Membrane Protein Production Using the Yeast SPP System

使用酵母 SPP 系统生产膜蛋白

基本信息

项目摘要

DESCRIPTION (provided by applicant): Membrane proteins play a critical role in dynamic cellular processes that are essential to maintain homeostasis and human health. Determining the structure of these proteins complements classical genetic and biochemical approaches to understanding their function, yet structural analysis has been hampered by difficulties in expressing and purifying proteins that are properly folded. The unfolded nature of eukaryotic proteins obtained from bacterial expression systems has been attributed to lack of post-translational modifications, the absence of chaperones or other eukaryotic processes that influence folding, and the possibility that the protein is intrinsically unfolded in nature. A first generation expression system for eukaryotic protein expression has been developed that has the potential to improve the expression and recovery of correctly folded proteins. This system, called the yeast SPP system, uses Saccharomyces cerevisiae and a bacterial toxin called MazF to impart a state of growth arrest that allows continued expression of recombinant protein without the toxicity that is frequently caused by overexpression, resulting in an increased yield of the target protein coupled with reduced background of yeast proteins. The long-term goal is to establish the yeast SPP system to produce biologically-important membrane proteins for structural studies. Filling this gap in structure-function studies represents an important key to developing new therapeutic approaches for diseases linked to membrane proteins. This proposal will establish proof-of-concept that this novel approach is capable of achieving robust production of properly folded eukaryotic proteins through the accomplishment of three specific aims. Aim 1 will optimize the first generation yeast SPP system using Saccharomyces cerevisiae and the MazF toxin by producing human eotaxin as a model target protein. Comparison with NMR data from eotaxin produced by yeast SPP vs. conventional methods will establish the utility of this technology. Aim 2 will further adapt the SPP system for the expression and purification of selected yeast and human membrane proteins where structural information is available. Heteronuclear Single Quantum Coherence (HSQC) and backbone resonance assignment analysis will validate proteins produced by the yeast SPP system. Aim 3 will extend these studies further to produce biologically-important membrane proteins involved in glucose transport. These studies fit with the mission of the NIH Structural Biology Roadmap by providing a significant advance in technology that will advance the study of membrane protein structure. PUBLIC HEALTH RELEVANCE: Understanding the structure of proteins found on the surface of cells, called membrane proteins, can provide important insight into their role in health and disease. However, it is difficult to produce and purify these proteins in their natural form. This project will develop new technology using yeast to produce human membrane proteins that can be studied to advance our knowledge of how they function, thereby generating new approaches to treat disease.
描述(由申请人提供):膜蛋白在动态细胞过程中发挥关键作用,对维持体内平衡和人类健康至关重要。确定这些蛋白质的结构补充了经典的遗传和生物化学方法来理解它们的功能,但结构分析受到表达和纯化正确折叠的蛋白质的困难的阻碍。从细菌表达系统获得的真核蛋白质的未折叠性质归因于缺乏翻译后修饰、不存在影响折叠的分子伴侣或其他真核过程以及蛋白质在自然界中本质上未折叠的可能性。已经开发了用于真核蛋白表达的第一代表达系统,其具有改善正确折叠蛋白的表达和回收的潜力。该系统称为酵母SPP系统,使用酿酒酵母和称为MazF的细菌毒素来赋予生长停滞状态,该生长停滞状态允许重组蛋白的持续表达而没有通常由过表达引起的毒性,从而导致靶蛋白的产量增加以及酵母蛋白的背景减少。长期目标是建立酵母SPP系统,以生产生物学上重要的膜蛋白用于结构研究。填补结构-功能研究中的这一空白是开发与膜蛋白相关疾病的新治疗方法的重要关键。该提案将建立概念验证,即这种新方法能够通过实现三个特定目标来实现正确折叠的真核蛋白的稳健生产。目的1以人嗜酸性粒细胞趋化因子(eotaxin)为模型蛋白,优化第一代酵母SPP系统。比较NMR数据从嗜酸性粒细胞趋化因子产生的酵母SPP与传统的方法将建立这种技术的实用性。目的2将进一步调整SPP系统,用于表达和纯化结构信息可用的选定酵母和人膜蛋白。异源单量子相干(HSQC)和骨架共振分配分析将验证由酵母SPP系统产生的蛋白质。目标3将进一步扩展这些研究,以产生参与葡萄糖转运的生物重要的膜蛋白。这些研究符合NIH结构生物学路线图的使命,提供了一个重大的技术进步,将推进膜蛋白结构的研究。 公共卫生相关性:了解在细胞表面发现的蛋白质(称为膜蛋白)的结构,可以为了解它们在健康和疾病中的作用提供重要的见解。然而,很难以其天然形式生产和纯化这些蛋白质。该项目将开发使用酵母生产人类膜蛋白的新技术,这些蛋白可以被研究,以提高我们对它们如何发挥作用的认识,从而产生治疗疾病的新方法。

