Structural characterization of energy transduction by Tol proteins

Tol 蛋白能量转导的结构表征

基本信息

项目摘要

Expression and purification of Tol proteins for structural studies We have cloned the tolQ and tolR genes from various Gram-negative bacteria into several expression vectors. The TolQ protein construct contains a C-terminal histidine tag while the TolR protein contains an N-terminal strep tag. Complementation and physiological tests with tolQ and tolR deletion mutants result in a restored wild-type phenotype and the tags do not interfere with function. However, expression levels have not yet been optimized for structural studies. Future work includes cloning tolQ, tolR and tolA into expression vectors using different promoters to try to obtained functional, membrane-inserted protein in good yields. We have initiated small scale purification experiments with the tolQR construct using a Ni affinity column. Our preliminary results show that when TolQ and TolR are overexpressed, they form a complex with chromosomally expressed TolA, resulting in small yields of the triple complex. These encouraging results will be scaled up once the expression system has been optimized. We are also studying expression systems for ExbB (a TolQ homolog) and have obtained an expression system that produces many milligrams of membrane-inserted protein suitable for structural studies. Initial purification experiments show two major proteolytic fragments which are under analysis currently. We intend to correct proteolysis by deletion or substitution mutagenesis and then proceed with purification and crystallization experiments on ExbB.
用于结构研究的Tol蛋白的表达和纯化 我们已经将来自各种革兰氏阴性菌的tolQ和tolR基因克隆到几种表达载体中。TolQ蛋白构建体含有C-末端组氨酸标签,而TolR蛋白含有N-末端链球菌标签。使用tolQ和tolR缺失突变体的互补和生理测试导致恢复的野生型表型,并且标签不干扰功能。然而,表达水平尚未优化的结构研究。未来的工作包括使用不同的启动子将tolQ、tolR和托拉克隆到表达载体中,试图以高产率获得功能性的膜插入蛋白。 我们已经使用Ni亲和柱用tolQR构建体开始了小规模纯化实验。我们的初步结果表明,当TolQ和TolR过表达时,它们与染色体表达的托拉形成复合物,导致三重复合物的产量很小。一旦表达系统得到优化,这些令人鼓舞的结果将扩大规模。 我们还在研究ExbB(TolQ同源物)的表达系统,并获得了一种表达系统,该系统可产生许多毫克适合结构研究的膜插入蛋白。初步纯化实验显示了目前正在分析的两个主要蛋白水解片段。我们打算通过缺失或取代诱变来校正蛋白水解,然后对ExbB进行纯化和结晶实验。

项目成果

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Susan Buchanan其他文献

Susan Buchanan的其他文献

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{{ truncateString('Susan Buchanan', 18)}}的其他基金

Structural characterization of OM proteins from Gram-negative pathogens
革兰氏阴性病原体 OM 蛋白的结构表征
  • 批准号:
    8741336
  • 财政年份:
  • 资助金额:
    $ 34.02万
  • 项目类别:
structural characterization of iron uptake from human transferrin
人转铁蛋白吸收铁的结构特征
  • 批准号:
    8741420
  • 财政年份:
  • 资助金额:
    $ 34.02万
  • 项目类别:
structural characterization of iron uptake from human transferrin
人转铁蛋白吸收铁的结构特征
  • 批准号:
    8553451
  • 财政年份:
  • 资助金额:
    $ 34.02万
  • 项目类别:
Structural characterization of OM proteins from Gram-negative pathogens
革兰氏阴性病原体 OM 蛋白的结构表征
  • 批准号:
    8939481
  • 财政年份:
  • 资助金额:
    $ 34.02万
  • 项目类别:
structural characterization of bacterial secretion channels
细菌分泌通道的结构特征
  • 批准号:
    10000710
  • 财政年份:
  • 资助金额:
    $ 34.02万
  • 项目类别:
structural characterization of bacterial secretion channels
细菌分泌通道的结构特征
  • 批准号:
    10248132
  • 财政年份:
  • 资助金额:
    $ 34.02万
  • 项目类别:
structural characterization of bacterial secretion channels
细菌分泌通道的结构特征
  • 批准号:
    7593557
  • 财政年份:
  • 资助金额:
    $ 34.02万
  • 项目类别:
Structural characterization of outer membrane proteins from Yersinia pestis
鼠疫耶尔森氏菌外膜蛋白的结构表征
  • 批准号:
    7733943
  • 财政年份:
  • 资助金额:
    $ 34.02万
  • 项目类别:
structural characterization of bacterial secretion channels
细菌分泌通道的结构特征
  • 批准号:
    8148751
  • 财政年份:
  • 资助金额:
    $ 34.02万
  • 项目类别:
structural characterization of bacterial secretion channels
细菌分泌通道的结构特征
  • 批准号:
    8741419
  • 财政年份:
  • 资助金额:
    $ 34.02万
  • 项目类别:
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