Department of Biological Sciences and Graduate School of Nanoscience & Technology (WCU), KAIST, Yuseong-Gu, Daejon, South Korea.
Appl Microbiol Biotechnol. 2011 Mar;89(6):1917-28. doi: 10.1007/s00253-011-3106-9. Epub 2011 Feb 1.
In order to understand the intracellular responses in recombinant CHO (rCHO) cells producing antibody in serum-free medium (SFM) supplemented with optimized hydrolysates mixtures, yielding the highest specific growth rate (μ, SFM#S1) or the highest specific antibody productivity (q(Ab,) SFM#S2), differentially expressed proteins in rCHO cells are measured by two-dimensional gel electrophoresis combined with nano-LC-ESI-Q-TOF tandem MS. The comparative proteomic analysis with basal SFM without hydrolysates revealed that the addition of hydrolysate mixtures significantly altered the profiles of CHO proteome. In SFM#S1, the expression of metabolism-related proteins, cytoskeleton-associated proteins, and proliferation-related proteins was up-regulated. On the other hand, the expression of anti-proliferative proteins and pro-apoptotic protein was down-regulated. In SFM#S2, the expression of various chaperone proteins and proliferation-linked proteins was altered. 2D-Western blot analysis of differentially expressed proteins confirmed the proteomic results. Taken together, identification of differentially expressed proteins in CHO cells by a proteomic approach can provide insights into understanding the effect of hydrolysates on intracellular events and clues to find candidate genes for cell engineering to maximize the protein production in rCHO cells.
为了了解在含有优化水解混合物的无血清培养基（SFM）中生产抗体的重组 CHO（rCHO）细胞中的细胞内反应，以获得最高的比生长速率（μ，SFM#S1）或最高的比抗体产率（q（Ab，）SFM#S2），通过二维凝胶电泳结合纳升 LC-ESI-Q-TOF 串联 MS 测量 rCHO 细胞中的差异表达蛋白。与不含水解物的基础 SFM 的比较蛋白质组学分析表明，水解混合物的添加显著改变了 CHO 蛋白质组的图谱。在 SFM#S1 中，与代谢相关的蛋白质、细胞骨架相关的蛋白质和增殖相关的蛋白质的表达上调。另一方面，抗增殖蛋白和促凋亡蛋白的表达下调。在 SFM#S2 中，各种伴侣蛋白和与增殖相关的蛋白的表达发生改变。差异表达蛋白的 2D-Western blot 分析证实了蛋白质组学结果。总之，通过蛋白质组学方法鉴定 CHO 细胞中的差异表达蛋白可以深入了解水解物对细胞内事件的影响，并为寻找用于最大化 rCHO 细胞中蛋白质生产的细胞工程候选基因提供线索。
