Identification of Na+binding to novel Na+/H+antiporter UPF0118
UPF0118 is a novel Na+(Li+)/H+antiporter,which belongs to the AI-2E superfamily and has been classified into the Na+(Li+)/H+antiporter subfamily.However,there is no direct evidence that UPF0118 can bind to Na+.In order to identify whether UPF0118 had Na+binding function,the coding sequence of ufp0118 was amplified by PCR and it was inserted into the prokaryotic expression vector pETDuet-1 by restriction enzyme cutting sites,Eco R I and Pst I.The resultant constructs were trans-formed into E.coli BL21*(DE3)competent cells.The protein expressed after induction was subjected to a two-step chromatography,Ni2+affinity chromatography followed by gel filtration chromatography.The purified protein was titrated by isothermal titration calorimetry.The results showed that the dropping of Na+at pH 5.5 led to a significant exothermic phenomenon in the protein solution.The results showed that when UPF0118 was titrated at pH 5.5 with Na+and the reaction was exothermic.UPF0118 had Na+binding function and the binding reaction was an exothermic reaction.The evidence of the Na+binding function of UPF0118 laid a foundation for the study of its functional units and molecular mechanisms.
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