Pyrrolysyl-tRNA synthetase
(PylRS)/tRNAPyl pairs from Methanosarcina mazei and Methanosarcina barkeri 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 Nε-((((E)-cyclooct-2-en-1-yl)oxy)carbonyl)-l-lysine (TCO*Lys), by using Methanomethylophilus alvus PylRS. First, based on the crystal structure of M. alvus 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 M. mazei PylRS, or by mutating the outer layer of the amino acid-binding
pocket. Thus, we achieved full productivity even for TCO*Lys. 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.