Exploring Protein-Protein Ligation Approaches for the Cytosolic Delivery of Antigens Using AIP56

Background/Objectives: The intracellular delivery of biologics, particularly large cargoes like proteins, remains a challenge in biotechnology and biomedicine. The modular structure of well-characterized AB toxins allows different cargoes to be grafted, creating a target-specific biotechnological tool capable of cytosolic delivery. Methods: In this study, we employed protein-protein fusion strategies - SpyCatcher003, SnoopCatcher, and SnoopLigase - to generate chimeras between the delivery region of AIP56 (AIP56L258-N497) and β-lactamase and performed functional delivery assays. Results: The chimeras were successfully obtained using these strategies and in vitro β-lactamase activity assays confirmed the catalytic activity of the chimeras. Importantly, we show that the chimera BlaL19-W286::SnoopAIP56L258-N497 delivered β-lactamase into the cytosol of J774.A1 macrophages. Conclusions: AIP56 delivery region transporting other cargo directly to the cytosol of antigen-presenting cells might be a promising platform for antigen delivery. This study highlights the potential of protein-protein fusion strategies to create versatile, antigenically distinct toxin-based delivery systems for therapeutic applications.