期刊
ACS SYNTHETIC BIOLOGY
卷 9, 期 4, 页码 718-732出版社
AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.9b00288
关键词
cell-free protein synthesis; non-canonical amino acid; pyrrolysyl-tRNA synthetase; Methanomethylophilus alvus; structure-based protein engineering; Herceptin-Fab
资金
- Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED [JP17am0101081]
- Leading Advanced Projects for Medical Innovation (LEAP) from AMED [JP19gm0010001]
- Takeda Science Foundation
- MEXT [16K05859, 24550203]
- Grants-in-Aid for Scientific Research [24550203, 16K05859] Funding Source: KAKEN
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 Ne-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.
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