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

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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.