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Bimolecular Fluorescence Complementation to Visualize Protein-Protein Interactions in Human Cells Based on Gateway
Adriana Lepur1,2, Oliver Vugrek3
1BUCRO BIOCentre, Zagreb, Croatia. alepur@irb.hr.
Methods in Molecular Biology (Clifton, N.J.)
|June 2, 2018
Summary
Bimolecular fluorescence complementation (BiFC) visualizes protein-protein interactions in living cells. This study enhances BiFC with Gateway cloning for efficient, high-throughput discovery of new interactions.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Bimolecular fluorescence complementation (BiFC) is a sensitive method for detecting protein-protein interactions (PPIs) in live cells.
- The technique relies on reconstituting a split fluorescent protein (e.g., YFP) when fused proteins interact.
- BiFC can capture weak and transient interactions due to its irreversible complementation.
Purpose of the Study:
- To enhance the BiFC system for efficient and high-throughput investigation of PPIs.
- To integrate Gateway cloning into BiFC vectors for faster vector construction.
- To provide a protocol for BiFC-based PPI surveys in human cells.
Main Methods:
- Fusion of split fluorescent protein fragments to interacting proteins.
- Expression of fusion proteins in living human cells via non-viral transfection.
- Utilizing Gateway-based cloning for rapid vector assembly.
- Visualization of reconstituted fluorescence to confirm PPIs.
Main Results:
- Successful implementation of Gateway cloning into the BiFC system.
- Enabled time-efficient vector construction for BiFC assays.
- Facilitated high-throughput screening of numerous protein-protein interactions.
- Demonstrated visualization and localization of PPIs in living cells.
Conclusions:
- The enhanced BiFC system with Gateway cloning significantly improves efficiency for PPI discovery.
- This approach allows for rapid, high-throughput investigation of protein interactions.
- The protocol facilitates comprehensive PPI surveys in human cells.
Keywords:
BiFCComplementationFluorescent microscopyFluorescent proteinGatewayLive cellsMammalianPPIsProtein interactionsVenusMore Related Videos
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