Making Bacterial Toxins into Virus-Like Particles

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James Ross, Bruce Turnbull, Mike Webb, Emanuele Paci

University of Leeds, United Kingdom

The re-design and controlled self-assembly of natural systems into non-natural functional products is a quickly developing area of Synthetic Biology. Specifically, the manipulation of existing and the introduction of new protein-protein interactions will allow great advances in bionanotechnology. In nature protein-protein assemblies mediate many cellular processes and exhibit complex and efficient functions. It is thus rational to assume human guided biomolecular assemblies could house equally complex functionality designed to address our current needs, including such devices as molecular diagnostic tools and therapeutic drug delivery systems. The goal of our project is the design and production of a capsid-like protein cage of dodecahedral symmetry and diameter of 15 nm or 32 nm, with an internal cargo-holding space, by the de novo design of a protein-protein interface between subunits. Firstly we used scaffolding molecules to tether our protein into the correct proximity for dodecahedral assembly. A combination of current computational methods was then used to suggest mutations which reduced the G of interaction across the interface. These designs were then experimentally characterised and the simulations optimised. These methods should be appropriate for generic application to the design of self-assembling protein systems. The dodecahedral particle is assembled from Cholera Toxin B-subunit, a natural homopentamer with an inbuilt cell targeting and endocytic triggering mechanism. Future applications could therefore use our capsid as a drug delivery vehicle to transport protected therapeutic agents to targeted cell types.