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Functional analysis of Enterococcus faecalis pheromone receptor TraA family proteins

粪肠球菌信息素受体TraA家族蛋白的功能分析

基本信息

批准号：
12670247
负责人：
FUJIMOTO Shuhei
金额：
$ 2.11万
依托单位：
Gunma University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12670247/
关键词：
Enterococcus faecalis plasmid conjugation pheromone receptor TraA peptide binding DNA binding

项目摘要

We investigated relationship between structure and function of Enterococcus faecalis pheromone receptor TraA family proteins employing chimera protein formation between different members of the protein family. Fusion of pPD1 TraA and pAD1 TraA genes as tagged protein with strep-tag was performed with a conventional restriction enzyme cleavage and ligation method and Overlap Extension (OLE) PCR method. We developed the peptide associated protein-tag chromatography (PPAC) method to observe peptide pheromone binding of the tagged proteins using unmodified non-radioactive peptide. We observed the DNA binding, the pheromone binding, and the pheromone-to-DNA-binding signaling of the chimera proteins independently by DPAC (DNA associated protein-tag chromatography) and PPAC. A pPD1 TraA sustained its DNA binding properties when 149 aa from its N-terminal were kept on the chimera protein. It sustained its pheromone binding properties when more than 146 aa from its N-terminal were kept on chimera proteins. The DNA binding domain of pPD1 TraA was supposed to reside on N-terminus (Ca. 150 aa) and the pheromone binding domain was supposed to reside on C-terminus (Ca. 170 aa). We purified pAD1 TraA protein, pPD1 TraA protein, and PrgX of pCF10 as tagged proteins and observed DNA and pheromone binding specificities of these proteins by DPAC and PPAC. These proteins bound exclusively to their own cognate DNA fragments and pheromones. We investigated the pheromone response of clinical isolates of vancomycin resistant enterococci (VRE). A strain showed clumping response against two different synthetic pheromones cAD1 and cCF10. We constructed new E. faecalis- E. coli shuttle vectors and showed that pAD1 TraA protein works in-trans in vivo.

我们研究了粪肠球菌信息素受体Traa家族蛋白的结构和功能之间的关系，利用该蛋白家族不同成员之间的嵌合体蛋白形成。将pPD1TraA和PAD1TraA基因作为标记蛋白，用常规的限制性内切酶酶切连接法和重叠延伸(OLE)聚合酶链式反应(OLE)进行融合。我们建立了多肽相关蛋白-标签层析(PPAC)的方法，以观察未修饰的非放射性多肽与标记蛋白的多肽信息素结合。分别用DPAC和PPAC观察嵌合体蛋白的DNA结合、信息素结合和信息素与DNA结合的信号。当pPD1TraA的N-端保留149个氨基酸时，该嵌合体蛋白保持其与DNA的结合特性。当嵌合体蛋白上保持其N端超过146个氨基酸时，它保持了信息素结合特性。推测pPD1TraA的DNA结合区位于N-末端(Ca.150aa)，信息素结合区可能位于C-末端(Ca.170 AA)。我们纯化了pCF10的PAD1Traa蛋白、pPD1Traa蛋白和PrGX作为标记蛋白，并用DPAC和PPAC观察了这些蛋白的DNA和信息素结合的特异性。这些蛋白质只与它们自己的同源DNA片段和信息素结合。我们调查了临床分离的万古霉素耐药肠球菌(VRE)的信息素反应。一株菌株对两种不同的合成信息素cAD1和cCF10表现出聚集反应。我们构建了新的粪便大肠杆菌穿梭载体，并证明了PAD1TraA蛋白在体内起反式作用。