Precrystallization Screening Of Muscarinic Acetylcholine Receptors

Acetylcholine is a neurotransmitter in the CNS, PNS and autonomic ganglia, which is involved in the pathophysiology of many diseases such as Alzheimer's disease (AD) and smooth muscle disorders. Modulation of the muscarinic acetylcholine receptors (mAChRs), members of the G-protein coupled receptor family, could be beneficial for these disorders. In the case of Alzheimer's disease, the currently available treatment is non-selective with side effects. There is a need to design more selective therapies involving the stimulation of mAchRs through the design of selective agonists. To design selective receptor agonists we need a template, or the protein's structure. Hence, we have begun to generate mAChR protein and test the receptor's stability outside of the plasma membrane. Muscarinic AChR cDNA was subcloned into an insect expression system vector, pFastBac, for protein expression in the Spodoptera frugiperda (SF9) cell line. The mAChR protein was generated after 48 hours incubation and isolated using affinity chromatography. The isolated protein was assessed using SDS-PAGE analysis. We observed mAChR protein expression from the insect cell expression system and isolated protein migrated half the length of a gradient SDS-PAGE gel, corresponding to a 52 kDa protein. We have begun large-scale protein generation and are working towards protein crystallization trials. From the results obtained thus far, we can conclude that the insect expression system will be suitable for generating large quantities of mAChR, consistent with other GPCRs. We anticipate that we will be able to generate suitable quantities of mAChR for identifying protein crystallization conditions and 3-dimensional structure determination.