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首页> 外文学位 >The production of foreign proteins from cultured plant cells: Methods for increased production and improved monitoring.
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The production of foreign proteins from cultured plant cells: Methods for increased production and improved monitoring.

机译：从培养的植物细胞中生产外源蛋白质：增加产量和改善监测的方法。

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In this dissertation, aspects of foreign protein production from plant cells are addressed. To provide background, plant culture behavior and the performance of plant cells as a host system are reviewed. After a discussion of gene transfer techniques, strategies to overcome production limitations are presented along with implications for the future development of this technology.; In the first investigation, human granulocyte macrophage-colony stimulating factor (GM-CSF) was produced by tobacco cells. A leader sequence from the tobacco etch virus was added to the transgene to increase transcriptional and translational efficiency. For purification, a 6-his tag was also added to the protein. ELISA and Western analysis confirmed GM-CSF production. Plant produced GM-CSF was biologically active, and could bind to a nickel affinity matrix. Adding stabilizing proteins and salt to the growth media increased the recovery of secreted GM-CSF.; In the second investigation, an affinity chromatography bioreactor (ACBR) was developed for production and in situ purification of proteins. Optimizing flow rate, pH, aeration method, and resin type could increase product recovery. Semi-continuous protein removal using the ACBR increased levels of recoverable protein two to seven-fold as compared with batch yields by isolating the product from degrading influences and inhibition pathways.; In the third investigation, an image analysis technique was developed to provide quantitative growth data for plant cells grown on solid media. Properties obtained from image analysis were correlated with the observed mass of plant callus using multiple linear regression analysis. Image analysis was used to estimate cell mass, specific growth rate, and cell viability under various growth conditions. This work demonstrates that image analysis is a flexible and useful tool for monitoring and characterizing cultures.; In the fourth investigation, a modeling approach, based on the probability of gene suppression, was used to explain the loss of protein productivity over successive generations. Although plant cell lines were relatively stable, the production level could decrease significantly after a large number of generations. Motivated by this problem, a dispersion method was developed to isolate productive cells from existing cultures. Applying this method, it was possible to recover high producing cell lines.
机译：本文探讨了植物细胞产生外源蛋白质的各个方面。为了提供背景，综述了植物培养行为和植物细胞作为宿主系统的性能。在讨论了基因转移技术之后，提出了克服生产限制的策略以及对该技术未来发展的影响。在第一个研究中，烟草细胞产生了人类粒细胞巨噬细胞集落刺激因子（GM-CSF）。来自烟草蚀刻病毒的前导序列被添加到转基因中，以提高转录和翻译效率。为了纯化，还将6-his标签添加到蛋白质中。 ELISA和Western分析证实了GM-CSF的产生。植物产生的GM-CSF具有生物活性，并且可以与镍亲和基质结合。向生长培养基中加入稳定的蛋白质和盐可以增加分泌的GM-CSF的回收率。在第二项研究中，开发了亲和色谱生物反应器（ACBR）用于生产和原位纯化蛋白。优化流速，pH，通气方法和树脂类型可以提高产品回收率。通过将产物从降解影响和抑制途径中分离出来，使用ACBR进行半连续去除蛋白质使可回收蛋白质的水平与批量生产相比增加了2到7倍。在第三次调查中，开发了一种图像分析技术，以提供在固体培养基上生长的植物细胞的定量生长数据。使用多元线性回归分析，从图像分析获得的特性与观察到的植物愈伤组织质量相关。图像分析用于估计各种生长条件下的细胞质量，比生长速率和细胞活力。这项工作表明，图像分析是监测和表征文化的灵活而有用的工具。在第四项研究中，基于基因抑制的概率的建模方法被用来解释连续几代蛋白质生产力的损失。尽管植物细胞系相对稳定，但经过大量的世代生产后产量可能会显着下降。受此问题的驱使，开发了一种分散方法以从现有培养物中分离生产性细胞。应用该方法，可以回收高产细胞系。

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