Biotin Ligase-Based Reporter for Detecting Protease Activity

Area of Science:

• Biochemistry
• Molecular Biology
• Cellular Biology

Background:

• Protease activity is vital for cellular signaling pathways and viral replication.
• Existing methods for detecting protease activity have limitations in certain cellular contexts.

Purpose of the Study:

• To develop a novel, proximity-dependent sensor for detecting protease activity.
• To create a biotin ligase-based reporter system for protease cleavage events.
• To enable sensitive and versatile detection of protease function in biological systems.

Main Methods:

• Engineering a proximity-dependent biotin ligase (TurboID) into a sensor named BAGELS (Biotin Annotating Genetically Encoded Ligase-based Sensor).
• Testing the sensor's signal-to-background ratio in response to protease activity.
• Validating the sensor's performance in neurons and HEK293T cells.
• Demonstrating applications in protease inhibitor characterization and multiplexed imaging.

Main Results:

• BAGELS demonstrated a signal-to-background ratio of approximately 48.
• The sensor successfully linked protease activity to proximal protein biotinylation.
• The reporter was validated in both neuronal and HEK293T cell lines.
• The system proved effective for characterizing protease inhibitors and for multiplexed imaging.

Conclusions:

• BAGELS is the first protease activity sensor utilizing a biotin ligase readout in neurons and HEK293T cells.
• This sensor offers a versatile, enzymatically amplified, biotin-based detection method.
• The reporter system is compatible with various biotin-based detection techniques, expanding its utility.