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

该计划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。具有合适性质的蛋白质将被进一步的生物工程和/或化学修饰，以调整它们的传感能力以适应特定的应用。背景：病毒门户蛋白是天然的生物纳米孔，设计用于在病毒包装过程中将病毒基因组dsDNA转移到预先形成的衣壳中。几个实验室的研究表明，这些蛋白质具有在生物技术中应用于纳米孔的有益性质，特别是肽/蛋白质传感和测序，表现出比目前可用的其他类型的生物纳米孔更好的性质。来自几种病毒的门户蛋白，包括嗜热噬菌体，已经在约克进行了生化和结构特征分析。梦想资本正专注于为生物分子开发新的纳米孔传感应用，这些应用可能受益于门户蛋白的使用。该项目将通过识别和表征新的纳米孔来扩大现有技术的范围。目标：从几种耐热病毒(YOCK)中制备以前未被表征的门户蛋白。表征这些蛋白质的生物物理性质(YORK)。通过X射线结晶学或低温电子显微镜(YORK)确定有希望的PORTAL蛋白的三维结构。通过生物和/或化学工程将PORTAL变异体插入纳米孔传感装置(YOKK/DreamPore)。控制蛋白质纳米孔的插入和在脂质和或固态膜中的稳定性(DreamPore)。检测和表征蛋白质或多肽生物标记物通过单分子水平上的电信号对不同的门户蛋白通道(梦幻孔)进行测序，利用蛋白质模型(梦幻孔)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(约克和梦幻)。有趣的是：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革新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。及时性：基于纳米孔的传感正在迅速发展，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 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).