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

背景：基于蛋白质的隔室如病毒样颗粒（VLP）、细菌纳米隔室（BMCs）和最近的细菌微隔室（BMC）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具有广泛的应用。它们完全是自组装的，可以通过基因工程进行改造。VLP类似于病毒，由于表位在表面上的重复呈递，它们具有高度免疫原性，因此被开发为疫苗和治疗剂。尽管VLP是稳健的结构，但当将外源表位插入VLP中以在表面上展示时，稳定的所谓“嵌合VLP”的形成可能会显著减少（Peyret et al. 2015，Kazaks et al. 2017）。目前稳定VLP的方法包括在VLP蛋白和二硫键上添加羧基末端多组氨酸标签（Schumacher et al. 2018）。该项目正在研究稳定B病毒核心抗原（HBcAg）VLP的新策略，即使用蛋白质纳米结构作为支架，以改善VLP颗粒的组装和稳定性。这种纳米支架可能包括易形成的葡聚糖，在宽pH范围内非常稳定，并且可以通过基因工程产生融合蛋白（Giessen 2016）。这些性质将用于促进与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。随着人们对开发用于VLP组装、合成电路测试和酶工程的无细胞蛋白表达系统的兴趣日益浓厚，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，该项目还将研究是否可以在无细胞环境中容易地形成更复杂的多蛋白质组装体，如细菌微区室（BMC）。BMC是兆道尔顿蛋白质组装体，由10-20种类型的10，000 - 20，000个多肽组成，这些多肽以模块化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。可以修饰和组合天然存在的BMC的组分，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内生产和测试的，目的是在体内组织酶。无细胞系统可以为工程化的BMCs提供高通量的测试平台，并且可以允许在无细胞系统中的酶支架。目标：-在体内和无细胞条件下稳定组装BMCs（1-12个月）-从工程化的BMCs开发纳米支架（13-24个月）-在多蛋白结构上自组装酶支架（25-36个月）

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)