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

该项目的总体目标是将植物细胞生物反应器培养物发展成为生产高价值重组蛋白的更有效的系统。本研究的具体目标如下：1)测试可诱导的细胞周期控制，以最大限度地延长有用的培养寿命和目标蛋白的产量；2)采用经过测试的分子策略来补充细胞周期控制，以进一步提高目标蛋白的表达水平；3)通过在线GFP融合监测和高密度培养中的动态过程优化来优化蛋白质生产。为了实现这些目标，首席调查员将着手以下研究任务：(1)研究细胞周期抑制物在植物悬浮培养中对细胞增殖的影响；(2)研究靶基因和细胞周期基因的协调表达以提高靶蛋白的产量；(3)利用合适的靶向和内质网(ER)保留信号来促进分泌和稳定靶蛋白；(4)将GFP作为双功能融合标签用于过程监测和蛋白质纯化；(5)研究在线检测GFP-融合蛋白荧光的信号处理；(6)制定有效的利用在线荧光数据的动态过程优化方案；(7)发展高密度灌流和补料分批培养，结合受控增殖技术和融合蛋白监测/优化方案。烟草将被用作示范东道主系统。本研究选择融合了GFP的人粒细胞巨噬细胞集落刺激因子(GMCSF)作为模型蛋白。这项拟议的研究将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。这也应该有助于阐明植物细胞周期的调控以及植物细胞中外源基因表达的细胞周期依赖性。

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.