Monitoring regulated protein-protein interactions using split TEV

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• Cellular signaling relies on protein-protein interactions (PPIs) for signal transduction.
• Monitoring these interactions in real-time within living cells is crucial for understanding cellular processes.

Purpose of the Study:

• To develop a novel biological assay for monitoring protein-protein interactions in living mammalian cells.
• To provide a flexible system for detecting both constitutive and regulated PPIs.

Main Methods:

• Engineered inactive fragments of the tobacco etch virus (TEV) NIa protease.
• Fused these fragments to interacting proteins of interest.
• Utilized reporter systems (fluorescent, luminescent, or transcription-coupled) to detect reconstituted TEV protease activity.

Main Results:

• Successfully developed the split TEV assay for monitoring PPIs in living cells.
• Demonstrated the assay's ability to detect functional reconstitution of TEV protease activity.
• Applied split TEV to monitor neuregulin-induced ErbB2/ErbB4 receptor tyrosine kinase heterodimerization.

Conclusions:

• Split TEV is a versatile and flexible assay for studying protein-protein interactions in real-time.
• The assay can be adapted with various reporter systems for diverse readout strategies.
• This method offers a valuable tool for investigating signaling pathways and molecular interactions within living cells.