Characterizing and utilizing the large peroxisomes of Ogataea parapolymorpha for heterologous protein compartmentalization

表征并利用 Ogataea parapolymorpha 的大过氧化物酶体进行异源蛋白质区室化

• 批准号: 2104261
• 负责人: John Dueber
• 金额: $ 80万
• 依托单位: University of California-Berkeley
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2104261&HistoricalAwards=false
• 关键词: Characterizing; utilizing; large; peroxisomes; Ogataea

Organelles are cellular compartments surrounded by a membrane. Cells of higher organisms use organelles to separate reactions from the rest of the cell. One example is the peroxisome. Inside this organelle, the cell uses oxygen to generate hydrogen peroxide, which is then used to degrade molecules without damaging other important cellular proteins. It is a good model system because it hosts a variety of reactions. The yeast Ogataea parapolymorpha can create very large peroxisomes, which in this project will be used to compartmentalize a variety of proteins. The capacity of the peroxisomes to house cargo proteins, and proteins of different types, will be characterized. As a collaboration between U.C. Berkeley and San Francisco State University, graduate and undergraduate students from both institutions will be trained and conduct research as part of this project.The model yeast, O. parapolymorpha, can produce peroxisomes with high protein cargo capacity. In this project, induction of peroxisome proliferation will be quantified, rewired, and utilized to compartmentalize heterologous proteins. Fluorescence microscopy data will be used to accurately calculate the volume and surface area of the peroxisome. Shotgun proteomics will be employed to determine the percentage of protein compartmentalized in the peroxisomes with and without induction. Synthetic control over peroxisome proliferation will be gained by engineering transcription factors devoid of native regulation. This will allow achievement of high cargo capacity in the presence of glucose. Ultimately, the plasticity of the peroxisome for high-density compartmentalization of heterologous cargo proteins will be directly tested using a model protein of commercial interest, human serum albumin. The effect on expression level, growth rate, and the protection of human serum albumin from degradation from proteases will be determined.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

细胞器是被膜包围的细胞室。高等生物的细胞利用细胞器将反应与细胞的其他部分分开。过氧化物酶体就是一个例子。在这个细胞器内部，细胞利用氧气产生过氧化氢，然后过氧化氢被用来降解分子，而不会破坏其他重要的细胞蛋白质。它是一个很好的模型系统，因为它承载了各种各样的反应。酵母Ogataea parapolymorpha可以产生非常大的过氧化物酶体，在这个项目中，它将被用来划分各种蛋白质。过氧化物酶体容纳货物蛋白质和不同类型蛋白质的能力将被表征。作为加州大学伯克利分校和旧金山州立大学的合作项目，来自两所大学的研究生和本科生将接受培训，并作为该项目的一部分进行研究。模型酵母，O. parapolymorpha，可以产生具有高蛋白载货能力的过氧化物酶体。在这个项目中，诱导过氧化物酶体增殖将被量化，重新连接，并用于区隔异种蛋白。荧光显微镜数据将用于精确计算过氧化物酶体的体积和表面积。散弹枪蛋白质组学将用于确定有和没有诱导的过氧化物酶体中区隔的蛋白质百分比。对过氧化物酶体增殖的合成控制将通过缺乏天然调控的工程转录因子来实现。这将允许在葡萄糖存在下实现高载货能力。最终，过氧化物酶体对异种货物蛋白高密度区隔化的可塑性将直接使用具有商业价值的模型蛋白——人血清白蛋白进行测试。对表达水平、生长速度和保护人血清白蛋白免受蛋白酶降解的影响将被确定。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。