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Engineering a new tripartite split-ccGFP system from Corynactis californica for detecting protein–protein interactions
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Engineering a new tripartite split-ccGFP system from Corynactis californica for detecting protein–protein interactions
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Journal Article
Engineering a new tripartite split-ccGFP system from Corynactis californica for detecting protein–protein interactions
2025
Overview
Protein-protein interactions (PPIs) are critical to a range of biological processes and, consequently, aberrant interactions are implicated in many disorders. The study of the complex networks of PPIs promises to elucidate undiscovered roles in cellular processes and the mechanisms of disease. To accomplish this, tools to effectively sense PPIs are necessary. Effective PPI sensors must rapidly detect interactions in real-time with high sensitivity without perturbing the proteins of interest (POIs) under study. Split fluorescent proteins have previously been used to successfully monitor PPIs, in part due to the small size of the tags. Here, we developed an optimized tripartite split GFP system based on
Corynactis californica
GFP (ccGFP) to detect PPIs in vitro. In this sensor system, ccGFP fragments ccGFP10 and ccGFP11 are tagged to two POIs. PPIs can then be detected
via
fluorescence by complementation to the third fragment, ccGFP1-9, which reconstitutes functional ccGFP. The optimized ccGFP system shows improved detection kinetics and pH and temperature stability compared to a previous system. We then validated the sensor by monitoring PPIs in two model systems: attractive/repulsive coiled-coils and rapamycin-inducible FRB/FKBP heterodimerization. Finally, we developed an anti-tripartite ccGFP single-chain variable fragment (scFv), which could enable versatile detection of identified protein-protein complexes.
Publisher
Nature Publishing Group UK,Nature Publishing Group,Nature Portfolio
Subject
/ 631/1647
/ 631/45
/ 631/61
/ Animals
/ Antibody
/ Green fluorescent protein (GFP)
/ Green Fluorescent Proteins - chemistry
/ Green Fluorescent Proteins - genetics
/ Green Fluorescent Proteins - metabolism
/ Humanities and Social Sciences
/ Protein fragment complementation
/ Protein Interaction Mapping - methods
/ Protein-protein interactions
/ Proteins
/ Science
/ Sensors
