6jp2

Publication Abstract from PubMed

Pyrrolysyl-tRNA synthetase (PylRS)/tRNA(Pyl) 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 N(epsilon)-((((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.

Fully Productive Cell-Free Genetic Code Expansion by Structure-Based Engineering of Methanomethylophilus alvus Pyrrolysyl-tRNA Synthetase.,Seki E, Yanagisawa T, Kuratani M, Sakamoto K, Yokoyama S ACS Synth Biol. 2020 Apr 17;9(4):718-732. doi: 10.1021/acssynbio.9b00288. Epub, 2020 Mar 17. PMID:32182048^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.