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

EPIMAX-RCR：翻译后修饰蛋白在真核细胞中的受控表达 共价翻译后修饰(Ptm)对许多真核蛋白质的功能至关重要，其中许多 它们在调控不当时会导致人类疾病。PTM发病机制的分子机制研究 功能将对生物医学研究产生深远的影响，并最终可以拯救生命，改善人类健康。 然而，技术障碍阻碍了这方面的进展。与细菌表达的体外研究不同 携带所需PTM的重组蛋白帮助研究人员详细剖析了PTM的功能， 由于缺乏生产感兴趣蛋白质的有效技术，体内研究远远落后。 在真核细胞中携带所需的PTM。 本次R21技术开发探索性研究旨在打造以真核细胞为基础的 能够生产含有所需PTM的蛋白质用于功能研究的技术。EPIMAX- 本文将要开发的RCR系统是基于细菌蛋白相互作用模块辅助的功能X (PIMAX)方法由PI的小组设计(Sui等人，2015，Mol.牢房。蛋白质组学14：251-62)。PIMAX使用 一对异源二聚蛋白质-蛋白质相互作用模块(PIM)，以促进蛋白质之间的结合 兴趣蛋白(POI)及其修饰酶。PIMAX系统已被证明是非常有效和具体的。 目前的项目旨在将PIMAX概念应用于真核系统。以适应需要和 此外，为了利用真核蛋白质表达系统，在PIMAX中添加了新的成分 最终实现对POI的具体和有效修改的系统，同时最大限度地减少 修饰酶异位表达的不良影响。在这个项目的过程中，我们将 实现三个目标。首先，我们将用荧光法测试ePIMAX-RCR核心设计的可行性 共振能量转移(FRET)。其次，我们将使用与生物医学相关的具有代表性的蛋白质 以检验该方法的适用性。第三，我们将建立稳定的ePIMAX-RCR应用细胞系， 这将为动物模型开发的后续R01项目提供支持。这项ePIMAX-RCR技术将 深刻影响生物医学研究。

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.