Bacterial expression of functional, biotinylated peripheral cannabinoid receptor CB2

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• The peripheral cannabinoid receptor (CB2) is a G protein-coupled receptor (GPCR) involved in various physiological processes.
• Efficient purification and immobilization of membrane proteins like CB2 are crucial for structural and functional studies.
• Biotinylation is a powerful tool for protein labeling, purification, and immobilization.

Purpose of the Study:

• To develop a method for efficient biotinylation of the CB2 receptor.
• To enable purification and oriented immobilization of the CB2 receptor for structural characterization.

Main Methods:

• A biotin-protein ligase recognition site (BRS) was engineered into a fusion protein containing CB2.
• Recombinant CB2 fusion proteins were expressed in Escherichia coli.
• Biotinylation was achieved both in vivo and in vitro using BirA biotin-protein ligase.
• Cleavage with tobacco etch virus protease and immobilization on avidin beads were performed.

Main Results:

• A CB2-fusion protein with a C-terminal BRS (CB2-109) showed complete in vivo biotinylation and retained ligand-binding capacity.
• A CB2-fusion protein with an N-terminal BRS (CB2-112) required in vitro biotinylation but was successfully biotinylated after purification.
• Biotinylated CB2 fusion proteins were competent for streptavidin binding and could be immobilized on avidin beads.

Conclusions:

• Engineered biotinylation sites allow for efficient purification and oriented immobilization of the CB2 receptor.
• This method facilitates structural characterization of the CB2 receptor by providing a homogeneously oriented protein on a solid support.