FIGURE 1.

Protein ligating enzymes mediate a covalent conjugation of RBCEVs with peptides. (a) Design of an EGFR‐targeting (ET) peptide with a sortase binding site and biotin (bi) conjugation (bi‐ET^S peptide). Sortagging reaction occurs between the bi‐ET^S peptide and proteins with N‐terminal Glycine (G) on RBCEVs, mediated by Sortase A. (b) Western blot (WB) analysis of biotin following an SDS‐PAGE separation of RBCEV proteins conjugated with the bi‐ET^S peptide. Sortase intermediates were removed in three washes with PBS. Biotin was detected using HRP‐conjugated streptavidin. Molecular weights (kDa) of protein markers are shown on the left. (c) Design of a typical OaAEP1‐ligase‐mediated reaction between a biotinylated ET peptide with a ligase binding site (bi‐ET^L peptide) and proteins containing N‐terminal GL (preferred but not required) on RBCEVs. (d) Western blot analysis of biotin resulted from the OaAEP1‐ ligase‐mediated conjugation of RBCEVs with the bi‐ET^L peptide, similar to (b). (e) Western Blot analysis of RBCEVs from three different donors (D1‐D3) ligated with a biotinylated control peptide using OaAEP1 ligase. Dibiotinylated HRP was used as a reference for quantification, and a particle analyzer was used to obtain the number of ligated EVs loaded per well. (f) Average number of peptides ligated to each EV ± SEM (n = 8 donors).