Extended Data Fig. 2: Engineering bioorthogonal aminoacyl-tRNA synthetase for site-specific incorporation of FnFSYs into GFP.
From: Intratumoural vaccination via checkpoint degradation-coupled antigen presentation
a, Targeted saturation mutagenesis sites on PylRS. The chFSYRS V366A PylRS variant, identified after four rounds of positive selection, enables bio-orthogonal recognition of all FnFSYs. (PDB: 3QTC). b, Comparison of FnFSY incorporation into full-length GFP in E. coli by the chFSYRS V366A variant versus the original chFSYRS. PrUAA: proximity-reactive unnatural amino acids. Data are representative of three biological replicates. For gel source data, see Supplementary Fig. 1. c–e, Sequencing results of four targeted sites: (c) before selection, (d) after three rounds of positive selection, and (e) after a final round of positive selection. f,g, Quantitative analysis of EGFP expression by fluorescence: (f) Representative fluorescence imaging normalized by OD600, measured with a Typhoon FLA 9500 Fluorescence Imager. (g) Fluorescence intensity per group, measured by plate reader and normalized to FSY (n = 3 biological replicates). Data are the mean ± s.d. Statistics: two-way ANOVA (g) with Tukey’s multiple comparisons test.
