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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：
10263311
负责人：
Min-Hao Kuo
金额：
$ 19.56万
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 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10263311
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 Proteins Proteome Proteomics Recombinant Proteins Recombinants Research Research Personnel Risk Scheme System TP53 gene Technology Testing Time Ubiquitin alpha Tubulin animal model development base design feasibility testing 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：翻译后修饰蛋白在真核生物中的控制表达