项目成果

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NANCY ANN WOYCHIK其他文献

NANCY ANN WOYCHIK的其他文献

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{{ truncateString('NANCY ANN WOYCHIK', 18)}}的其他基金

Repurposing Mycobacterium tuberculosis tRNase toxins for cancer chemotherapy
重新利用结核分枝杆菌 tRNase 毒素进行癌症化疗
  • 批准号:
    10532244
  • 财政年份:
    2021
  • 资助金额:
    $ 30万
  • 项目类别:
Repurposing Mycobacterium tuberculosis tRNase toxins for cancer chemotherapy
重新利用结核分枝杆菌 tRNase 毒素进行癌症化疗
  • 批准号:
    10354376
  • 财政年份:
    2021
  • 资助金额:
    $ 30万
  • 项目类别:
Genome exploration through toxin-mediated ribosome stalling
通过毒素介导的核糖体停滞进行基因组探索
  • 批准号:
    10396107
  • 财政年份:
    2020
  • 资助金额:
    $ 30万
  • 项目类别:
Genome exploration through toxin-mediated ribosome stalling
通过毒素介导的核糖体停滞进行基因组探索
  • 批准号:
    10159845
  • 财政年份:
    2020
  • 资助金额:
    $ 30万
  • 项目类别:
Genome exploration through toxin-mediated ribosome stalling
通过毒素介导的核糖体停滞进行基因组探索
  • 批准号:
    10616690
  • 财政年份:
    2020
  • 资助金额:
    $ 30万
  • 项目类别:
Proteome reprogramming by tRNA-cleaving toxins
通过 tRNA 裂解毒素进行蛋白质组重编程
  • 批准号:
    10112828
  • 财政年份:
    2020
  • 资助金额:
    $ 30万
  • 项目类别:
Genome exploration through toxin-mediated ribosome stalling
通过毒素介导的核糖体停滞进行基因组探索
  • 批准号:
    10034312
  • 财政年份:
    2020
  • 资助金额:
    $ 30万
  • 项目类别:
Transcriptome and proteome remodeling by Mycobacterium tuberculosis MazF toxins
结核分枝杆菌 MazF 毒素的转录组和蛋白质组重塑
  • 批准号:
    10307528
  • 财政年份:
    2019
  • 资助金额:
    $ 30万
  • 项目类别:
Transcriptome and proteome remodeling by Mycobacterium tuberculosis MazF toxins
结核分枝杆菌 MazF 毒素的转录组和蛋白质组重塑
  • 批准号:
    10062823
  • 财政年份:
    2019
  • 资助金额:
    $ 30万
  • 项目类别:
Transcriptome and proteome remodeling by Mycobacterium tuberculosis MazF toxins
结核分枝杆菌 MazF 毒素的转录组和蛋白质组重塑
  • 批准号:
    10530645
  • 财政年份:
    2019
  • 资助金额:
    $ 30万
  • 项目类别:

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职业:阐明聚糖纳米​​材料与细菌蛋白质、毒素和细胞的协同纳米级和碳水化合物相互作用
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开发机器学习方法,用于自动设计细菌蛋白质的新生物功能。
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    2600923
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    Studentship
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细菌蛋白作为配方成分。
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生产难以表达的必需细菌蛋白
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细菌蛋白质的细胞表面展示
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    2016
  • 资助金额:
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分泌细菌蛋白的磷酸化和乙酰化:新的调控
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  • 财政年份:
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奈瑟氏球菌的蛋白质 O-糖基化途径:细菌蛋白质 O-糖基化的模型系统,具有生物技术的潜在用途
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  • 财政年份:
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细菌蛋白 YjeE、YeaZ 和 YgjD 的表征以及作为潜在新型抗菌靶点的评估
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