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

提出的项目是生产新的嗜热噬菌体门蛋白，以表征其结构和物理化学性质，并在电传感实验中定义其作为纳米孔的功能。具有适当性质的蛋白质将进一步进行生物工程和/或化学修饰，以调整其特定应用的传感能力。背景：病毒门静脉蛋白是一种天然的生物纳米孔，用于在病毒包装过程中将病毒基因组dsDNA转运到预先形成的衣壳中。几个实验室的研究表明，这些蛋白质对纳米孔在生物技术中的应用具有有益的特性，特别是在肽/蛋白质传感和测序方面，比目前可用的其他类型的生物纳米孔具有优越的性能。来自几种病毒的门蛋白，包括嗜热噬菌体，已经在约克进行了生化和结构表征。Dreampore专注于开发新的纳米孔传感应用于生物分子，这些应用可能受益于门脉蛋白的使用。该项目将通过识别和表征适合先进纳米传感应用的具有新特性的新纳米孔来扩展当前技术的范围。目的：从几种耐热病毒中产生以前未表征的门蛋白（York）。描述这些蛋白质的生物物理特性（约克）通过x射线晶体学或冷冻电镜（York）确定有前途的门脉蛋白的三维结构。生物和/或化学工程入口变体插入纳米孔传感装置（York/Dreampore）。控制蛋白质纳米孔在脂质和/或固态膜（Dreampore）内的插入和稳定性。通过电信号在单分子水平上检测和表征不同门静脉蛋白通道（Dreampore）的蛋白质或多肽生物标志物。设计、生产、表征和测试具有特定纳米孔传感应用前景的门脉蛋白的新变体（York和Dreampore）。新颖性：由于像G20c这样的嗜热门蛋白比它们的中温同源物更稳定，它们为下一代纳米传感器提供了潜在的来源，这些纳米传感器比现有的纳米传感器更坚固，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志物的检测，不仅在Dreampore，而且在英国/欧盟以及其他地区的生物技术领域。及时性：基于纳米孔的传感技术正在迅速发展，超越了已建立的DNA测序应用，是当前小型化纳米传感技术蓬勃发展的一部分，这将彻底改变个人医疗和健康管理。实验方法：将克隆嗜热噬菌体门脉蛋白（如phiOH， GBSV和GVE2）基因并优化其表达可溶性门脉蛋白组装体。可溶性门脉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），然后使用已发表的技术插入纳米孔设备以定义电传感电位（在Dreampore）。在一个迭代的过程中，特定的传感特性将被设计成合适的门蛋白，在约克生产和表征，随后在Dreampore进行传感应用测试。

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).