Liu Zhenke, Dai Shujia, Bones Jonathan, Ray Somak, Cha Sangwon, Karger Barry L, Li Jingyi Jessica, Wilson Lee, Hinckle Greg, Rossomando Anthony
Barnett Inst. and Dept. of Chemistry and Chemical Biology, Northeastern University, Boston, MA, 02115.
Dept. of Statistics, University of California, Los Angeles, CA, 90095.
Biotechnol Prog. 2015 Jul-Aug;31(4):1026-38. doi: 10.1002/btpr.2090. Epub 2015 Apr 29.
A goal in recombinant protein production using Chinese hamster ovary (CHO) cells is to achieve both high specific productivity and high cell density. Addition of glucose to the culture media is necessary to maintain both cell growth and viability. We varied the glucose concentration in the media from 5 to 16 g/L and found that although specific productivity of CHO-DG44 cells increased with the glucose level, the integrated viable cell density decreased. To examine the biological basis of these results, we conducted a discovery proteomic study of CHO-DG44 cells grown under batch conditions in normal (5 g/L) or high (15 g/L) glucose over 3, 6, and 9 days. Approximately 5,000 proteins were confidently identified against an mRNA-based CHO-DG44 specific proteome database, with 2,800 proteins quantified with at least two peptides. A self-organizing map algorithm was used to deconvolute temporal expression profiles of quantitated proteins. Functional analysis of altered proteins suggested that differences in growth between the two glucose levels resulted from changes in crosstalk between glucose metabolism, recombinant protein expression, and cell death, providing an overall picture of the responses to high glucose environment. The high glucose environment may enhance recombinant dihydrofolate reductase in CHO cells by up-regulating NCK1 and down-regulating PRKRA, and may lower integrated viable cell density by activating mitochondrial- and endoplasmic reticulum-mediated cell death pathways by up-regulating HtrA2 and calpains. These proteins are suggested as potential targets for bioengineering to enhance recombinant protein production.
利用中国仓鼠卵巢（CHO）细胞进行重组蛋白生产的一个目标是实现高比生产率和高细胞密度。向培养基中添加葡萄糖对于维持细胞生长和活力是必要的。我们将培养基中的葡萄糖浓度从5 g/L变化到16 g/L，发现尽管CHO-DG44细胞的比生产率随葡萄糖水平升高而增加，但活细胞综合密度却降低了。为了研究这些结果的生物学基础，我们对在分批培养条件下于正常（5 g/L）或高（15 g/L）葡萄糖中培养3天、6天和9天的CHO-DG44细胞进行了发现性蛋白质组学研究。针对基于mRNA的CHO-DG44特异性蛋白质组数据库，可靠地鉴定出了约5000种蛋白质，其中2800种蛋白质用至少两个肽段进行了定量。使用自组织映射算法对定量蛋白质的时间表达谱进行反卷积分析。对变化蛋白质的功能分析表明，两种葡萄糖水平下生长的差异是由葡萄糖代谢、重组蛋白表达和细胞死亡之间的串扰变化引起的，从而提供了对高葡萄糖环境反应的整体情况。高葡萄糖环境可能通过上调NCK1和下调PRKRA来增强CHO细胞中的重组二氢叶酸还原酶，并可能通过上调HtrA2和钙蛋白酶激活线粒体和内质网介导的细胞死亡途径来降低活细胞综合密度。这些蛋白质被认为是生物工程增强重组蛋白生产的潜在靶点。
