Affinity Chromatography Medium Preparation And Purification Of Housefly Gaba Receptor

γ-Aminobutyric acid (GABA) receptor is the concerned target for manyinsecticides. The structure of GABA receptor is closely related to the insecticideresistance and toxicology. Therefore, isolation/purification of GABA receptors andfurther study on the spatial structure will shed light on the development ofhigh-efficient, low toxic and safe insecticide as well as overcoming the pesticideresistance. This paper presents a thorough study of affinity separation method forpurifying housefly GABA receptor.This paper outlined the reported GABA receptor isolation/purificationmethods, analysed the influence of the detergents on extraction efficiency of GABAreceptor, and then determined the ligand of affinity chromatography to be Ro7-1986/1,basing on the computational analysis result of the organic molecules inhibited toGABA receptor in the literature. The affinity chromatography media was preparedthrough matrix activation, coupling and closure process, at the same time, theapplication conditions of affinity chromatography was determined.Matrix activation: Select Sepharose6B as affinity chromatography matrix,1,4-butanediol diglycidyl ether as activator and directly connected on Sepharose6B.The density of epoxy groups contained in activated Sepharose6B was65.27μmol/ggel. The influence of activation temperature, time, pH and other conditions on theefficiency of activation was also studied. The optimal active conditions of Sepharose6B with1,4-butanediol diglycidyl ether as following: the mixture of5.0g Sepharose6B,5.0ml0.3mol/L sodium borohydride,10mg NaBH4, and5ml1,4-butanedioldiglycidyl ether, at35℃, stirring at170rpm for8h.Coupling and closing: The immobilization of Ro7-1986/1on activatedSepharose6B was realized through the reaction of the terminal primary amine ofRo7-1986/1with the epoxy group of activated Sepharose6B, and then the residualepoxy group was closed with1mol/L ethanolamine.The coulping degree ofRo7-1986/1on Sepharose6B reached62.09μmol/g gel.Affinity chromatography: The received affinity chromatography media was transported to a column, and used it for the the separation and purification of GABAreceptor from housefly brain. After affinity chromatography and ion exchangechromatography, the concentration of GABA receptor was7.3μg/mL, and its totalproduct was182.5μg. SDS-PAGE image showed three band, molecular weight at45kD,50kD and80kD respectively. The three bands may be the α, β and γ subunits ofhousefly GABA receptor, and this result was close to the corresponding subunits ofother organisms reported in the literature.The innovation of this paper was successfully received GABA receptor fromhousefly brain by using affinity chromatography for the first time, and identified themolecular weight of each subunit.