Developing new nanopores: molecular engineering of thermostable pore containing proteins for nanosensing applications

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The proposed project is to produce novel thermophilic bacteriophage portal proteins in order to characterise their structure and physicochemical properties and define their function as nanopores in electrical sensing experiments. Proteins with suitable properties will be further bioengineered and/or chemically modified to tune their sensing capabilities for specific applications.Background: Viral portal proteins are natural biological nanopores designed to translocate viral genomic dsDNA into preformed capsids during the viral packaging process. Research from several laboratories has indicated that these proteins have beneficial properties for nanopore applications in biotechnology, in particular peptide/protein sensing and sequencing, presenting superior properties over other types of biological nanopores available at present. Portal proteins from several viruses, including thermophilic bacteriophages, have been biochemically and structurally characterised at York. Dreampore are focusing on the development of new nanopore sensing applications for biomolecules that could benefit from the use of portal proteins. This project would expand the scope of current technologies by identifying and characterising new nanopores with novel properties suitable for advanced nanosensing applications.Objectives:To produce previously uncharacterised portal proteins from several thermostable viruses (York).To characterize biophysical properties of these proteins (York).To determine 3D structures of promising portal proteins by X-ray crystallography or Cryo-EM (York).To biologically and/ or chemically engineer portal variants for insertion into nanopore sensing devices (York/Dreampore).To control protein nanopore insertion and stability within lipid and or solid-state membranes (Dreampore).To detect and characterize protein or peptides biomarkers by electrical signals at single molecular level for the different portal protein channels (Dreampore)To investigate the sequencing potential of these channels with a protein model (Dreampore).To design, produce, characterise and test new variants of portal portal proteins with promising properties for specific nanopore sensing applications (York and Dreampore).Novelty:Since thermophilic portal proteins, such as G20c, are more stable than their mesophilic homologues, they provide a potential source of next-generation nanosensors that are more robust and easily engineered than those currently available. As such, these proteins offer a unique opportunity to revolutionise sequencing applications and detection of biomarkers, not only at Dreampore, but across the biotech sectors in the UK/EU, and beyond.Timeliness:Nanopore based sensing is advancing rapidly beyond the well-established DNA sequencing applications and is part of the current boom in miniaturised nanosensing technology that will revolutionise personal medicine and health management.Experimental Approach:The genes for thermophilic bacteriophage portal proteins (such as phiOH, GBSV and GVE2) will be cloned and optimised for expression of soluble portal protein assemblies. Soluble portal proteins will be produced to high purity and characterized biophysically and structurally (at York), before being inserted, using published technology, into nanopore devices to define the electrical sensing potential (at Dreampore). Specific sensing properties will be engineered, in an iterative process, into suitable portal proteins produced and characterized (at York) and subsequently tested for sensing applications (at Dreampore).