High-Throughput Aminoacyl-tRNA Synthetase Engineering for Genetic Code Expansion in Yeast.

ABSTRACT: Protein expression with genetically encoded noncanonical amino acids (ncAAs) benefits a broad range of applications, from the discovery of biological therapeutics to fundamental biological studies. A major factor limiting the use of ncAAs is the lack of orthogonal translation systems (OTSs) that support efficient genetic code expansion at repurposed stop codons. Aminoacyl-tRNA synthetases (aaRSs) have been extensively evolved in Escherichia coli but are not always orthogonal in eukaryotes. In this work, we use a yeast display-based ncAA incorporation reporter platform with fluorescence-activated cell sorting to screen libraries of aaRSs in high throughput for (1) the incorporation of ncAAs not previously encoded in yeast; (2) the improvement of the performance of an existing aaRS; (

SUBMITTER: Stieglitz JT 

PROVIDER: S-EPMC10065163 | biostudies-literature | 2022 Jul

REPOSITORIES: biostudies-literature