Split-GFP-based three fragment recombination protein system designation and functional study

doi:10.3877/cma.j.issn.2095-7157.2025.04.009

Chinese Journal of Gastrointestinal Endoscopy(Electronic Edition) ›› 2025, Vol. 12 ›› Issue (04): 273-279. doi: 10.3877/cma.j.issn.2095-7157.2025.04.009

• Original Article • Previous Articles

Split-GFP-based three fragment recombination protein system designation and functional study

Yiyan Zhou¹, Kaimin Jia², Wenjun Zhang², Mingzhou Guo³^,^†()

¹Department of Molecular and Cell Biology, University of California, Berkeley
²Department of Chemical and Biomolecular Engineering; University of California, Berkeley
³Department of Gastroenterology, The First Medical Centre of Chinese PLA General Hospital, Beijing 100853, China

Received:2025-10-19 Online:2025-11-15 Published:2026-01-12
Contact: Mingzhou Guo

Abstract

Abstract:

Objective

Green fluorescent protein (GFP) can be split into multiple fragments that reconstitute fluorescence upon spatial proximity, making it a powerful tool for probing biomolecular interactions.This study aimed to design and optimize a tripartite split-GFP system for monitoring natural product-target protein interactions in vivo.

Methods

The high-affinity interaction between rapamycin and FKBP12 was used as a model system to evaluate split-GFP reconstitution efficiency in yeast cells.A chloroalkane-modified rapamycin derivative was employed to enable HaloTag labeling, and fluorescence output was assessed.To improve system performance, solubility tags were introduced to enhance the stability of the GFP1-9 fragment, and the HaloTag labeling system was replaced with a streptavidin-biotin system with higher binding affinity.

Results

Chemical modification of rapamycin with a chloroalkane moiety significantly enhanced fluorescence intensity without compromising HaloTag labeling efficiency. Nevertheless, the tripartite split-GFP system exhibited substantial background fluorescence in vivo, resulting in weak effective signal and limiting its applicability in high-throughput screening. Improving the solubility and structural stability of the GFP1-9 core fragment increased reconstitution efficiency with the other GFP fragments. Furthermore, replacing the HaloTag system with the higher-affinity streptavidin-biotin labeling system led to a pronounced enhancement of fluorescence signal.

Conclusion

This study establishes and optimizes a tripartite split-GFP platform for detecting natural product-target protein interactions.By improving GFP fragment solubility and incorporating a high-affinity labeling system, signal intensity was significantly enhanced while background interference was reduced, supporting the potential application of this system in high-throughput screening and chemical biology research.GFP reconstitution has been well studied and applied widely as a report system.Since natural products are promising drug candidates, a robust split GFP report system can be valuable for drug development.

Key words: Small molecule report system, Split GFP system, Natural products

Yiyan Zhou, Kaimin Jia, Wenjun Zhang, Mingzhou Guo. Split-GFP-based three fragment recombination protein system designation and functional study[J]. Chinese Journal of Gastrointestinal Endoscopy(Electronic Edition), 2025, 12(04): 273-279.

/ / Recommend

Add to citation manager EndNote|Ris|BibTeX

URL: https://zhwcnjdzzz.cma-cmc.com.cn/EN/10.3877/cma.j.issn.2095-7157.2025.04.009

https://zhwcnjdzzz.cma-cmc.com.cn/EN/Y2025/V12/I04/273

Figures/Tables 6

References 13

[1]	Atanasov AG，Zotchev SB，Dirsch VM，et al.Natural Products in Drug Discovery：Advances and Opportunities[J]．Nat Rev Drug Discov，2021，20(3)：200-216.
[2]	Vane JR.Inhibition of Prostaglandin Synthesis as a Mechanism of Action for Aspirin-like Drugs[J]．Nature New Biol，1971，231 (25)：232- 235.
[3]	Vane JR，Botting RM.The Mechanism of Action of Aspirin[J]．Thromb Res，2003，110(5-6)：255-258.
[4]	Jordan MA，Wilson L．Microtubules as a Target for Anticancer Drugs [J]．Nat Rev Cancer，2004, 4(4)：253-265.
[5]	Tsien RY.The Green Fluorescent Protenin[J]．Annu Rev Biochem, 1998，67(1)：509-544.
[6]	Paulmurugan R，Gambhir SS.Monitoring Protein-Protein Interactions Using Split Synthetic Renilla Luciferase Protein-Fragment-Assisted Complementation[J]．Anal Chem，2003，75(7)：1584-1589.
[7]	Lukyanov KA.Fluorescent proteins for a brighter science[J]．Biochem Biophys Res Commun，2022，10(633)：29-32.
[8]	Park Ki Sung，Cha Hanvit，Niu Jia，et al.DNA-controlled protein fluorescence: Design of aptamer-split peptide hetero-modulator for GFP to respond to intracellular ATP levels[J]．Nucleic Acids Res，2024，52(14)：8063-8071.
[9]	Chen J，Zheng XF，Brown EJ，et al.Identification of an 11-kDa FKBP12- Rapamycin-Binding Domain within the 289-kDa FKBP12- Rapamycin-Associated Protein and Characterization of a Critical Serine Residue[J]．Proc Natl Acad Sci，1995，92(11)：4947-4951.
[10]	Brown EJ，Albers MW，Bum Shin T，et al.A Mammalian Protein Targeted by G1-Arresting Rapamycin-Receptor Complex[J]．Nature，1994, 369(6483)：756-758.
[11]	Cabantous S，Nguyen HB，Pedelacq JD，et al.A New Protein-Protein Interaction Sensor Based on Tripartite Split-GFP Association[J]．Sci Rep, 2013，3(1)：2854.
[12]	Kapust RB，Waugh DS.Escherichia Coli Maltose-binding Protein Is Uncommonly Effective at Promoting the Solubility of Polypeptides to Which It Is Fused[J]．Protein Sci，1999，8(8)：1668-1674.
[13]	Erdmann RS，Baguley SW，Richens JH，et al.Labeling Strategies Matter for Super-Resolution Microscopy：A Comparison between HaloTags and SNAP-Tags[J]．Cell Chem Biol，2019，26(4)：584-592.e6.

Please choose a citation manager

Content to export

Split-GFP-based three fragment recombination protein system designation and functional study

RichHTML

PDF (PC)

Knowledge