Electrostatic Encoding Enables Simultaneous Dual Labeling of Proteins by a Single Sortase A Enzyme
Year: 2026
Type: preprint
Abstract: Site-specific dual labeling of proteins provides access to multifunctional biomolecular constructs for applications ranging from imaging to the development of next-generation therapeutics. Currently, enzymatic dual labeling strategies rely on workflows involving multiple (semi-)orthogonal enzymes or temporary protection/deprotection schemes. Here, we present a one-pot strategy for protein dual labeling that relies on the single sortase A pentamutant (SrtA-5M) enzyme. By rationally designing complementary charged modules on the protein substrate and labeling reagent, we exploit electrostatic interactions to selectively guide the simultaneous installation of two distinct labels at the N- and C-termini of recombinant proteins without the need for multiple ligases, protecting groups, intermediate purification steps, or large reagent excesses. The efficiency of the approach is demonstrated on protein substrates and affords dually labeled products with high selectivity under mild reaction conditions. Beyond its practical utility, this study provides new insights into the influence of the P2 residue x within the sortase recognition motif (LPxTG) relative to glycyl nucleophiles, revealing an important role in governing sortase-mediated transpeptidation outcomes.
Source: ChemRxiv
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W7211891365