Department of Chemistry, Boston College, 2609 Beacon Street, Chestnut Hill, MA 02467, USA.
Department of Chemistry, Boston College, 2609 Beacon Street, Chestnut Hill, MA 02467, USA.
J Mol Biol. 2022 Apr 30;434(8):167304. doi: 10.1016/j.jmb.2021.167304. Epub 2021 Oct 13.
We report the development of a robust user-friendly Escherichia coli (E. coli) expression system, derived from the BL21(DE3) strain, for site-specifically incorporating unnatural amino acids (UAAs) into proteins using engineered E. coli tryptophanyl-tRNA synthetase (EcTrpRS)-tRNA pairs. This was made possible by functionally replacing the endogenous EcTrpRS-tRNA pair in BL21(DE3) E. coli with an orthogonal counterpart from Saccharomyces cerevisiae, and reintroducing it into the resulting altered translational machinery tryptophanyl (ATMW-BL21) E. coli strain as an orthogonal nonsense suppressor. The resulting expression system benefits from the favorable characteristics of BL21(DE3) as an expression host, and is compatible with the broadly used T7-driven recombinant expression system. Furthermore, the vector expressing the nonsense-suppressing engineered EcTrpRS-tRNA pair was systematically optimized to significantly enhance the incorporation efficiency of various tryptophan analogs. Together, the improved strain and the optimized suppressor plasmids enable efficient UAA incorporation (up to 65% of wild-type levels) into several different proteins. This robust and user-friendly platform will significantly expand the scope of the genetically encoded tryptophan-derived UAAs.
我们报告了一种强大的、易于使用的大肠杆菌（E. coli）表达系统的开发，该系统源自 BL21(DE3) 菌株，可使用工程化的大肠杆菌色氨酰-tRNA 合成酶（EcTrpRS）-tRNA 对蛋白质进行定点掺入非天然氨基酸（UAAs）。这是通过功能上取代 BL21(DE3) E. coli 中的内源性 EcTrpRS-tRNA 对，并用来自酿酒酵母的正交对来替换，并将其作为正交无意义抑制子重新引入到产生的改变的翻译机制色氨酰（ATMW-BL21）E. coli 菌株中实现的。该表达系统受益于 BL21(DE3) 作为表达宿主的有利特征，并且与广泛使用的 T7 驱动的重组表达系统兼容。此外，表达无意义抑制工程 EcTrpRS-tRNA 对的载体被系统地优化，以显著提高各种色氨酸类似物的掺入效率。改良的菌株和优化的抑制子质粒共同使几种不同蛋白质的 UAA 掺入效率提高（高达野生型水平的 65%）。这个强大且易于使用的平台将显著扩展遗传编码的色氨酸衍生的 UAAs 的范围。
