‘ABC’ toxin complexes (Tcs) are large multi-subunit, pore-forming protein complexes secreted by numerous insecticidal bacteria and some mammalian pathogens. ABC toxins are composed of three major subunits: TcA, TcB and TcC. The TcA subunit is the injection apparatus that delivers the cytotoxic protein and determines the host cell specificity. The TcB and TcC subcomplex fold cooperatively into a cocoon-like structure that encapsulates the variable toxin domain of TcC. The hypervariability of the toxin domain suggests the system can be engineered to deliver non-native cargo proteins instead of the native toxic payload. Thus, the modularity of ABC toxins makes it an attractive candidate as a designer protein delivery machine.
The TcA subunit secreted by the soil bacterium Yersinia entomophaga MH96 are decorated with accessory chitinases (Chi1 and Chi2). The hypothesised action of chitinases in Yen-TcA is to degrade the chitin-rich peritrophic membrane of insect hosts, enabling access to midgut epithelial cells. Alternatively, the Chi1 subunits may play an additional role in cell surface recognition based on its outward rotation and proximity to the membrane surface following the protrusion of the Yen-TcA translocation pore. By replacing the associated chitinases with different ligand-binding proteins, targeting of non-native cell types is conceivable.
Here we outline our progress in producing an engineered version of Yen-TcA. Based on our structure of Chi1 and sequence similarity analyses, we inserted an epidermal growth factor receptor (EGFR)-binding peptide in the variable surface loops of Chi1. This epitope peptide is known to bind to EGFR overproducing cells. We successfully expressed the recombinant Chi1-peptide fusion protein in E. coli Rosetta2 cells. The purified proteins will be used for future proof-of-concept binding experiments to the A431 carcinoma cell line.