Developing protein scaffolds for VLP assembly and enzyme organisation in cell-free protein synthesis

开发用于无细胞蛋白质合成中 VLP 组装和酶组织的蛋白质支架

• 批准号: 2073430
• 金额: --
• 依托单位: University College London
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2018
• 资助国家: 英国
• 起止时间: 2018 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2073430
• 关键词: Developing; protein; scaffolds; VLP; assembly

Background:Protein-based compartments such as Virus-like-particles (VLPs), bacterial nanocompartments (encapsulins) and more recently, bacterial microcompartments (BMCs) are attractive nano-scaffolds with broad applications in biomedicine, nano-biotechnology, biocatalysis and material sciences. They are entirely self-assembling and can be genetically engineered to incorporate modifications. VLPs resemble viruses, they are highly immunogenic due to the repetitive presentation of epitopes on the surface and are therefore developed as vaccines and therapeutics. Although VLPs are robust structures, when foreign epitopes are inserted into the VLP for display on the surface, the formation of stable so-called 'chimeric VLPs' can be dramatically decreased (Peyret et al. 2015, Kazaks et al. 2017). Current approaches to stabilising VLPs include addition of carboxy-terminal polyhistidine tags on VLP proteins and disulphide-bridges (Schumacher et al. 2018).This project is investigating novel strategies for the stabilisation of hepatitis B virus core antigen (HBcAg) VLPs, namely the use of protein nano-structures as scaffolds for improved assembly and stability of VLP particles. Such nano-scaffolds may include encapsulins, which form readily, are extremely stable at a wide pH range and can be genetically engineered to create fusion proteins (Giessen 2016). These properties will be utilised to facilitate interactions with VLP proteins with the aim to provide mechanical and chemical stability for chimeric VLPs. With a growing interest in the development of cell-free protein expression systems for VLP assembly, synthetic circuit testing and enzyme engineering, multi-protein assembly and spatial organisation in cell-free systems is important to understand (Lu 2017). In this respect, the project will also investigate if more complex, multi-protein assemblies such as bacterial microcompartments (BMCs) can be readily formed in a cell-free environment. BMCs are megadalton protein assemblies, composed of 10,000-20,000 polypeptides of 10-20 types that self-assemble in a modular fashion and house enzymes up to mM concentration, providing an environment for high local enzyme concentration. Components of naturally occurring BMCs can be modified and combined to permit the construction of large protein scaffolds and bioreactors with novel functions (Kerfeld and Erbilgin 2015). So far these are produced and tested within bacterial cells with the aim to organise enzymes in vivo. Cell-free systems could provide a high-throughput test-platform for engineered BMCs and may allow for enzyme scaffolding in cell-free systems.Objectives:- Stable assembly of encapsulins in vivo and cell-free (1-12 months)- Development of nano-scaffolds from engineered encapsulins (13-24 months)- Self-assembling on multi-protein structures for enzyme scaffolding (25-36 months)

背景：基于蛋白质的隔间，如病毒样颗粒（vlp）、细菌纳米隔间（胶囊）和最近的细菌微隔间（BMCs）是有吸引力的纳米支架，在生物医学、纳米生物技术、生物催化和材料科学中有着广泛的应用。它们完全是自组装的，可以通过基因工程进行改造。VLPs类似于病毒，由于表位在表面的重复呈现，它们具有高度的免疫原性，因此被开发为疫苗和疗法。虽然VLPs是坚固的结构，但当外源表位插入到VLP中并在表面显示时，稳定的所谓“嵌合VLPs”的形成可以显着减少（Peyret et al. 2015, Kazaks et al. 2017）。目前稳定vllp的方法包括在VLP蛋白和二硫化物桥上添加羧基端多组氨酸标签（Schumacher et al. 2018）。该项目正在研究稳定乙型肝炎病毒核心抗原（HBcAg） vllp的新策略，即使用蛋白质纳米结构作为支架来改善VLP颗粒的组装和稳定性。这种纳米支架可能包括易于形成的胶囊，在很宽的pH范围内非常稳定，并且可以通过基因工程来制造融合蛋白（Giessen 2016）。这些特性将被用于促进与VLP蛋白的相互作用，目的是为嵌合VLP提供机械和化学稳定性。随着人们对开发无细胞蛋白表达系统用于VLP组装、合成电路测试和酶工程的兴趣日益浓厚，了解无细胞系统中的多蛋白组装和空间组织非常重要（Lu 2017）。在这方面，该项目还将研究更复杂的多蛋白组合，如细菌微室（BMCs），是否可以在无细胞环境中轻易形成。bmc是由10-20种类型的10,000-20,000多肽组成的兆道尔顿蛋白质组件，以模块化的方式自组装，并容纳高达mM浓度的酶，为高局部酶浓度提供环境。天然存在的bmc成分可以被修改和组合，以允许构建具有新功能的大型蛋白质支架和生物反应器（Kerfeld和Erbilgin 2015）。到目前为止，这些都是在细菌细胞中生产和测试的，目的是在体内组织酶。无细胞系统可以为工程bmc提供高通量测试平台，并且可以在无细胞系统中允许酶支架。目标：-在体内和无细胞中稳定组装胶囊蛋白（1-12个月）-从工程胶囊蛋白中开发纳米支架（13-24个月）-酶支架的多蛋白结构自组装（25-36个月）