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Fully Productive Cell-Free Genetic Code Expansion by Structure-Based Engineering of Methanomethylophilus alvus Pyrrolysyl-tRNA Synthetase.
ACS Synth Biol ; 9(4): 718-732, 2020 04 17.
Article em En | MEDLINE | ID: mdl-32182048
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.
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Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Engenharia de Proteínas / Euryarchaeota / Código Genético / Aminoacil-tRNA Sintetases Idioma: En Revista: ACS Synth Biol Ano de publicação: 2020 Tipo de documento: Article

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Engenharia de Proteínas / Euryarchaeota / Código Genético / Aminoacil-tRNA Sintetases Idioma: En Revista: ACS Synth Biol Ano de publicação: 2020 Tipo de documento: Article