Issue 2, 2020

Reporter system architecture affects measurements of noncanonical amino acid incorporation efficiency and fidelity

Abstract

The ability to genetically encode noncanonical amino acids (ncAAs) within proteins supports a growing number of applications ranging from fundamental biological studies to enhancing the properties of biological therapeutics. Currently, our quantitative understanding of ncAA incorporation systems is confounded by the diverse set of characterization and analysis approaches used to quantify ncAA incorporation events. While several effective reporter systems support such measurements, it is not clear how quantitative results from different reporters relate to one another, or which details influence measurements most strongly. Here, we evaluate the quantitative performance of single-fluorescent protein reporters, dual-fluorescent protein reporters, and cell surface-displayed protein reporters of ncAA insertion in response to the TAG (amber) codon in yeast. While different reporters support varying levels of apparent readthrough efficiencies, flow cytometry-based evaluations with dual reporters yielded measurements exhibiting consistent quantitative trends and precision across all evaluated conditions. Further investigations of dual-fluorescent protein reporter architecture revealed that quantitative outputs are influenced by stop codon location and N- and C-terminal fluorescent protein identity. Both dual-fluorescent protein reporters and a “drop-in” version of yeast display support quantification of ncAA incorporation in several single-gene knockout strains, revealing strains that enhance ncAA incorporation efficiency without compromising fidelity. Our studies reveal critical details regarding reporter system performance in yeast and how to effectively deploy such reporters. These findings have substantial implications for how to engineer ncAA incorporation systems—and protein translation apparatuses—to better accommodate alternative genetic codes for expanding the chemical diversity of biosynthesized proteins.

Graphical abstract: Reporter system architecture affects measurements of noncanonical amino acid incorporation efficiency and fidelity

Supplementary files

Article information

Article type
Paper
Submitted
14 8 2019
Accepted
13 1 2020
First published
23 1 2020

Mol. Syst. Des. Eng., 2020,5, 573-588

Author version available

Reporter system architecture affects measurements of noncanonical amino acid incorporation efficiency and fidelity

P. K. A., S. J. T., L. M. and V. D. J. A., Mol. Syst. Des. Eng., 2020, 5, 573 DOI: 10.1039/C9ME00107G

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