Bio-nanomanufacturing of Protein Therapeutics Using Membrane Microfluidics

治疗性蛋白质的市场价值每年接近1400亿美元。这些治疗药物中有许多是糖蛋白，它们需要通过蛋白质糖基化过程在蛋白质的精确位置添加特定的糖，称为“聚糖”。聚糖影响蛋白质的折叠和功能，并确保其保持其治疗效力。在自然界中，糖蛋白是通过细胞内一系列连续的反应产生的。由于各种原因，在细胞内制造治疗性糖蛋白是具有挑战性的，并且需要大量和昂贵的纯化步骤来收获治疗材料。凭借该奖项，将在微流体装置中构建无细胞糖基化网络，该装置可以在空间和时间上分离反应，从而在优化单个反应和构建具有高特异性的聚糖方面具有极高的灵活性。这种制造模式对社会的好处是降低了这些药物的成本，并为科学家提供了设计和开发合成药物化合物的途径，这些合成药物化合物可能存在于自然界中，也可能不存在于自然界中，以治疗疾病。相关的教育计划为一名高中女生创建了一个亲身实践的生物纳米制造活动，由pi和他们的学生学员组织，使年轻女性了解生物技术的力量，并受到激励，追求这些职业道路。无细胞蛋白质合成在生产高价值的生物治疗纳米材料方面具有很大的前景，无需细胞培养并受益于化学制造技术。在这里，原料和生物酶混合生产生物制品。这种方法的缺点是相互竞争的反应、副作用和低收率。细胞通过将反应定位在亚细胞区室内并编排反应序列来避免这些缺点。赋予最终分子其基本翻译后特征的生物催化剂被分隔在膜中。在酶的天然脂质环境之外处理酶可以大大降低它们的活性。因此，在体外，顺序的生物酶反应从未以无细胞的方式实现。研究目的是模拟细胞在微流控生物膜装置中使用的优雅的区隔化策略，以适当的空间和时间顺序组织生物反应。这些装置将产生真正的糖基化蛋白质。通过对纳米结构产品结构的评估，这项工作将促进对纳米材料制造和发现的纳米尺度现象和过程的理解。这种无细胞装置的概念将使糖基化蛋白的生产易于优化，并为理解实验条件如何影响产品产量和质量提供了一个框架，该框架广泛适用于几乎任何翻译后修饰蛋白的生物纳米制造。

The market for therapeutic proteins is valued near $140 billion annually. Many of these therapeutics are glycoproteins, which require the addition of specific sugars, called 'glycans' at an exact position on the protein through a process called protein glycosylation. The glycan affects protein folding and function and ensures it retains its therapeutic potency. In nature, glycoproteins are produced through a series of sequential reactions inside a cell. Making therapeutic glycoproteins within cells is challenging for a variety of reasons, and extensive and costly purification steps are required to harvest the therapeutic material. With this award, a cell-free glycosylation network will be constructed in a microfluidic device that separates reactions in space and time, giving supreme flexibility in optimizing individual reactions and constructing glycans with high specificity. The benefits of this manufacturing paradigm to society are reducing the cost of these drugs and providing scientists an avenue to design and develop synthetic drug compounds that may or may not exist in nature to treat disease. The related education plan creates a hands-on bio-nanomanufacturing activity for a high school girls organized by the PIs and their student trainees, so that young women will understand the power of biotechnology and be inspired to pursue these career paths. Cell-free protein synthesis holds great promise for producing high-value, biotherapeutic nanomaterials without cell culture and benefitting from chemical manufacturing know-how. Here, raw materials and biological enzymes are mixed to produce biological products. Shortcomings of this approach are competing reactions, side products, and low yields. Cells avoid these shortcomings by localizing reactions within subcellular compartments and orchestrating the reaction sequences. The biocatalysts that give the final molecule its essential posttranslational features are compartmentalized in membranes. Handling enzymes outside of their native lipid environment can drastically reduce their activity. Thus, in vitro, sequential, bio-enzymatic reactions have never been achieved in a cell-free manner. The research objective is to mimic the elegant compartmentalization strategies used by cells in a microfluidic biomembrane device that organizes biological reactions in proper spatial and temporal sequence. These devices will generate authentically glycosylated proteins. Through assessment of nanostructure product architectures, this work will advance understanding of nanoscale phenomena and processes for nanomaterials manufacture and discovery. This cell-free device concept will enable facile optimization of glycosylated protein production, and provide a framework for understanding how experimental conditions affect product yield and quality that is broadly applicable to the bio-nanomanufacturing of virtually any posttranslationally-modified protein.