyl)-L-lysine (TMSK), 7-fluoro-L-tryptophan (7FTrp), m-cyanopyridylalanine (mCNP), and p-cyanopyridylalanine (pCNP). Novel applications of GCE methods are introduced including alteration of protein-protein interactions, protein structural probing by NMR spectroscopy, ligand binding detection, and bioorthogonal conjugation. An optimized expression system recruiting an orthogonal pair of the pyrrolysyl aaRS derived from methanogenic archaeon ISO4-G1 and its cognate tRNA (G1PylRS/G1tRNAPyl) is also described in our stories. The aptitude of G1PylRS with broad substrate diversity (towards TFA-Lys, 7FTrp, mCNP, and pCNP) is elucidated together with the first solved crystal structure of a G1PylRS mutant that specifically recognize mCNP