Enhance Heterologous Protein Production in High-Density Plant Suspension Cultures using Cell-Cycle Engineering and GFP-based On-line Optimization

使用细胞周期工程和基于 GFP 的在线优化增强高密度植物悬浮培养中的异源蛋白产量

• 批准号: 0126191
• 负责人: Wei Wen Su
• 金额: $ 24.97万
• 依托单位: University of Hawaii
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-15 至 2006-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0126191&HistoricalAwards=false
• 关键词: Enhance; Heterologous; Protein; Production; Density

The overall goal of this project is to develop plant cell bioreactor cultures into a more effective system for producing high-value recombinant proteins. The specific objectives of the present research are as follows: 1) Test inducible cell-cycle control to maximize the useful culture life and yield of the target protein; 2) Employ tested molecular strategies that complement the cell-cycle control to further enhance the level of target protein expression; 3) Optimize protein production using on-line GFP-fusion monitoring and dynamic process optimization in high-density cultures. To accomplish these objectives, the Principal Investigators (PIs) will embark on the following research tasks: (1) examine the effect of expressing a cell-cycle inhibitor on cell proliferation in plant suspension cultures; (2) study coordinated expression of the target gene and the cell-cycle gene to enhance the target protein production; (3) exploit suitable targeting and endoplasmic reticulum (ER)-retention signals to promote secretion and to stabilize the target protein; (4) engineer GFP as a dual function fusion tag for process monitoring and protein purification; (5) investigate signal processing of on-line detected fluorescence of GFP-fusion proteins; (6) devise an effective dynamic process optimization scheme that draws on the on-line fluorescence data; and (7) develop high-density perfusion and fed-batch cultures integrated with the controlled proliferation technology and the fusion protein monitoring/optimization schemes. Tobacco will be used as the model host system. Human granulocyte macrophage colony-stimulating factor (GMCSF) with GFP fusion is chosen as a model protein in this research. The proposed research should lead to development of a more effective plant cell culture process for recombinant protein production. It should also shed light on regulation of the plant cell cycle and cell-cycle dependence of foreign gene expression in plant cells.

该项目的总体目标是将植物细胞生物反应器培养物开发成更有效的系统，用于生产高价值重组蛋白。本研究的具体目标如下： 1）测试诱导细胞周期控制，以最大限度地延长目标蛋白的有用培养寿命和产量； 2) 采用经过测试的补充细胞周期控制的分子策略，进一步提高靶蛋白表达水平； 3) 在高密度培养物中使用在线 GFP 融合监测和动态过程优化来优化蛋白质生产。 为了实现这些目标，主要研究者（PI）将开展以下研究任务：（1）研究细胞周期抑制剂的表达对植物悬浮培养物中细胞增殖的影响； (2) 研究目的基因与细胞周期基因的协调表达，以提高目的蛋白的产量； (3) 利用合适的靶向和内质网（ER）保留信号来促进分泌并稳定靶蛋白； (4) 将 GFP 设计为双功能融合标签，用于过程监控和蛋白质纯化； (5) 研究在线检测的GFP融合蛋白荧光的信号处理； (6)利用在线荧光数据设计有效的动态过程优化方案； (7) 开发与受控增殖技术和融合蛋白监测/优化方案相结合的高密度灌注和补料分批培养。烟草将用作模型宿主系统。本研究选择具有 GFP 融合的人粒细胞巨噬细胞集落刺激因子 (GMCSF) 作为模型蛋白。 拟议的研究应该会导致开发一种更有效的植物细胞培养工艺来生产重组蛋白。它还应该阐明植物细胞周期的调节和植物细胞中外源基因表达的细胞周期依赖性。