新型Na^(+)/H^(+)逆向转运蛋白UPF0118的Na^(+)结合鉴定

Identification of Na^(+) binding to novel Na^(+)/H^(+) antiporter UPF0118/LI

UPF0118蛋白是一种新型Na^(+)(Li^(+))/H^(+)逆向转运蛋白,属于AI-2E超家族,目前已被划分至Na^(+)/H^(+)逆向转运蛋白亚家族,但无直接证据表明该蛋白可与Na+产生相互作用。为鉴定UPF0118蛋白是否具有Na^(+)结合功能,利用PCR技术扩增upf0118基因编码序列,通过Eco RⅠ和PstⅠ酶切位点构建至原核表达载体p ETDuet-1,转化至E. coli BL21*(DE3),对诱导后大量表达的蛋白进行Ni^(2+)亲和层析和凝胶过滤层析,利用等温滴定量热技术对纯化蛋白进行滴定。结果表明,在pH 5.5条件下,Na^(+)滴入导致蛋白溶液产生明显放热现象。在pH 5.5条件下该蛋白具有Na^(+)结合功能,证明该蛋白Na+结合功能为其功能单元与分子机制的研究奠定基础。

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 transformed into E.coli BL21*(DE3)competent cells.The protein expressed after induction was subjected to a two-step chromatography,Ni^(2+)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.