Genetic Code Expansion of Unnatural Amino Acids in Mammalian Cells and Pseudomonas putida KT2440

Genetic code expansion of unnatural amino acids (unAAs) into proteins in prokaryotic and eukaryotic cells pave a novel route to study and alter protein structures and functions, probe, sense, and control biological processes, as well as to develop new therapeutics. In Chapter 1, a brief overview of genetic code expansion of unAAs and recent advances were given, including the development of the most commonly used orthogonal aminoacyl-tRNA synthetases (aaRS) and tRNA pairs. Genetic code expansion of unAAs can be utilized to study posttranslational modifications (PTMs) on many proteins, especially in eukaryotes. To facilitate the study of protein tyrosine O-sulfation (PTS), we successfully develop an approach to site-specially encode sulfotyrosine (sTyr) into proteins in mammalian cells (Chapter 2). This method enables us the ability to investigate the biological mechanisms of PTS on secreted and membrane-bound proteins in live mammalian cells. In Chapter 3, we reported the first genetic incorporation of p-carboxymethyl-L-phenylalanine (pCMF), a nonhydrolyzable phosphotyrosine (pTyr) analog, into proteins in mammalian cells. The ability to synthesize full-length proteins containing nonhydrolyzable pTyr analog in eukaryotes can contribute to the study of the biological processes of tyrosine phosphorylation in mammalian cells, and can accelerate biomedical applications that target protein tyrosine phosphorylation. In Chapter 4, we reported the first genetic code expansion of four unAAs in Pseudomonas putida KT2440, which is famous for its ability to degrade and utilize aromatic compounds. The ability to genetically encode unAAs into proteins in P. putida KT2440 offers us a novel approach to uncovering and controlling protein functions in P. putida KT2440