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

• Published in: ACS synthetic biology Vol. 9; no. 4; pp. 718 - 732
• Main Authors: Seki, Eiko, Yanagisawa, Tatsuo, Kuratani, Mitsuo, Sakamoto, Kensaku, Yokoyama, Shigeyuki
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 17.04.2020
• Subjects: Amino Acids - genetics; Amino Acids - metabolism; Amino Acyl-tRNA Synthetases - chemistry; Amino Acyl-tRNA Synthetases - genetics; Amino Acyl-tRNA Synthetases - metabolism; Archaeal Proteins - chemistry; Archaeal Proteins - genetics; Archaeal Proteins - metabolism; Binding Sites; Cell-Free System; Euryarchaeota - enzymology; Euryarchaeota - genetics; Genetic Code - genetics; Immunoglobulin Fab Fragments - genetics; Models, Molecular; Protein Engineering - methods; Trastuzumab - genetics; non-canonical amino acid; Methanomethylophilus alvus; cell-free protein synthesis; pyrrolysyl-tRNA synthetase; structure-based protein engineering; Herceptin-Fab
• ISSN: 2161-5063; 2161-5063
• DOI: 10.1021/acssynbio.9b00288

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 N ε-(((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.