Engineering an AB5 Protein Carrier

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.

Review of Newly Identified Functions Associated With the Heat-Labile Toxin of Enterotoxigenic Escherichia coli

“…The LTA2 domain together with a fused fluorescent protein can be transported into cells by an A2 domain-mediated transmembrane transporting process similar to a cell-penetrating peptide (CPP). Recently, Lichtenstein and Hocker identified the requisite sequence and associated structures of LTA2 required for the efficient and stable co-assembly of protein cargo with the non-toxic LTB subunit (Lichtenstein and Höcker, 2018). These results suggest that the LT toxin or the LTA2 domain of LTA alone could be used as an intracellular trafficking vehicle for the delivery of biologically active proteins and drugs into cells; in the future, these strategies might be exploited for the treatment of cancer.…”

Sortase-Modified Cholera Toxoids Show Specific Golgi Localization

“…A wide range of studies have explored enhancing the activity [47][48][49] and changing the specificity of this enzyme [50][51][52]. In addition, a variety The intrinsic ability of bacterial toxins to induce endocytosis has been exploited in several studies to transport biomolecules and probes into mammalian cells both in vivo and in vitro [24][25][26][27][28][29][30]. Intracellular delivery has predominantly been achieved through the use of bacterial toxin chimeras [29,[31][32][33], but this approach requires a gene construct to be created for every novel protein fusion, and it is quite likely that expression optimization would also be necessary.…”