Engineering a new tripartite split-ccGFP system from Corynactis californica for detecting protein–protein interactions

Groseclose, Thomas Michael; Kober, Erin; Zupancic, Jennifer Marie; McLelland, Claire K.; Esmith Lujan, Lexy Amber; Kunde, Yuliya A.; Mozden, Sarah C.; Velappan, Nileena; Lillo, Antonietta Maria; Solomon, Emilia; Yoder, Jacob; Waldo, Geoffrey S.; Nguyen, Hau Thi Bich

doi:10.1038/s41598-025-24657-6

Engineering a new tripartite split-ccGFP system from Corynactis californica for detecting protein–protein interactions

Journal Article · Wed Nov 19 00:00:00 EST 2025 · Scientific Reports

DOI:https://doi.org/10.1038/s41598-025-24657-6· OSTI ID:3007513

^[1]; Kober, Erin ^[1]; ^[1]; McLelland, Claire K. ^[1]; Esmith Lujan, Lexy Amber ^[1]; ^[1]; Mozden, Sarah C. ^[1]; ^[1]; ^[1]; Solomon, Emilia ^[1]; Yoder, Jacob ^[1]; Waldo, Geoffrey S. ^[1]; ^[1]

Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)

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.

View Accepted Manuscript (DOE)

Research Organization:: Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)

Sponsoring Organization:: Defense Threat Reduction Agency (DTRA); Los Alamos National Laboratory Directed Research and Development program; USDOE National Nuclear Security Administration (NNSA)

Grant/Contract Number:: 89233218CNA000001

OSTI ID:: 3007513

Report Number(s):: LA-UR--25-25469; 10.1038/s41598-025-24657-6; 2045-2322

Journal Information:: Scientific Reports, Journal Name: Scientific Reports Journal Issue: 1 Vol. 15; ISSN 2045-2322

Publisher:: Nature Publishing GroupCopyright Statement

Country of Publication:: United States

Language:: English

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Related Subjects

59 BASIC BIOLOGICAL SCIENCES
Antibody
Directed evolution
Green fluorescent protein (GFP)
Protein detection
Protein engineering
Protein fragment complementation
Protein tagging
Protein-protein interactions
Single-chain variable fragment (scFv)
Split fluorescent protein

Engineering a new tripartite split-ccGFP system from Corynactis californica for detecting protein–protein interactions

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