Thermophage portal proteins for nanopore biotechnology

该项目旨在制备新型的嗜热噬菌体门户蛋白，以表征其结构和物理化学性质，并确定其在电传感实验中作为纳米孔的功能。几个实验室的研究表明，这种门户蛋白具有在生物技术中应用于纳米孔的有利性质，特别是肽/蛋白质传感和测序，表现出优于目前可用的其他类型的生物纳米孔的性质。行业合作伙伴梦幻波尔，在开发生物分子和特定疾病或健康相关生物标志物的新纳米孔传感应用方面处于世界领先地位这个项目将通过识别和表征具有适用于先进纳米传感应用的新特性的新纳米孔来扩大现有技术的范围。目标：从耐热病毒中生产门户蛋白(约克)。表征蛋白质的生物物理性质(约克)。通过X射线结晶学或冷冻-EM(约克)确定有希望的门户蛋白的三维结构。生物和/或化学工程门户变体以插入纳米孔传感设备(约克)。控制蛋白质纳米孔在脂质和固体中的插入和稳定性-状态膜(梦想孔)通过单分子水平上的电信号来检测和表征不同门户蛋白通道的蛋白质或多肽生物标记物(梦想孔)，以利用蛋白质模型(梦想孔)来研究这些通道的测序潜力。生产、鉴定和测试新的门户蛋白变体，这些变体具有用于特定纳米孔传感应用的前景(York和DreamPore)。新的由于嗜热门户蛋白，如G20c，比它们的中温同系物更稳定，它们提供了一个潜在的下一代纳米传感器的来源，比目前可用的更强大和更容易设计。因此，这些蛋白质提供了一个独特的机会来彻底改变测序应用和生物标记物的检测，不仅在梦幻波尔，而且在英国/欧盟和其他地区的生物技术部门。基于时间的纳米孔传感正在迅速发展，超越了成熟的DNA测序应用，是当前微型纳米传感技术繁荣的一部分，这将给个人医学和健康管理带来革命性的变化。实验方法将克隆和优化嗜热噬菌体门户蛋白的基因，以表达可溶的门户蛋白组件。可溶性门户蛋白将被生产到高纯度，并在生物物理和结构上进行表征(在约克)，然后使用已公布的技术插入纳米孔设备中，以确定电子传感潜力(在梦幻波尔)。特定的传感特性将在迭代过程中被改造成适当的门户蛋白(在约克)，并随后进行传感应用测试(在梦幻波尔)。

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).