Lee Dongheon, Kim Ja Gyung, Kim Tae Wan, Choi Jong-Il
Department of Biotechnology and Bioengineering, Chonnam National University, Gwangju, 61186, Republic of Korea.
AMB Express. 2024 May 24;14(1):60. doi: 10.1186/s13568-024-01706-3.
Genetic code expansion involves introducing non-canonical amino acids (ncAAs) with unique functional groups into proteins to broaden their applications. Orthogonal aminoacyl tRNA synthetase (aaRS), essential for genetic code expansion, facilitates the charging of ncAAs to tRNA. In this study, we developed a new aaRS mutant from Methanosaeta concilii tyrosyl-tRNA synthetase (Mc TyrRS) to incorporate para-azido-L-phenylalanine (AzF). The development involved initial site-specific mutations in Mc TyrRS, followed by random mutagenesis. The new aaRS mutant with amber suppression was isolated through fluorescence-activated cell sorting. The M. concilii aaRS mutant structure was further analyzed to interpret the effect of mutations. This research provides a novel orthogonal aaRS evolution pipeline for highly efficient ncAA incorporation that will contribute to developing novel aaRS from various organisms.
遗传密码扩展涉及将带有独特官能团的非标准氨基酸（ncAAs）引入蛋白质中，以拓宽其应用范围。正交氨酰-tRNA合成酶（aaRS）是遗传密码扩展所必需的，它促进了ncAAs与tRNA的氨酰化。在本研究中，我们从嗜甲基产甲烷丝菌酪氨酰-tRNA合成酶（Mc TyrRS）开发了一种新的aaRS突变体，用于掺入对叠氮基-L-苯丙氨酸（AzF）。该开发过程包括在Mc TyrRS中进行初始位点特异性突变，然后进行随机诱变。通过荧光激活细胞分选分离出具有琥珀抑制作用的新aaRS突变体。进一步分析了嗜甲基产甲烷丝菌aaRS突变体的结构，以解释突变的影响。本研究为高效掺入ncAA提供了一种新型正交aaRS进化途径，这将有助于从各种生物体开发新型aaRS。
