Fig. 3: Identifying a chimeric histidyl-tRNA synthetase.

a Cartoon structures of chimeric histidyl-tRNA synthetases (chHisRSs) in which the sequence from pylRS is coloured in green and the sequence from hisRS is coloured in blue. The pylRS TD mutant form carries IPYE mutations. ChHisRS-4 and chHisRS-5 carry a chimeric pylRS TD comprising residues 1-149 from Mb.pylRS and 185-240 from Mm.pylRS, which is reported to promote amber suppression efficiency⁴⁰. b Analysis of amber suppression activity of the generated chHisRSs by GFP reporter assay at 30 °C (n = 4). c Analysis of amber suppression activity of the generated pylRSs by GFP reporter assay in the presence of 2 mM Boc-L-lysine (BocK) (n = 3). d Mass spectrometry characterises the fidelity of His incorporation into GFP. e Analysis of amber suppression activity of the chHisRS under different promoters by GFP reporter assay and a native in-gel fluorescence imaging assay. (Supplementary Fig. 3D) GFP-190CAT (GFP-190His) and GFP-WT (GFP-190Asp) are used as controls (n = 3 for GFP-190ACT, n = 4 for GFP-190TAG glnS promoter and n = 5 for others). f Flow cytometry analysis of amber suppression efficiency in mammalian cells transfected with the indicated orthogonal systems. For the pylRS system, BocK (2 mM) is added to the medium of HEK 293T cells. For the chHisRS system, the addition of UAA is not necessary, and His in the medium is used as the substrate. Error bars represented ±standard error of the mean. Source data are available in the Source Data file.