A Robust Platform for Unnatural Amino Acid Mutagenesis in E. coli Using the Bacterial Tryptophanyl-tRNA synthetase/tRNA pair.

ABSTRACT: 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^Trp pairs. This was made possible by functionally replacing the endogenous EcTrpRS-tRNA^Trp 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 recombi

SUBMITTER: Ficaretta ED 

PROVIDER: S-EPMC9005579 | biostudies-literature | 2022 Apr

REPOSITORIES: biostudies-literature