Development of genetically encoded Sortase A substrates in mammalian cells

Protein-protein interactions and cell-cell communication are central to the regulation of complex biological processes. Proximity-labelling and contact-dependent labelling systems have emerged as powerful tools for capturing interaction events in living cells. While poweful, these methods suffer from lack of multiplexing ability to record different interactions occurring in heterogeneous settings. In this thesis, we developed a Sortase A-based engineered cellular platform designed to display its genetically encoded substrate so that the entire enzyme-substrate complex is presented on cell membrane ready to label interacting partners. By equipping the substrate with a peptide tag, we can assign a precise identity to enzyme-expressing cells. We explored the feasibility of this platform by generating lentiviral vectors to direct the substrate into different cellular compartments (membrane-bound, secreted, soluble). By transducing Sortase A-expressing cells, we determined that only membrane-bound substrate was correctly expressed on cell surface and we proceeded to enrich the positive population. Overall, this work established a mammalian engineered cell line expressing a surface-displayed Sortase A substrate, providing a functional framework for future multiplexed cell-surface labelling applications.