A sensitive split luciferase assay for detecting olfactory receptor response to odorants

Area of Science:

• Biochemistry
• Molecular Biology
• Assay Development

Background:

• Olfactory receptors (ORs) play a crucial role in detecting odorants.
• Developing sensitive assays for OR ligand identification is essential for understanding olfaction.
• Existing assays often lack sufficient sensitivity for detecting low-affinity ligands.

Purpose of the Study:

• To develop a highly sensitive split luciferase-based assay for identifying olfactory receptor (OR) ligands.
• To optimize the cell surface expression and assembly of fusion proteins for enhanced luminescence.
• To improve the detection sensitivity of OR-ligand interactions compared to conventional methods.

Main Methods:

• Constructed fusion proteins of ORs and Golf with split luciferase components (LgBiT and SmBiT).
• Engineered a membrane-localized Golf fusion protein (V1N-G4S3-SmBiT-G4S3-Mini-Golf) to enhance luminescence.
• Co-expressed fusion proteins and receptor transporting protein 1 short form (RTP1S) in HEK293T cells.
• Utilized AlphaFold3 for structural predictions of protein interactions.

Main Results:

• Initial fusion proteins showed low luminescence; engineered membrane-localized Golf fusion significantly increased signal.
• Co-expression of RTP1S markedly enhanced cell surface expression of the OR-LgBiT fusion protein.
• The optimized assay demonstrated a 1000- to 3000-fold increase in sensitivity for detecting OR ligands compared to cAMP assays.
• AlphaFold3 predicted an interaction between RTP1S N-terminal domain and the LgBiT tag.

Conclusions:

• Optimizing the cell membrane assembly of split luciferase components is critical for sensitive OR ligand detection.
• The developed split luciferase assay offers a powerful tool for investigating ligands of both known and orphan ORs.
• This enhanced assay system significantly advances the field of olfactory receptor research and drug discovery.