Detection of the expression and function of m_1AChR-G_(11) fusion protein

AIM To generate m 1AChR-G 11 fusion protein in baculovirus-Sf9 cells system and detect the effects of various muscarinic ligands. METHODS m 1AChR-G 11 fused DNA was generated through a two-step PCR and then expressed in Sf9 cells to produce fusion protein. QNB and GTPγS binding experiment as performed to study the function of m 1AChR-G 11 fusion protein. RESULTS The expression level of m 1AChR-G 11 was (45.38±2.26) pmol·mg -1 protein. The affinity of GDP to G 11 partner changed in the presence of different muscarinic ligands. IC 50 values of GDP in the presence of ACh, CCh, AF-102B, Isopropamide, R-(+)-hyoscyamine and Atropine were 131.82?61.66?28.18?3.24?6.02 and 5.25 μmol·L -1 respectively,and that in the absence of muscarinic ligand was 16.60 μmol·L -1. CONCLUSION The m 1AChR-G 11 fusion protein has the pharmacological specificity of m 1 receptor and the efficient coupling interaction of the two partner. Affinity of GDP to ligand-bound fusion protein represents the species of muscarinic ligands.This is helpful in screening and detecting the new specific ligands to muscarinic receptors.