Interaction of Peripheral Cannabinoid Receptor CB2 with Antibodies Studied by Surface Plasmon Resonance (SPR)

Tuesday, October 09, 2012 — Poster Session I
1:00 p.m. – 3:00 p.m	Natcher Conference Center, Building 45	NIAAA	BIOCHEM-9

Authors

• S.C. Locatelli-Hoops
• I. Gorshkova
• K. Gawrisch
• A.A. Yeliseev

Abstract

Cannabinoid receptor CB2 is a G protein-coupled receptor involved in immune response. To exploit its potential as a therapeutic target a better understanding of its molecular structure and mode of action is required The goal of this study is to develop SPR-based assays for monitoring interactions of CB2 with binding partners in real time. Functionally active CB2 was produced in E coli as a fusion with maltose binding protein (MBP), purified by tandem chromatography using small affinity tags, and MBP proteolytically removed during purification. Either CB2 fusion protein or purified receptor, containing a C-terminal nonapeptide (1D4), were captured on the CM4 sensor chip surface (Biacore) pre-coated with anti-1D4 Ab. Capture was specific and quantitative, and binding of a monoclonal antibody against CB2 was demonstrated. A surface regeneration protocol was developed. Furthermore, purified CB2 reconstituted into liposomes was immobilized onto L1 sensor chip. Specific binding of single domain antibodies that recognize functional conformations of CB2 was demonstrated using temperature-inactivated receptor as a reference, and KD values determined. To conclude, immobilization of CB2 in detergent micelles or liposomes on a sensor chip and feasibility of studying its interaction with binding partners by SPR has been demonstrated.

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