Fig. 6. Site-specific incorporation of two different bioconjugation handles into EGFP-39-TAG-151-TGA. (A) Expression of the full length EGFP reporter, measured by its characteristic fluorescence in cell-free extract, upon transfecting HEK293T cells with pAcBac3-EcTyrTGA-EGFP** and pAcBac1-PylTAG (for hPrK + AzF, AzK + AcF, and CpK + AzF), or pAcBac3-EcTyrTAG-EGFP** and pAcBac1-PylTGA (for AzK + PrY) in the presence or absence of indicated ncAAs. (B) Structures of DBCO-TAMRA and Tet-fluorescein. (C) Treating EGFP-39CpK-151AzF with DBCO-TAMRA or Tet-fluorescein leads to appropriate fluorescence-labeling, as shown by fluorescence imaging following SDS-PAGE. Wild-type EGFP fails to undergo labeling under identical conditions. (D) ESI-MS analysis reveals complete single or dual labeling of EGFP-39CpK-151AzF upon treatment with DBCO-TAMRA or Tet-fluorescein or both reagents.
