大肠杆菌YggG与结合蛋白Era的相互作用研究  

Interaction between YggG and Era in Escherichia coli

作　　者：黄勇[1] 陈苏民[2] 黄庆生[1] 梅其炳[1] 

机构地区：[1]西北工业大学生命科学院,西安710072 [2]第四军医大学生物化学与分子生物学教研室,西安710072

出　　处：《中国生物工程杂志》2009年第11期36-40,共5页China Biotechnology

基　　金：陕西省自然科学基金资助项目(200900277)

摘　　要：Era是细菌生长必须的一高度保守的GTPase。yggG是从大肠杆菌全基因组文库中钓取并克隆的Era结合蛋白基因,进一步的研究表明该基因在大肠杆菌中的表达与环境应激相关,提示yggG基因产物参与细菌的应激调控。为了阐明YggG蛋白与Era蛋白间的相互关系,利用所构建的双启动子表达载体pDH2-YggG-Ptac-Era在同一细胞中同时表达YggG与Era蛋白,并通过免疫共沉淀实验检测细菌裂解产物YggG与Era蛋白间的相互作用;在此基础上,构建并表达纯化了GST融合的Era蛋白氨基端截短肽和Era羧基端截短肽,通过GSTPull-down检测了Era不同功能区域与YggG蛋白间的相互作用。结果显示,Era/YggG复合物仅存在于同时过表达Era和YggG蛋白的细菌细胞内,不诱导Era或者不诱导YggG蛋白过表达,均检测不到Era/YggG复合物存在;纯化的GST不能Pull-down YggG蛋白,而纯化的GST融合的Era蛋白、Era氨基端截短肽及Era羧基端截短肽均可以Pull-down YggG蛋白;GST融合Era氨基端截短肽和GST融合的Era蛋白对YggG蛋白结合作用明显高于GST融合的Era蛋白羧基端截短肽。上述结果说明,YggG是一大肠杆菌Era结合蛋白,YggG与Era的氨基端和羧基端的结合活性存在差异。Era is a highly conserved GTPase essential for bacterial growth, yggG gene was identified to encod a Era-binding protein when using a digoxigenin-labeled Era protein to screen a phage expression library of Escherichia coli genomic DNA. Subsequent study showed the regulation of yggG gene was associated with environment stress, indicating yggG expression products involved in the stress regulation of bacteria responding to environment changes. To confirm the interactions between Era and YggG proteins when overexpressed, E. coli cells were exposed to a double promoter plasmid, pDH2-YggG-Ptac-Era that was induced to express the Era and YggG proteins by different induction methods. Immunoprecipitation of cell lysates to detect YggG/Era complexes. Then expression and purification of GST fusion truncated peptide of Era N terminus, Era C-terminus and Era C-terminus and linker. Identified the interaction of YggG and different Era truncated peptides by GST Pull-down. YggG/Era complexes existed in the cell lysates overexpressed YggG and Era, which was not able to detect in ceils over expressed YggG or Era only. GST pull-down assay showed GST fused Era protein, GST fused Era N-terminus peptide and GST fused C-terminus peptide could pull-down YggG protein. The binding activity of YggG to Era C-terminus was same to Era, which was more strong than that to Era N-terminus. The results indicated that YggG is a Era-binding protein in E. coli, and the binding activiy of YggG to Era N-terminus and C-terminus are difference.

关 键 词：大肠杆菌 ERA YggG 

分 类 号：Q786[生物学—分子生物学]