Fully Productive Cell-Free Genetic Code Expansion by Structure-Based Engineering of Pyrrolysyl-tRNA Synthetase.

Pyrrolysyl-tRNA synthetase (PylRS)/tRNA pairs from and are widely used for site-specific incorporations of non-canonical amino acids into proteins (genetic code expansion). In this study, we achieved the full productivity of cell-free protein synthesis for difficult, bulky non-canonical amino acids, such as -(((()-cyclooct-2-en-1-yl)oxy)carbonyl)-l-lysine (TCOLys), by using PylRS. First, based on the crystal structure of PylRS, the productivities for various non-canonical amino acids were greatly increased by rational engineering of the amino acid-binding pocket. The productivities were further enhanced by using a much higher concentration of PylRS over that of PylRS, or by mutating the outer layer of the amino acid-binding pocket. Thus, we achieved full productivity even for TCOLys. The quantity and quality of the cell-free-produced antibody fragment containing TCO*Lys were drastically improved. These results demonstrate the importance of full productivity for the expanded genetic code.