Prokaryotic Expression And Purification Of Extracellular Domain Of Inhibitory Glutamate-Gated Chloride Ion Channel

Inhibitory glutamate-gated chloride ion channels (IGluCls) are members of anion channels in"Cys-loop"superfamily of ligand-gated ion channels. They are mainly expressed in neurons and extra-junctionally in muscles in invertebrates. IGluCls play important part in regulating swallow, locomotion and sense. They only exist in invertebrates. As the ideal insecticide targets with high selectivity and effective and safe characteristics, it has extensive application prospects to screen selective toxic insecticide from natural products libraries by establishing screening methods of the target in vitro. Drosophila melanogaster inhibitory glutamate-gated chloride ion channels (DmIGluClαECD) were the research objects in the study. DmIGluClαECD was subcloned and expressed in E.coli and Pichia Pastoris for study. The DmIGluClαECD expressed in E.coli was purified. Main results were as followed:Firstly, the DmIGluClαare consist of extracellular domain (ECD), four transmembrane domains (TM) and intracellular domain(ICD). In view of Pichia Pastoris expression system is eukaryotic expression system which has modification process such as posttranslational processing, we prefer to express gene in it. First of all, the gene encoded 313Aa between TM2-TM3 loop of DmIGluClαsubunit was cloned, using vector containing DmIGluClαfull length gene already constructed in our lab as template. The PCR products were insert into shuttle plasmid pPIC9K. The recombinant vector was successfully transformed to Pichia Pastoris strain GS115. Ralated research on expression were carry out.Secondly, DmIGluClαECD gene was subcloned. It was inserted into eukaryotic expression vector pPIC9K and prokaryotic expression vector pET-28a-c(+) simultaneously. Restriction digestion and sequence analysis suggested that the positive eukaryotic and prokaryotic expression vector were successfully constructed. The eukaryotic and prokaryotic expression vector was successfully transformed into Pichia Pastoris strain GS115 and E.coli strain Rosetta. Ralated research on expression were carry out.Thirdly, foreign DmIGluClαECD gene was induced and expressed in E.coli strain Rosetta. SDS-PAGE analysis show the result that foreign protein were expressed as the form of insoluble inclusion bodies which were exist in precipitation of broken cells. Optimization experiments were undertook. It is suggested that adding 0.1mM IPTG to the medium and inducing for 5 hours at 25℃was the best condition for the highest production of target protein. Through gel electrophoresis densitometric scan analysis, it is showed that the target protein account for 31.02% of the total expressed protein.Finally, 3M urea was used to wash and purify the inclusion. Under denaturation conditions, Ni2+ column was used for purification. DmIGluClαECD proteins were highly purified to an unique fraction.