Overcoming Near-Cognate Suppression in a Release Factor 1-Deficient Host with an Improved Nitro-Tyrosine tRNA Synthetase

• Published in: Journal of molecular biology Vol. 432; no. 16; pp. 4690 - 4704
• Main Authors: Beyer, Jenna N., Hosseinzadeh, Parisa, Gottfried-Lee, Ilana, Van Fossen, Elise M., Zhu, Phillip, Bednar, Riley M., Karplus, P. Andrew, Mehl, Ryan A., Cooley, Richard B.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 24.07.2020
• Subjects: active sites; amber; Amino Acyl-tRNA Synthetases - chemistry; Amino Acyl-tRNA Synthetases - genetics; Amino Acyl-tRNA Synthetases - metabolism; Catalytic Domain; Codon, Terminator; Computer Simulation; Crystallography, X-Ray; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli Proteins; genetic code; genetic code expansion; Genetic Engineering; Models, Molecular; Mutation; near-cognate suppression; nitro-tyrosine; Peptide Termination Factors - deficiency; Protein Binding; Protein Conformation; proteins; Release Factor 1; stop codon; tRNA synthetase; Tyrosine - analogs & derivatives; Tyrosine - metabolism; tyrosine-tRNA ligase; TCO-K; ncAA; GCE; genetic code expansion; Tet2.0; tRNA synthetase; Release Factor 1; aaRS; nitroTyr; AzF; Bpa; nitro-tyrosine; near-cognate suppression; sfGFP; RF1
• ISSN: 0022-2836; 1089-8638; 1089-8638
• DOI: 10.1016/j.jmb.2020.06.014

Genetic code expansion (GCE) technologies incorporate non-canonical amino acids (ncAAs) into proteins at amber stop codons. To avoid unwanted truncated protein and improve ncAA–protein yields, genomically recoded strains of Escherichia coli lacking Release Factor 1 (RF1) are becoming increasingly popular expression hosts for GCE applications. In the absence of RF1, however, endogenous near-cognate amber suppressing tRNAs can lead to contaminating protein forms with natural amino acids in place of the ncAA. Here, we show that a second-generation amino-acyl tRNA synthetase (aaRS)/tRNACUA pair for site-specific incorporation of 3-nitro-tyrosine could not outcompete near-cognate suppression in an RF1-deficient expression host and therefore could not produce homogenously nitrated protein. To resolve this, we used Rosetta to target positions in the nitroTyr aaRS active site for improved substrate binding, and then constructed of a small library of variants to subject to standard selection protocols. The top selected variant had an ~2-fold greater efficiency, and remarkably, this relatively small improvement enabled homogeneous incorporation of nitroTyr in an RF1-deficient expression host and thus eliminates truncation issues associated with typical RF1-containing expression hosts. Structural and biochemical data suggest the aaRS efficiency improvement is based on higher affinity substrate binding. Taken together, the modest improvement in aaRS efficiency provides a large practical impact and expands our ability to study the role protein nitration plays in disease development through producing homogenous, truncation-free nitroTyr-containing protein. This work establishes Rosetta-guided design and incremental aaRS improvement as a viable and accessible path to improve GCE systems challenged by truncation and/or near-cognate suppression issues. [Display omitted] •Near-cognate amber codon suppression in “recoded” E. coli expression hosts that lack Release Factor 1 creates a general fidelity problem for genetic code expansion (GCE).•Improving the efficiency of an amino-acyl tRNA synthetase/tRNA pair overcame these near-cognate suppression problems.•Improvements were made using Rosetta-guided library design and directed evolution strategies.•This new 3-nitro-tyrosine GCE system expands our ability to produce site-specifically nitrated proteins in E. coli.