Two-Tier Screening Platform for Directed Evolution of Aminoacyl-tRNA Synthetases with Enhanced Stop Codon Suppression Efficiency.

ABSTRACT: Genetic code expansion through amber stop codon suppression provides a powerful tool for introducing non-proteinogenic functionalities into proteins for a broad range of applications. However, ribosomal incorporation of noncanonical amino acids (ncAAs) by means of engineered aminoacyl-tRNA synthetases (aaRSs) often proceeds with significantly reduced efficiency compared to sense codon translation. Here, we report the implementation of a versatile platform for the development of engineered aaRSs with enhanced efficiency in mediating ncAA incorporation by amber stop codon suppression. This system integrates a white/blue colony screen with a plate-based colorimetric assay, thereby combining high-throughput capabilities with reliable and quantitative measurement of aaRS-dependent ncAA incorpor

SUBMITTER: Owens AE 

PROVIDER: S-EPMC5586079 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature