Identification of the Limitations on Recombinant Gene Expression in CHO Cell Lines With Varying Luciferase Production Rates
Identification of the Limitations on Recombinant Gene Expression in CHO Cell Lines With Varying Luciferase Production Rates
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DOI：
10.1002/bit.22201
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发表时间：
2009-04-15
影响因子：
3.8
通讯作者：
von der Haar, Tobias
中科院分区：
文献类型：
--
作者：
Mead, Emma J.;Chiverton, Lesley M.;von der Haar, Tobias
哺乳动物细胞系目前被用作表达基于重组蛋白的药物的主要细胞工厂之一。高产细胞系的建立通常从异质起始细胞群开始，通过经验方法从中选择最高产细胞。这种方法是耗时的，并且可能遇到由所讨论的细胞系的固有生物学强加的自然上限。在试图了解的性质的变异性的细胞群体转染重组蛋白编码DNA和生产力限制的自然机制，我们开发了详细的研究在这样的细胞系中的基因表达途径的协议。然后将这种新方法应用于一组克隆CHOKI细胞系，以不同的生产率生产重组荧光素酶。我们的研究结果表明，在这些细胞系中的初始限制是在转录水平上，然而，在最高产量的细胞系中，影响蛋白质周转和蛋白质折叠的翻译后机制变得严重限制，为提高重组蛋白生产水平的工程细胞的发展战略的影响进行了讨论。
Mammalian cell lines are Currently employed as one of the main cellular factories for the expression of recombinant protein-based drugs. The establishment of high-producing cell lines typically begins with a heterogeneous starter Population of cells, from which the highest producing cells are selected via empirical approaches. This approach is time consuming, and is likely to encounter natural upper limits imposed by the inherent biology of the cell lines in question. In an attempt to understand both the nature of the variability in populations of cells transfected with recombinant protein encoding DNA and the natural mechanisms of productivity limitation, we developed protocols for the detailed investigation of gene expression pathways in such cell lines. This novel approach was then applied to a set of clonal CHOKI cell lines producing recombinant luciferase with varying productivities. Our results show that the initial limitation in these cell lines is at the transcriptional level, however in the highest producing cell line post-translational mechanisms affecting both protein turnover and protein folding become severely limiting, The implications for the development of strategies to engineer cells for enhanced recombinant protein production levels are discussed.