Cell-Free Synthesis of Human Endothelin Receptors and Its Application to Ribosome Display

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• G-protein-coupled receptors (GPCRs) are a large protein family and major drug targets due to their critical roles in signaling pathways.
• Engineering GPCRs for enhanced stability or altered function is of significant interest for therapeutic development.

Purpose of the Study:

• To develop a high-throughput screening (HTS) method for GPCRs using an in vitro transcription-translation (IVTT) system.
• To investigate the role of phospholipids in the functional synthesis of GPCRs within an IVTT system.

Main Methods:

• Reconstituted human endothelin receptor type-B (ETBR) and type-A (ETAR) in nanodiscs (NDs) using specific phospholipids (POPG) within an IVTT system.
• Utilized ribosome display for in vitro directed evolution and screening of up to 10^12 GPCR mutants.
• Validated the method using a mock library for gene screening of ETBR.

Main Results:

• POPG-containing NDs were crucial for the functional folding and ET-1 binding of ETBR in the IVTT system.
• Both ETAR and ETBR were successfully synthesized in functional forms and subjected to ribosome display under optimized IVTT conditions.
• Demonstrated the feasibility of high-throughput screening and directed evolution of GPCRs in vitro.

Conclusions:

• The developed IVTT system with POPG-NDs enables the functional synthesis and high-throughput screening of GPCRs.
• This platform facilitates the directed evolution of GPCRs, paving the way for novel drug discovery targeting these important receptors.