The Specific Ligand Screening for M_2-G_（i1α） Fusion Protein Expressed in Sf9 Cells
- VernacularTitle:M_2-G_（i1α）融合蛋白在Sf9细胞中的表达及M_2AChR特异性药物的鉴别
- Author:
Xuewei ZHANG
;
Zhengdong GUO
;
Lichuan BAI
;
Aimin JIANG
;
Ke SUN
- Publication Type:Journal Article
- Keywords:
fusion protein;
muscarinic acetylcholine receptor;
G protein coupled receptor;
agonists;
antagonists
- From:
Journal of China Medical University
2010;(6):428-431
- CountryChina
- Language:Chinese
-
Abstract:
Objective Using M2-Gi1α fusion protein expressed by baculovirus-Sf9 cell system to find the specific ligand for M2 receptor and detect the interaction of the two parts of the fusion protein.Methods The fused M2-Gi1α cDNAs were generated in a two-step PCR and then expressed in Sf9 cells.[3H]QNB and[35S]GTPγS binding experiments were employed to study the function of M2-Gi1α fusion protein.Results The expression level of M2-Gi1α fusion protein was 8.44±0.39 nmol·g-1 protein.The affinity of GDP to the Gi1α part changed under the affection of different ligands.The IC50 value in the appearance of acetylcholine,oxotremorine,arecoline,atropine,fangchinoline,levitimide were 21.35 μmol·L-1,23.86 μmol·L-1,11.91 μmol·L-1,0.13 μmol·L-1,1.05 μmol·L-1,1.75 μmol·L-1,and 2.5 μmol·L-1 when there was no ligand.Conclusion The M2-Gi1α fusion protein expressed in baculovirus-Sf9 cell system has pharmacological specificity for M2 receptor and the efficient coupling function between the two parts.The M2-Gi1αfusion protein is a helpful tool for detecting the new specific ligands of the M2 receptor.
