Optimisation of heteromeric protein complex formation via co-ordinated mRNA localisation

通过协调 mRNA 定位优化异聚蛋白复合物的形成

• 批准号: 2449207
• 金额: --
• 依托单位: University of Manchester
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2020
• 资助国家: 英国
• 起止时间: 2020 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2449207
• 关键词: Optimisation; heteromeric; protein; complex; formation

The goal of the project is to understand how to optimally generate heteromeric protein complexes in yeast making use of cytosolic translation factories and the test case eIF3. Information gained will be applied to the production of heterologous heteromeric complexes that can be commercially important in yeast (e.g. haemoglobin and human gonadotropin). More widely though the lessons learned will be generally applicable to the production of heterologous proteins using yeast as a host organism. The biotechnology focus of this studentship makes it a good fit to the BBSRC high-level theme 2 "Tackling strategic challenges"More specificially, in this project a student will study the co-ordinated localisation and translation of mRNAs to translation factories. The mRNAs to be studied encode subunits of heteromeric protein complexes, and the key question to be addressed is whether the folding, assembly and biological function of the resulting protein complexes are regulated as a consequence of production at a defined site in a cytosolic translation factory. The eIF3, eIF2 and NatA complexes in yeast will be used as exemplar protein complexes where the mRNAs encoding the various subunits are localised to translation factories. eIF3 and eIF2 represent key complexes involved in the synthesis of proteins in the recruitment of the ribosome to the mRNA to be translated. NatA is an N-acetyl transferase complex responsible for the modification of proteins while they are being produced by the ribosome. All three complexes are highly abundant and important, so it is unsurprising that they form co-translationally at specific sites, but what is less clear is how such an arrangement is set up, organised and controlled. These studies will establish a set of rules that can be then applied to the production of biotechnologically relevant heterologous protein complexes - assessing whether targeting heterologous mRNAs to protein factories increases productivity and yield of protein complexes.A student will be trained in a plethora of different methods and assays as part of this project. Live cell MS2-based imaging techniques and single molecule fluorescent in situ hybridisation will be used to establish the location of mRNAs in cells. Innovative fluorescent assays will be used to study the site of translation of each mRNA, and polysome analysis will be used to characterise the efficiency of translation. Biochemical assays will be used to study the proportion of each subunit that is present in the soluble or protein aggregate fractions, and in vitro assays using yeast translation extracts will be used to dissect the eIF3 assembly process.The questions that will be addressed are: 1. What RNA elements and protein factors control the localisation and translation of the eIF3, eIF2 and NatA mRNAs?2. Is eIF3, eIF2 and NatA production affected by conditions and mutations that alter the localisation of the mRNAs?3. Does localisation to a translation factory alter the proportion of protein complex subunits that are present as soluble active forms versus insoluble aggregates especially under stress conditions?4. Can the information gleaned for eIF3, eIF2 and NatA complex formation be used to direct the formation of translation factories for heterologous protein expression?

该项目的目的是了解如何利用胞质翻译工厂和测试用例EIF3在酵母中最佳生成异源蛋白质复合物。获得的信息将应用于在酵母中可能在商业上至关重要的异源异源复合物的生产（例如血红蛋白和人促性腺激素）。尽管所学的经验教训通常适用于使用酵母作为寄主生物的异源蛋白质的生产。该学生奖学金的生物技术重点使其非常适合BBSRC高级主题2“解决战略挑战”，从而更具体地，在该项目中，学生将研究MRNA对翻译工厂的协调本地化和翻译。要研究的mRNA编码异质蛋白复合物的编码亚基，要解决的关键问题是，由于胞质翻译工厂的定义位点的生产而导致所得蛋白质复合物的折叠，组装和生物学功能是否受到调节。酵母中的EIF3，EIF2和NATA复合物将用作示例蛋白复合物，其中编码各种亚基的mRNA位于翻译工厂。 EIF3和EIF2代表与蛋白质合成核糖体合成核糖体向mRNA募集的关键复合物。 NATA是一种N-乙酰转移酶复合物，负责蛋白质在核糖体产生时的修饰。所有三个复合物都高度丰富且重要，因此它们在特定地点共译并不奇怪，但是尚不清楚的是如何建立，组织和控制这种安排。这些研究将建立一组规则，然后可以应用于生物技术相关的异源蛋白质复合物的产生 - 评估靶向异源mRNA对蛋白质的靶向是否会提高蛋白质复合物的生产率和产量。实时细胞MS2的成像技术和单分子荧光原位杂交将用于建立mRNA在细胞中的位置。创新的荧光测定将用于研究每个mRNA的翻译位点，多层分析将用于表征翻译的效率。生化测定将用于研究可溶性或蛋白质聚集部分中存在的每个亚基的比例，以及使用酵母翻译提取物的体外测定将用于解剖EIF3组装过程。将解决的问题是：1。rna元素和蛋白质因子的本地化和蛋白质因素控制EIF3的本地化和2 eif3，eIF3，eif3，eif2和nat AAT，MRN？ EIF3，EIF2和NATA的产生是否会受到改变mRNA的定位的条件和突变的影响。翻译工厂的定位是否会改变作为可溶性活性形式与不溶性骨料的蛋白质复合亚基的比例，尤其是在压力条件下？4。 EIF3，EIF2和NATA复合物的收集信息是否可以用于指导转化工厂的形成以进行异源蛋白表达？