2D-DIGE screening of high-productive CHO cells under glucose limitation Basic changes in the proteome equipment and hints for epigenetic effects
2D-DIGE screening of high-productive CHO cells under glucose limitation Basic changes in the proteome equipment and hints for epigenetic effects
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DOI：
10.1016/j.jbiotec.2015.01.005
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发表时间：
2015-03-27
影响因子：
4.1
通讯作者：
Hoffrogge, Raimund
中科院分区：
文献类型：
--
作者：
Wingens, Marc;Gaetgens, Jochem;Hoffrogge, Raimund
CHO衍生物（中国仓鼠卵巢）是工业重组蛋白生产中最重要的哺乳动物细胞。已经进行了许多努力来提高CHO细胞在生物反应器过程中的生产率和稳定性。在这里，我们跟踪了在过程优化中观察到的一个几乎不理解的现象：当葡萄糖（和乳酸盐）储备耗尽时，葡萄糖限制灌注培养后期的细胞比生产率显着增加。我们的目的是阐明与从葡萄糖过剩到葡萄糖限制阶段的代谢转变有关的细胞活动。利用2D-DIGE，我们比较了CHO细胞灌注培养中的三个阶段：初始生长时葡萄糖浓度高，乳酸产量低，第二阶段葡萄糖进入限制状态，乳酸水平高，最后葡萄糖限制状态，乳酸浓度低，但细胞比生产率增加。通过我们的蛋白质组学方法，我们能够证明葡萄糖限制对蛋白质表达机制的影响，这也可以对更高的重组蛋白质产量发挥作用。最有趣的是，我们检测到对参与组蛋白修饰的蛋白质（HDAC 11 -2，SET，RBBP 7，DDX 5）水平的表观遗传效应。随着能量代谢、细胞骨架和蛋白质表达的蛋白质库存的变化，出现了CHO细胞在长期葡萄糖限制下细胞设备的基本变化。(C)2015 Elsevier B. V.版权所有。
CHO derivates (Chinese hamster ovary) belong to the most important mammalian cells for industrial recombinant protein production. Many efforts have been made to improve productivity and stability of CHO cells in bioreactor processes. Here, we followed up one barely understood phenomenon observed with process optimizations: a significantly increased cell-specific productivity in late phases of glucose-limited perfusion cultivations, when glucose (and lactate) reserves are exhausted. Our aim was to elucidate the cellular activities connected to the metabolic shift from glucose surplus to glucose limitation phase. With 2D-DIGE, we compared three stages in a perfusion culture of CHO cells: the initial growth with high glucose concentration and low lactate production, the second phase with glucose going to limitation and high lactate level, and finally the state of glucose limitation and also low lactate concentration but increased cell-specific productivity. With our proteomic approach we were able to demonstrate consequences of glucose limitation for the protein expression machinery which also could play a role for a higher recombinant protein production. Most interestingly, we detected epigenetic effects on the level of proteins involved in histone modification (HDAC11-2, SET, RBBP7, DDX5). Together with shifts in the protein inventory of energy metabolism, cytoskeleton and protein expression, a picture emerges of basic changes in the cellular equipment under long-term glucose limitation of CHO cells. (C) 2015 Elsevier B.V. All rights reserved.