Membrane Protein Production Using the Yeast SPP System

使用酵母 SPP 系统生产膜蛋白

• 批准号: 8529714
• 负责人: NANCY ANN WOYCHIK
• 金额: $ 6.34万
• 依托单位: UNIV OF MED/DENT NJ-R W JOHNSON MED SCH
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-30 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8529714
• 关键词: Bacteria; Bacterial Proteins; Bacterial Toxins; Biochemical; Biological; Biological Assay; Biological Process; Cell Adhesion; Cell physiology; Cell surface; Cells; Centers for Disease Control and Prevention (U.S.); Complement; Coupled; Data; Databases; Development; Diabetes Mellitus; Disease; Endoribonucleases; Eotaxin; Epidemic; Escherichia coli; Family member; Foundations; GLUT2 gene; GLUT4 gene; Generations; Genetic; Genome; Glucose Transporter; Goals; Growth; Health; Homeostasis; Human; Incidence; Insulin; Knowledge; Life; Link; Malignant Neoplasms; Mediating; Membrane Proteins; Messenger RNA; Metabolic; Methods; Mission; Modeling; Molecular Chaperones; Nature; Noise; Non-Insulin-Dependent Diabetes Mellitus; Nuclear Magnetic Resonance; Organism; Pharmacologic Substance; Physiological; Play; Post-Translational Protein Processing; Process; Production; Property; Protein Analysis; Protein Biosynthesis; Proteins; Reagent; Recombinant Proteins; Recovery; Recruitment Activity; Resolution; Role; SLC2A1 gene; Saccharomyces cerevisiae; Signal Transduction; Structure; Subunit Vaccines; System; Technology; Testing; Toxic effect; Toxin; United States National Institutes of Health; Vertebral column; X-Ray Crystallography; Yeasts; animation; base; endoribonuclease; glucose transport; improved; insight; leukotriene-C4 synthase; new technology; novel strategies; novel therapeutic intervention; overexpression; protein expression; protein folding; protein function; protein structure; public health relevance; quantum; receptor; structural biology; structural genomics; success; yeast protein

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着关键作用，这对于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理解其功能的经典遗传和生化方法，但结构分析因表达和纯化正确折叠的蛋白质方面的困难而受到阻碍。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，以及蛋白质本质上是未折叠的可能性。已经开发出用于真核蛋白质表达的第一代表达系统，该系统具有改善正确折叠蛋白质的表达和回收的潜力。该系统称为酵母 SPP 系统，使用酿酒酵母和一种名为 MazF 的细菌毒素来赋予生长停滞状态，从而允许重组蛋白持续表达，而不会产生过度表达经常引起的毒性，从而提高目标产量蛋白质加上酵母蛋白质背景降低。长期目标是建立酵母SPP系统来生产具有生物学意义的膜蛋白，用于结构研究。填补结构功能研究中的这一空白是开发膜蛋白相关疾病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质的稳健生产。目标 1 将使用酿酒酵母和 MazF 毒素，通过产生人嗜酸细胞趋化因子作为模型靶蛋白来优化第一代酵母 SPP 系统。将酵母 SPP 产生的嗜酸细胞趋化因子与传统方法的 NMR 数据进行比较，将确定该技术的实用性。目标 2 将进一步调整 SPP 系统，用于表达和纯化选定的酵母和人膜蛋白（其中可获得结构信息）。异核单量子相干 (HSQC) 和骨架共振分配分析将验证酵母 SPP 系统产生的蛋白质。目标 3 将进一步扩展这些研究，以生产参与葡萄糖转运的具有生物学重要性的膜蛋白。这些研究通过提供显着的技术进步来推进膜蛋白结构的研究，符合 NIH 结构生物学路线图的使命。 公共健康相关性：了解细胞表面蛋白质（称为膜蛋白）的结构，可以提供有关其在健康和疾病中的作用的重要见解。然而，很难以天然形式生产和纯化这些蛋白质。该项目将开发利用酵母生产人类膜蛋白的新技术，通过研究这些蛋白可以增进我们对其功能的了解，从而产生治疗疾病的新方法。