Expression, Purification and Characterization of the Human Cannabinoid 1 Receptor

The human cannabinoid 1 receptor (hCB1) is involved in numerous physiological processes and therefore provides a wide scope of potential therapeutic opportunities to treat maladies such as obesity, cardio-metabolic disorders, substance abuse, neuropathic pain, and multiple sclerosis. Structure-based drug design using the current knowledge of the hCB1 receptor binding site is limited and requires purified active protein. Heterologous expression and purification of functional hCB1 has been the bottleneck for ligand binding structural studies using biophysical methods such as mass spectrometry, x-ray crystallography and NMR. We constructed several plasmids for in-cell or in vitro Escherichia coli (E. coli) based expression of truncated and stabilized hCB1 receptor (h Delta CB1 and h Delta CB1(T4L) ) variants and evaluated their competency to bind the CP-55,940 ligand. MALDI-TOF MS analysis of in vitro expressed and purified h Delta CB1(T4L) his6 variants, following trypsin digestion, generated similar to 80% of the receptor sequence coverage. Our data demonstrate the feasibility of a cell-free expression system as a promising part of the strategy for the elucidation of ligand binding sites of the hCB1 receptor using a Ligand Assisted Protein Structure (LAPS) approach.