A Robust and Quantitative Reporter System To Evaluate Noncanonical Amino Acid Incorporation in Yeast.

ABSTRACT: Engineering protein translation machinery to incorporate noncanonical amino acids (ncAAs) into proteins has advanced applications ranging from proteomics to single-molecule studies. As applications of ncAAs emerge, efficient ncAA incorporation is crucial to exploiting unique chemistries. We have established a quantitative reporter platform to evaluate ncAA incorporation in response to the TAG (amber) codon in yeast. This yeast display-based reporter utilizes an antibody fragment containing an amber codon at which a ncAA is incorporated when the appropriate orthogonal translation system (OTS) is present. Epitope tags at both termini allow for flow cytometry-based end point readouts of OTS efficiency and fidelity. Using this reporter, we evaluated several factors that influence amber suppression, including the amber codon position and different aminoacyl-tRNA synthetase/tRNA (aaRS/tRNA) pairs. Interestingly, previously described aaRSs that evolved from different parent enzymes to incorporate O-methyl-l-tyrosine exhibit vastly different behavior. Escherichia coli leucyl-tRNA synthetase variants demonstrated efficient incorporation of a range of ncAAs, and we discovered unreported activities of several variants. Compared to a plate reader-based reporter, our assay yields more precise bulk-level measurements while also supporting single-cell readouts compatible with cell sorting. This platform is expected to allow quantitative elucidation of principles dictating efficient stop codon suppression and evolution of next-generation stop codon suppression systems to further enhance genetic code manipulation in eukaryotes. These efforts will improve our understanding of how the genetic code can be further evolved while expanding the range of chemical diversity available in proteins for applications ranging from fundamental epigenetics studies to engineering new classes of therapeutics.

SUBMITTER: Stieglitz JT 

PROVIDER: S-EPMC6214617 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature