Fig. 2: Genetically encode MeaK into proteins in E. coli and mammalian cells.
From: Genetically encoding ε-N-methacryllysine into proteins in live cells
a Chemical structure of Meak. b Left: X-ray crystal structure of the MmPylRS complex with pyrrolysyl-AMP (PDB ID: 2Q7H), active-site residues for mutation are highlighted in green. Right: residue numbering is based on MbPylRS and MmPylRS. c SDS-PAGE gel of His-tag purified MBP-Z E24MeaK. Protein Z is IgG Fc-binding domain62, MBP-Z is maltose binding protein tagged protein Z. d Intact protein mass analysis of purified protein from E. coli expressed MBP-Z E24MeaK. e Tandem mass spectrum of MeaK incorporated peptide of EGFP D190MeaK. The red U represents MeaK incorporation site. f CycIm ion was mapped on the MS/MS spectrum of the MeaK incorporated peptide. g Genetically encode Meak into EGFP protein in mammalian cells. FACS analysis of HEK293T cells after MeaK incorporation into EGFP-151TAG. h Left: Fluorescence images of EGFP Y151MeaK showing incorporation of MeaK into EGFP in HEK293T cells. Right: Select images from different fields of view under a 10× objective lens, and use ImageJ software to calculate the mean fluorescence intensity (n = 5). Error bar represents ± one standard deviation (SD). i Intact protein mass analysis of HEK293T cells expressed EGFP Y151MeaK. j Tandem mass spectrum of MeaK incorporated peptide of EGFP Y151MeaK.
