Expression and purification of the human thrombin receptor

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• The human thrombin receptor (PAR1) plays a crucial role in various physiological processes.
• Efficient expression systems are needed for detailed studies of receptor structure and function.

Purpose of the Study:

• To overexpress and characterize the functional human thrombin receptor in insect cells.
• To establish a system for producing purified thrombin receptor for further investigation.

Main Methods:

• Human thrombin receptor gene cloned into a baculovirus vector for expression in Sf9 insect cells.
• Immunocytochemistry used to confirm cell surface receptor expression.
• Functional assays measuring intracellular calcium increase in response to thrombin and a specific agonist peptide.
• Immunoaffinity chromatography for receptor purification.
• SDS-PAGE and Western blotting to analyze purified receptor components.

Main Results:

• Successful cell surface overexpression of functional human thrombin receptor confirmed.
• Receptor responded to both thrombin (EC50 = 3.9 nM) and agonist peptide (EC50 = 2.7 µM) with intracellular calcium increases.
• Purified receptor yielded two species (48 and 90 kDa) recognized by specific antibodies.
• High yield of purified receptor (0.78 mg/L) achieved.

Conclusions:

• Baculovirus-mediated expression in Sf9 cells is an effective method for producing functional human thrombin receptor.
• The purified recombinant receptor is suitable for future structural and functional analyses.
• This system provides a valuable resource for studying thrombin receptor biology.