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Human M3 muscarinic acetylcholine receptor (M3R), present in both the central and the peripheral nervous system, is involved in several neurodegenerative and autoimmune diseases. Recently, M3R overexpression has been suggested to play a role in certain forms of cancer, showing promise as a new potential pharmacological target. However, the lack of structural information hampered to develop a new potent selective and potent antagonist. We describe here different strategies for overexpressing functional M3R on the perspective of future biophysical studies. To achieve this goal, four tagged M3R genes were engineered and codon optimized. Different heterologous expression systems, including mammalian cells and viral transfection, were employed to overexpress M3R. Although codon optimization resulted in only twofold to threefold increase of M3R expression, we found that epitope tagging of the synthetic M3R, especially with hemagglutinin and Flag epitope tags, could improve M3R expression levels. On the other hand, viral transfection led to a yield of 27 pmol/mg protein that is the highest level reported so far for this receptor subtype in mammalian cells. Taking together several of the strategies used can help increasing M3R expression, not only to start purification efforts but also for secondary structural analysis trial and functional analyses. V

Human M(3) muscarinic acetylcholine receptor (M3R), present in both the central and the peripheral nervous system, is involved in several neurodegenerative and autoimmune diseases. Recently, M3R overexpression has been suggested to play a role in certain forms of cancer, showing promise as a new potential pharmacological target. However, the lack of structural information hampered to develop a new potent selective and potent antagonist. We describe here different strategies for overexpressing functional M3R on the perspective of future biophysical studies. To achieve this goal, four tagged M3R genes were engineered and codon optimized. Different heterologous expression systems, including mammalian cells and viral transfection, were employed to overexpress M3R. Although codon optimization resulted in only twofold to threefold increase of M3R expression, we found that epitope tagging of the synthetic M3R, especially with hemagglutinin and Flag epitope tags, could improve M3R expression levels. On the other hand, viral transfection led to a yield of 27 pmol/mg protein that is the highest level reported so far for this receptor subtype in mammalian cells. Taking together several of the strategies used can help increasing M3R expression, not only to start purification efforts but also for secondary structural analysis trial and functional analyses.