Seki Eiko, Yanagisawa Tatsuo, Kuratani Mitsuo, Sakamoto Kensaku, Yokoyama Shigeyuki
ACS Synth Biol. 2020 Apr 17;9(4):718-732. doi: 10.1021/acssynbio.9b00288. Epub 2020 Mar 17.
Pyrrolysyl-tRNA synthetase (PylRS)/tRNA pairs from and 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 -(((()-cyclooct-2-en-1-yl)oxy)carbonyl)-l-lysine (TCOLys), by using PylRS. First, based on the crystal structure of 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 PylRS, or by mutating the outer layer of the amino acid-binding pocket. Thus, we achieved full productivity even for TCOLys. 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.
来自[具体来源1]和[具体来源2]的吡咯赖氨酸-tRNA合成酶（PylRS）/tRNA对被广泛用于将非天然氨基酸位点特异性掺入蛋白质中（遗传密码扩展）。在本研究中，我们通过使用[具体来源1]的PylRS实现了无细胞蛋白质合成对难溶性、大分子非天然氨基酸（如-(((()-环辛-2-烯-1-基)氧基)羰基)-L-赖氨酸（TCOLys）的完全生产力。首先，基于[具体来源1]的PylRS晶体结构，通过对氨基酸结合口袋进行合理工程改造，大大提高了各种非天然氨基酸的生产力。通过使用比[具体来源2]的PylRS浓度高得多的PylRS，或通过突变氨基酸结合口袋的外层，生产力进一步提高。因此，我们甚至对TCOLys也实现了完全生产力。含有TCO*Lys的无细胞产生的抗体片段的数量和质量得到了显著改善。这些结果证明了完全生产力对于扩展遗传密码的重要性。
