NMR characterization of recombinant transmembrane protein CB2 fragment CB2(180-233).

The expression of membrane proteins has been the bottleneck for their structural studies. Recently, we developed a method to obtain milligram quantities of isotope-labeled seven transmembrane G-protein coupled cannabinoid (CB) receptor fragment in E. coli. In order to verify this method and confirm the recombinant isotope-labeled CB2 fragment, 3D hetero-nuclear NMR techniques were used to analyze the structure of the fragment CB2(180-233) in DMSO-d6 solvent. The sequential assignments of TM5 and intra-cellular loop 3 were accomplished, which confirmed the experimental protocols of isotope-labeled recombinant protein expression, fusion protein cleavage, and membrane protein purification. The obtained structure also showed alpha-helix in the TM5 region, but it was interrupted by a disordered region (Gly204_ILe206). These results further revealed that our established approach is a promising method to express recombinant membrane proteins for their structural studies.