Developing protein scaffolds for VLP assembly and enzyme organisation in cell-free protein synthesis

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Background:Protein-based compartments such as Virus-like-particles (VLPs), bacterial nanocompartments (encapsulins) and more recently, bacterial microcompartments (BMCs) are attractive nano-scaffolds with broad applications in biomedicine, nano-biotechnology, biocatalysis and material sciences. They are entirely self-assembling and can be genetically engineered to incorporate modifications. VLPs resemble viruses, they are highly immunogenic due to the repetitive presentation of epitopes on the surface and are therefore developed as vaccines and therapeutics. Although VLPs are robust structures, when foreign epitopes are inserted into the VLP for display on the surface, the formation of stable so-called 'chimeric VLPs' can be dramatically decreased (Peyret et al. 2015, Kazaks et al. 2017). Current approaches to stabilising VLPs include addition of carboxy-terminal polyhistidine tags on VLP proteins and disulphide-bridges (Schumacher et al. 2018).This project is investigating novel strategies for the stabilisation of hepatitis B virus core antigen (HBcAg) VLPs, namely the use of protein nano-structures as scaffolds for improved assembly and stability of VLP particles. Such nano-scaffolds may include encapsulins, which form readily, are extremely stable at a wide pH range and can be genetically engineered to create fusion proteins (Giessen 2016). These properties will be utilised to facilitate interactions with VLP proteins with the aim to provide mechanical and chemical stability for chimeric VLPs. With a growing interest in the development of cell-free protein expression systems for VLP assembly, synthetic circuit testing and enzyme engineering, multi-protein assembly and spatial organisation in cell-free systems is important to understand (Lu 2017). In this respect, the project will also investigate if more complex, multi-protein assemblies such as bacterial microcompartments (BMCs) can be readily formed in a cell-free environment. BMCs are megadalton protein assemblies, composed of 10,000-20,000 polypeptides of 10-20 types that self-assemble in a modular fashion and house enzymes up to mM concentration, providing an environment for high local enzyme concentration. Components of naturally occurring BMCs can be modified and combined to permit the construction of large protein scaffolds and bioreactors with novel functions (Kerfeld and Erbilgin 2015). So far these are produced and tested within bacterial cells with the aim to organise enzymes in vivo. Cell-free systems could provide a high-throughput test-platform for engineered BMCs and may allow for enzyme scaffolding in cell-free systems.Objectives:- Stable assembly of encapsulins in vivo and cell-free (1-12 months)- Development of nano-scaffolds from engineered encapsulins (13-24 months)- Self-assembling on multi-protein structures for enzyme scaffolding (25-36 months)