Sortase A-mediated hydrazinolysis generates homogeneous N-terminal GL proteins for OaAEP1-catalyzed bioconjugation

OaAEP1-mediated ligation provides an effective platform for site-specific protein modification, but it needs to obtain homogeneous N-terminal Gly-Leu (GL) protein substrates. Here, we describe a Sortase A-mediated hydrazinolysis approach for generating homogeneous N-terminal GL-proteins. By incorporating additional leucine residues into the Sortase A recognition sequence, we identified an LLLPXTGL motif that facilitates hydrazinolysis while generating an OaAEP1-compatible N-terminal GL motif. This method enabled the preparation of homogeneous GL-Ub and GL-GFP, which were further applied in OaAEP1-catalyzed ligation to construct diubiquitin and an anti-EGFR nanobody-GFP conjugate. This strategy provides a concise and practical route to GL-protein substrates and expands the utility of OaAEP1-mediated protein bioconjugation.