Expression and characterization of recombinant human peripheral cannabinoid receptor CB2: exploring the Halo tag and 1D4-Rho tag as novel tools for purification and immobilization

Monday, October 24, 2011 — Poster Session I
Noon – 2:00 p.m.	Natcher Conference Center	NIAAA	BIOCHEM/CHEM-14

Authors

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

Abstract

Human peripheral cannabinoid receptor CB2 is a G protein-coupled receptor involved in regulation of the immune response. To obtain functional and structural information, essential to exploit its potential as a therapeutic target, pure and active protein is required. We aim at developing tools for efficient expression, purification and immobilization of CB2. Here, we explore usability of haloalkane dehalogenase protein (Halo tag) and rhodopsin C-terminal nanopeptide (1D4 Rho tag), that enables purification via covalent capture and specific antibody-binding respectively. Constructs expressing CB2 as a fusion with either of these affinity tags were prepared. Expression levels of the fusion protein and accessibility of tags were tested by Western blotting. Functional activity was evaluated by saturation binding and G-protein activation assay. Purification of CB2 from best performing constructs was performed by anti-1D4 antibody affinity chromatography, proteolytic removal of fusion partners and Ni-NTA affinity chromatography. Pure and functionally active protein was obtained. Purified 1D4-Rho tagged CB2 was specifically and quantitatively captured on a sensor chip for surface plasmon resonance, coated with anti-1D4 antibody. Binding of anti-CB2 monoclonal antibody confirmed immobilization of the receptor. In conclusion, we developed an efficient strategy for expression, purification and immobilization of functionally active CB2 suitable for further biophysical characterization.

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