Analysis of dynamic changes in the proteome of a Bcl-XL overexpressing Chinese hamster ovary cell culture during exponential and stationary phases
Analysis of dynamic changes in the proteome of a Bcl-XL overexpressing Chinese hamster ovary cell culture during exponential and stationary phases
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DOI：
10.1002/btpr.1534
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发表时间：
2012-05-01
影响因子：
2.9
通讯作者：
Hancock, William S.
中科院分区：
文献类型：
--
作者：
Carlage, Tyler;Kshirsagar, Rashmi;Hancock, William S.
用于生物制药生产的哺乳动物细胞培养随着时间的推移经历了各种动态的生物学变化，包括细胞培养过程中细胞从指数生长期过渡到稳定期。为了更好地了解细胞培养的动态方面，利用定量蛋白质组学方法确定了中国仓鼠卵巢(CHO)细胞培养过程中蛋白质表达的动态趋势，以生产重组单抗和过表达抗凋亡基因BclxL。使用iTRAQ标记、二维LC/MS和线性回归计算相结合的方法对样品进行分析，以确定蛋白质丰度的显著动态趋势。使用这种方法，59个蛋白质被鉴定出有显著的时间表达变化。路径分析工具被用来确定与细胞生长和凋亡相关的假定蛋白质网络。差异表达的蛋白质包括分子伴侣和异构酶，如GRP78和PDI，以及已报道的细胞生长标记MCM2和MCM5。此外，还鉴定了两种具有生长调节功能的蛋白质：转谷氨酰胺酶-2和聚集素。这些蛋白与肿瘤的增殖和凋亡有关，并在稳定期有较高水平的表达，Western blotting证实了这一点。本文描述的蛋白质组学方法提供了CHO分批细胞培养过程中蛋白质表达的动态视图，这可能有助于进一步阐明驱动哺乳动物细胞培养性能的生物学过程。(C)2012年美国化学工程师协会生物技术研究所。节目，2012年
Mammalian cell cultures used for biopharmaceutical production undergo various dynamic biological changes over time, including the transition of cells from an exponential growth phase to a stationary phase during cell culture. To better understand the dynamic aspects of cell culture, a quantitative proteomics approach was used to identify dynamic trends in protein expression over the course of a Chinese hamster ovary (CHO) cell culture for the production of a recombinant monoclonal antibody and overexpressing the antiapoptotic gene Bcl-xl. Samples were analyzed using a method incorporating iTRAQ labeling, two-dimensional LC/MS, and linear regression calculations to identify significant dynamic trends in protein abundance. Using this approach, 59 proteins were identified with significant temporal changes in expression. Pathway analysis tools were used to identify a putative network of proteins associated with cell growth and apoptosis. Among the differentially expressed proteins were molecular chaperones and isomerases, such as GRP78 and PDI, and reported cell growth markers MCM2 and MCM5. In addition, two proteins with growth-regulating properties, transglutaminase-2 and clusterin, were identified. These proteins are associated with tumor proliferation and apoptosis and were observed to be expressed at relatively high levels during stationary phase, which was confirmed by western blotting. The proteomic methodology described here provides a dynamic view of protein expression throughout a CHO fed-batch cell culture, which may be useful for further elucidating the biological processes driving mammalian cell culture performance. (C) 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012