Thermophage portal proteins for nanopore biotechnology

用于纳米孔生物技术的噬菌体门户蛋白

• 批准号: 2267465
• 金额: --
• 依托单位: University of York
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2019
• 资助国家: 英国
• 起止时间: 2019 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2267465
• 关键词: Thermophage; portal; proteins; nanopore; biotechnology

The proposed project aims 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. Research from several laboratories has indicated that such portal 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. The industrial partner, Dreampore, are world leaders in the development of new nanopore sensing applications for biomolecules and specific disease or health related biomarkers 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 portal proteins from thermostable viruses (York).To characterize biophysical properties of the 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).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).NoveltySince 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.TimelinessNanopore 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 ApproachThe 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).

该项目旨在生产新型嗜热噬菌体门户蛋白，以确定其结构和理化性质，并将其功能定义为电传感实验中的纳米孔。来自几个实验室的研究已经表明，这样的门户蛋白对于生物技术中的纳米孔应用具有有益的性质，特别是肽/蛋白质传感和测序，呈现出优于目前可用的其他类型的生物纳米孔的上级性质。工业合作伙伴Dreampore是开发生物分子和特定疾病或健康相关生物标志物的新型纳米孔传感应用的世界领导者。该项目将通过识别和表征具有适用于高级纳米传感应用的新特性的新型纳米孔来扩大现有技术的范围。目标：从耐热病毒（约克）中制备门脉蛋白，并对其生物物理性质进行表征，通过X-射线衍射确定有希望的门脉蛋白的三维结构。射线晶体学或Cryo-EM（约克）。生物和/或化学工程化门户变体以插入纳米孔传感装置（约克）。控制脂质和/或固态膜内的蛋白质纳米孔插入和稳定性（Dreampore）通过单个分子水平的电信号检测和表征不同门脉蛋白通道的蛋白质或肽生物标志物（Dreampore）用蛋白质模型研究这些通道的测序潜力（Dreampore）。设计、生产、验证和测试具有特定纳米孔传感应用的有希望特性的门户蛋白的新变体（约克和Dreampore）.新颖性由于嗜热门户蛋白，如G20 c，比它们的嗜温同源物更稳定，它们提供了下一代纳米传感器的潜在来源，这些纳米传感器比目前可用的纳米传感器更鲁棒且更容易工程化。因此，这些蛋白质提供了一个独特的机会，彻底改变测序应用和生物标志物的检测，不仅在Dreampore，但在整个生物技术部门在英国/欧盟，基于纳米孔的传感技术正在迅速发展，超越了井-已建立的DNA测序应用程序，是目前蓬勃发展的纳米传感技术的一部分，这将彻底改变个人医疗和健康管理。对于嗜热噬菌体的门蛋白（如phiOH、GBSV和GVE 2），将被克隆和优化以表达可溶性门蛋白组装体。可溶性门户蛋白将被生产至高纯度并在生物药理学和结构上表征（在约克），然后使用公开的技术插入纳米孔装置中以限定电感测电位（在Dreampore）。将在迭代过程中将特定的传感特性工程化到生产和表征的合适的门户蛋白中（在约克），随后测试传感应用（在Dreampore）。