Functional analysis of Enterococcus faecalis pheromone receptor TraA family proteins

我们研究了粪肠球菌信息素受体TraA家族蛋白质的结构和功能之间的关系，采用蛋白质家族的不同成员之间的嵌合蛋白质形成。用常规的限制性内切酶切割和连接方法和重叠延伸（OLE）PCR方法进行pPD 1 TraA和pAD 1 TraA基因的融合，作为具有strep-标签的标签蛋白。我们开发了肽相关蛋白标签色谱（PPAC）方法，观察肽信息素结合的标签蛋白使用未修饰的非放射性肽。我们分别用DPAC和PPAC技术观察了嵌合体蛋白的DNA结合、信息素结合和信息素-DNA结合信号。pPD 1 TraA在其N-末端的149个氨基酸保留在嵌合体蛋白上时保持其DNA结合特性。当其N-末端超过146个氨基酸保留在嵌合体蛋白上时，它维持其信息素结合特性。pPD 1 TraA的DNA结合结构域应该位于N-末端（Ca. 150 aa）和信息素结合结构域应该驻留在C-末端（Ca. 170 aa）。我们纯化了pAD 1 TraA蛋白、pPD 1 TraA蛋白和pCF 10的PrgX作为标记蛋白，并通过DPAC和PPAC观察了这些蛋白的DNA和信息素结合特异性。这些蛋白质只与它们自己的同源DNA片段和信息素结合。我们调查了万古霉素耐药肠球菌（VRE）的临床分离株的信息素反应。一个菌株对两种不同的合成信息素cAD 1和cCF 10表现出聚集反应。我们构建了新的E。粪肠球菌E. coli穿梭载体，证明pAD 1 TraA蛋白在体内是反式表达的。

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.