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

• 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
• 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

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：真核生物中后修饰蛋白质的受控表达 共价翻译后修饰（PTM）对于许多真核生物蛋白质的功能是至关重要的， 当这些基因失调时会导致人类疾病。PTM机制的分子见解 生物医学研究将产生深远的影响，最终可以挽救生命，改善人类健康。 然而，技术障碍阻碍了这方面的进展。与体外研究不同， 携带所需PTM的重组蛋白已经帮助研究人员详细分析PTM功能， 由于缺乏生产感兴趣蛋白质的有效技术， 在真核细胞中携带所需的PTM。 这项R21技术开发探索性研究旨在创建一种基于真核细胞的 该技术使得能够生产具有用于功能研究的所需PTM的蛋白质。ePIMAX- 本文所要开发的RCR系统是基于细菌蛋白质相互作用模块辅助功能X （PIMAX）方法（Sui等人，2015年，Mol. Cell. Proteomics 14：251-62）。PIMAX使用 一对异源二聚化蛋白质-蛋白质相互作用模块（PIM），以促进蛋白质之间的缔合 感兴趣的（POI）及其修饰酶。PIMAX系统已被证明是非常有效和具体的。 目前的项目旨在采用PIMAX概念的真核系统。为了满足需要， 此外，为了利用真核蛋白表达系统，将新的组分添加到PIMAX中， 该系统将最终实现POI的具体和有效的修改，同时最大限度地减少 修饰酶异位表达的副作用。在这个项目中，我们将 实现三个目标。首先，我们将通过荧光测试ePIMAX-RCR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 以测试该方法的适用性。第三，我们将建立用于ePIMAX-RCR应用的稳定细胞系， 为后续R 01项目动物模型开发提供支持。该ePIMAX-RCR技术将 对生物医学研究产生深远影响。