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

细胞器是被膜包围的细胞室。高等生物的细胞使用细胞器将反应与细胞的其他部分分开。Peroxisome就是一个例子。在这个细胞器内，细胞利用氧气产生过氧化氢，然后用来降解分子，而不会损害其他重要的细胞蛋白质。这是一个很好的模型系统，因为它承载了各种反应。酵母菌Ogataea parapolypha可以产生非常大的过氧化物体，在这个项目中，这些过氧化物体将被用来区分各种蛋白质。将对过氧化体容纳货物蛋白质和不同类型蛋白质的能力进行表征。作为加州大学伯克利分校和旧金山州立大学的合作项目，这两个机构的研究生和本科生将接受培训并进行研究，作为该项目的一部分。模型酵母O.parapolypha可以产生具有高蛋白质运载能力的过氧化物体。在这个项目中，过氧化物体增殖的诱导将被量化，重新连接，并用于区分异源蛋白。荧光显微镜数据将被用来准确计算过氧化物体的体积和表面积。鸟枪式蛋白质组学将被用来确定在诱导和未诱导的情况下，过氧化体中的蛋白质区隔的百分比。通过改造不受天然调控的转录因子，可以实现对过氧化物体增殖的综合控制。这将允许在葡萄糖存在的情况下实现高载货能力。最终，将使用商业上感兴趣的模型蛋白-人血清白蛋白直接测试用于高密度分隔异源货物蛋白的过氧化体的可塑性。将确定对表达水平、生长速度和保护人血清白蛋白免受蛋白酶降解的影响。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.