Extended Data Fig. 2: Screening and verification of PylRS variants for UAA recognition.
From: Genetic code expansion reveals aminoacylated lysine ubiquitination mediated by UBE2W
a, The schematic illustration of the screening process. The GFP-190TAG gene was used as the reporter gene in the assay. And the amber suppression efficiency of the PylRS variants showed in Extended Data Fig. 1 was assayed in the presence or absence of UAA. b, Screening of PylRS variants for MetK recognition. The MetKRS (variant 5) used in this study is highlighted in red. c, Screening of PylRS variants PraK recognition. The potential candidates (variant 4 and 41 for PraK) are shown in bold. d, Assessment of amber suppression efficiency of variants 4 and 41 for PraK recognition. The PraKRS used in this study is highlighted in red. e, The amino acid mutations of MetKRS and PraKRS used in this study. f, Analysis of amber suppression efficiency of MetKRS and PraKRS in the presence or absence of the corresponding UAAs in E. coli. Mass spectrometry characterization of the fidelity of MetK (g) and PraK (h) incorporation on GFP. i, the FACS gating method in Fig. 2d sorts the EGFP+ mCherry+ population of HEK293T cells. FSC and SSC are used to exclude cell debris. Cells expressing EGFP or mCherry fluorescent proteins are used to set the gain and determine the threshold for positive and negative cells in each channel. For d and f, data are presented as mean values ± SEM (n = 3 biologically independent experiments). Source data are provided as a Source Data file.
