Characterization of an engineered water-soluble variant of the full-length human mu opioid receptor.

A water-soluble variant of the transmembrane domain of the human mu opioid receptor (wsMOR-TM) was previously characterized. This study explored whether the full-length version of the engineered water-soluble receptor, (wsMOR-FL), could be overexpressed in Escherichia coli and if it would retain water solubility, binding capability and thermostability. wsMOR was over-expressed and purified in E. coli BL21(DE3) cells (EMD/Novagen) as we reported previously for the wsMOR-TM. Both native N and C termini were added back to the highly engineered wsMOR-TM. Six His-tag was added in the N terminus for purification purposes. The wsMOR-FL was characterized using atomic force microscope for its monomeric state, circular dichroism for its secondary structure and thermostability. Its binding with naltrexone is also determined. Compared to the native human MOR, wsMOR-FL displays similar helical secondary structure content and comparable affinity (nM) for the antagonist naltrexone. The secondary structure of the receptor remains stable within a wide range of pH (6?9). In contrast to the transmembrane portion, the secondary structure of full-length receptor tolerated a wide range of temperature (10?90??C). The receptor remains predominantly as a monomer in solution, as directly imaged using atomic force microscopy. This study demonstrated that functional full-length water-soluble variant of human mu receptor can be over-expressed and purified using an E. coli over-expression system. This provides a novel tool for the investigation of structural and functional properties of the human MOR. N- and C-termini strengthened the thermostability of the protein in this specific water soluble variant. Communicated by Ramaswamy H. Sarma