ePIMAX-RCR: Controlled expression of post-translationally modified proteins in eukaryotes

ePIMAX-RCR：真核生物中翻译后修饰蛋白的受控表达

基本信息

批准号：
10095625
负责人：
Min-Hao Kuo
金额：
$ 23.48万
依托单位：
MICHIGAN STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-15 至 2022-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10095625
关键词：
Acetylation Adopted Adverse effects Amino Acid Substitution Amino Acids Animal Model Aspartic Acid Bacterial Proteins Biomedical Research Cell Line Cells Charge Complex Development Dimerization Ectopic Expression Elements Environment Enzymes Eukaryota Eukaryotic Cell Event Fluorescence Resonance Energy Transfer Foundations Glutamine Health Heterodimerization Human In Vitro Leucine Zippers Measurable Mediating Methods Modification Molecular Nature Outcome Pathologic Peptide Hydrolases Phosphorylation Phosphoserine Post-Translational Protein Processing Production Protein p53 Proteins Proteome Proteomics Recombinant Proteins Recombinants Research Research Personnel Risk Scheme System Technology Testing Time Ubiquitin alpha Tubulin animal model development base design follow-up high reward human disease improved in vivo inducible gene expression innovation insight interest mimetics new technology novel overexpression pleiotropism protein expression protein protein interaction reconstitution spatiotemporal stable cell line technology development technology research and development three dimensional structure

项目摘要

ePIMAX-RCR: Controlled expression of post-translationally modified proteins in eukaryotes Covalent posttranslational modifications (PTM) are crucial for the function of numerous eukaryotic proteins, many of which contribute to human diseases when dysregulated. Molecular insights into the mechanism of PTM function will impact biomedical research profoundly, and can ultimately save lives and improve human health. However, technical hurdles hinder the progress in this front. Unlike in vitro studies in which bacterially expressed recombinant proteins bearing the desired PTM have helped researchers dissect PTM function to great details, in vivo studies are far behind due to the lack of an effective technology that produces the protein of interest bearing the desired PTM in eukaryotic cells. This R21 Exploratory Research for Technology Development aims to create a eukaryotic cell-based technology enabling the production of proteins bearing the desired PTM for functional studies. The ePIMAX- RCR system to be developed herein is based on the bacterial protein interaction modules-assisted function X (PIMAX) approach devised by the PI’s group (Sui et al., 2015, Mol. Cell. Proteomics 14:251-62). PIMAX uses a pair of heterodimerizing protein-protein interaction modules (PIMs) to facilitate the association between a protein of interest (POI) and its modifying enzyme. The PIMAX system has been proven highly effective and specific. The current project aims to adopt the PIMAX concept for eukaryotic systems. To accommodate the need and also to take advantage of eukaryotic protein expression systems, novel components are added to the PIMAX system that will eventually enable specific and efficient modification of the POI, and at the same time minimizes adverse effects from ectopic expression of the modifying enzyme. During the course of this project, we will achieve three aims. Firstly, we will test the feasibility of the core design of ePIMAX-RCR by fluorescence resonance energy transfer (FRET). Secondly, we will use representative proteins with biomedically relevant PTM to test the applicability of this method. Thirdly, we will establish stable cell lines for ePIMAX-RCR application, which will support the follow-up R01 project for animal model development. This ePIMAX-RCR technology will impact biomedical research profoundly.

Epimax-RCR：真核生物中翻译后修饰的蛋白的受控表达共价翻译后修饰（PTM）对于众多真核蛋白的功能至关重要，许多真核蛋白当失调失调时，其中有助于人类疾病。对PTM机制的分子见解功能将对生物医学研究产生深远的影响，并最终可以挽救生命并改善人类健康。但是，技术障碍阻碍了这方面的进步。与细菌表达的体外研究不同带有所需PTM的重组蛋白已帮助研究人员将PTM功能剖析到很好的细节中，由于缺乏产生感兴趣蛋白质的有效技术，体内研究远远落后在真核细胞中带有所需的PTM。 R21技术开发研究旨在创建基于真核生物的细胞技术可以生产具有所需PTM进行功能研究的蛋白质。 epimax- 本文开发的RCR系统基于细菌蛋白相互作用模块辅助函数x （PIMAX）PI组设计的方法（Sui等，2015，Mol。Cell。Proteomics14：251-62）。 Pimax使用一对杂二聚体蛋白质 - 蛋白质相互作用模块（PIM），以促进蛋白质之间的关联感兴趣的（POI）及其修饰酶。 PIMAX系统已被证明是高效且具体的。当前的项目旨在采用真核系统的PIMAX概念。满足需求和为了利用真核蛋白表达系统，新型组件也添加到PIMAX中最终将实现POI的特定和高效修改的系统，同时最小化修饰酶的回声表达产生的不利影响。在这个项目过程中，我们将实现三个目标。首先，我们将通过荧光测试Epimax-RCR的核心设计的可行性共振能量转移（FRET）。其次，我们将使用具有生物医学相关PTM的代表性蛋白质测试此方法的适用性。第三，我们将建立用于Epimax-RCR应用的稳定细胞系，这将支持动物模型开发的后续R01项目。这种Epimax-RCR技术将深刻影响生物医学研究。