Expression, surface immobilization, and characterization of functional recombinant cannabinoid receptor CB2

Area of Science:

• Biochemistry
• Molecular Biology
• Immunology

Background:

• The human peripheral cannabinoid receptor CB2 (a G protein-coupled receptor) is crucial for immune response regulation and a key pharmaceutical target.
• Structural and functional studies of CB2 require its immobilization on surfaces with controlled density and orientation.

Purpose of the Study:

• To develop a universal strategy for preparing functional recombinant CB2 and immobilizing it onto solid interfaces.
• To enable detailed structural and functional investigations of CB2 for drug discovery.

Main Methods:

• Expression of CB2 as a Rho-tag fusion protein in E. coli.
• Purification using sequential chromatography (1D4 and Ni-NTA resins).
• Surface Plasmon Resonance (SPR) for characterizing receptor-antibody interactions and immobilization orientation.

Main Results:

• Highly pure and functional CB2 was successfully obtained.
• Quantitative and uniformly oriented immobilization of CB2 onto a 1D4-coated sensor chip was achieved.
• SPR confirmed the accessibility of the immobilized CB2's extracellular surface and its interaction with a novel antibody (NAA-1).

Conclusions:

• An integrated strategy for CB2 purification and surface immobilization has been established.
• This method facilitates ligand- and antibody-binding studies of functional CB2.
• The developed approach supports pharmaceutical research targeting the CB2 receptor.