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

细胞器是由膜包围的细胞隔室。高等生物的细胞使用细胞器将反应与细胞的其余部分分开。一个例子是过氧化物酶体。在这个细胞器内，细胞利用氧气产生过氧化氢，然后用过氧化氢降解分子，而不损害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，它将用于区分各种蛋白质。将表征过氧化物酶体容纳货物蛋白和不同类型蛋白的能力。作为U.C.伯克利和弗朗西斯科州立大学的研究生和本科生将接受培训，并进行研究，作为这个项目的一部分。类多形藻（parapolymorpha）可以产生具有高蛋白质货物容量的过氧化物酶体。在这个项目中，过氧化物酶体增殖的诱导将被量化，重新连接，并用于区室化异源蛋白。荧光显微镜数据将用于准确计算过氧化物酶体的体积和表面积。将采用鸟枪蛋白质组学来确定在有和没有诱导的情况下过氧化物酶体中区室化的蛋白质的百分比。对过氧化物酶体增殖的合成控制将通过工程化缺乏天然调节的转录因子来获得。这将允许在葡萄糖的存在下实现高负载容量。 最后，过氧化物酶体的可塑性高密度区室化的异源货物蛋白将直接测试使用模型蛋白的商业利益，人血清白蛋白。该奖项反映了NSF的法定使命，并已被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。

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.