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
李红洋 1华贝杰 1姜巨全1
作者信息
- 1. 东北农业大学生命科学学院,哈尔滨 150030
- 折叠
摘要
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pETDuet-1,转化至E.coli BL21*(DE3),对诱导后大量表达的蛋白进行Ni2+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.
Abstract
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.
关键词
UPF0118/Na+/H+逆向转运蛋白/等温滴定量热法/蛋白纯化Key words
UPF0118/Na+/H+antiporter protein/isothermal titration calorimetry/protein purification引用本文复制引用
基金项目
国家自然科学基金联合基金重点项目(U23A20143)
国家自然科学基金面上项目(32070031)
国家自然科学基金面上项目(31770051)
出版年
2024
NETL
NSTL
万方数据