Abstract
The promise of biologic therapeutics is hindered by the challenge to deliver their activity to biochemically relevant sites within diseased cells. The favourable application of the natural protein carriers of the AB5 toxin family to this challenge has been restricted owing to still unresolved requirements for assembling non-native cargo into carrier complexes. Here, we clarify the properties of fusion peptides which allow co-assembly of a selected fluorescent protein cargo with the non-toxic B subunit of a heat-labile enterotoxin. We establish the influence of sequence length, sequence identity and secondary structure of these linking domains on the assembly and disassembly of the complexes. Through our engineering framework we identify several non-native, reduced length fusion sequences that robustly assemble with the native carriers, maintain their ability to deliver protein cargo to cells, and demonstrate substantially refined in vitro properties. Constructs based upon these sequences should prove directly applicable to a variety of protein delivery challenges, and the described design framework should find immediate application to other members of the AB5 protein carrier family.
Highlights
The promise of biologic therapeutics is hindered by the challenge to deliver their activity to biochemically relevant sites within diseased cells
Initial attempts to co-express heat labile enterotoxin I B subunits (LTIB) and non-native cargo fused to the native A2 subdomain were complicated by highly inconsistent protein expression levels
The previously used full length wt A2 sequence, which is sufficient for assembly of the LTIB protein carriers and delivery of non-native cargo, comes with toxicity and specificity issues
Summary
The promise of biologic therapeutics is hindered by the challenge to deliver their activity to biochemically relevant sites within diseased cells. Through our engineering framework we identify several non-native, reduced length fusion sequences that robustly assemble with the native carriers, maintain their ability to deliver protein cargo to cells, and demonstrate substantially refined in vitro properties. Constructs based upon these sequences should prove directly applicable to a variety of protein delivery challenges, and the described design framework should find immediate application to other members of the AB5 protein carrier family. The efficiencies of endosomal escape can vary widely, once released, the protein cargoes can localize to the cytoplasmic and nuclear compartments Significantly, they have no immediate access to the Golgi and ER affording little means to address the increasingly recognized diseases associated with deficiencies in these organelles[11].
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