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Abstract

Applications of externally introduced proteins in both therapeutics and synthetic biology have been limited by lack of readily accessible protein targeting and delivery carriers. Common approaches like antibodies or receptor ligands only get the protein cargo as far as the cell surface, precluding applications which function in the cytoplasm. These established motifs stand in contrast to the known bacterial AB₅ toxins, which bear structurally and functionally diverse cargo into eukaryotic cells. The carrier B subunits of these proteins are solely responsible for both targeting diverse glycosylated structures on cell surfaces and delivering their cargo to internal cell environments, and are an attractive starting point for creating a platform of protein delivery tools. Using heat labile enterotoxin I (LTI) as a model system for this family of proteins, we identify the sequence and structural parameters necessary to effect co-assembly of the carrier B subunits with non-native protein cargo. As a result, cargo-carrier complexes derived from optimized sequences based upon these principles reveal improved assembly and kinetic stability over the ‘wildtype’ complexes. These variations in in vitro stability of the complexes in turn correlates with the efficacy of cellular internalization of a fluorescent protein cargo. This improved tool is immediately useful for the delivery of protein cargoes, and on-going efforts seek to extend its scope both in function and cell targeting.