Dahodwala Hussain, Kaushik Prashant, Tejwani Vijay, Kuo Chih-Chung, Menard Patrice, Henry Michael, Voldborg Bjorn G, Lewis Nathan E, Meleady Paula, Sharfstein Susan T
College of Nanoscale Science and Engineering, SUNY Polytechnic Institute, Albany, NY, USA.
National Institute for Cellular Biotechnology, Dublin City University, Dublin 9, Ireland.
Curr Res Biotechnol. 2019 Nov;1:49-57. doi: 10.1016/j.crbiot.2019.09.001. Epub 2019 Oct 5.
Most therapeutic monoclonal antibodies in biopharmaceutical processes are produced in Chinese hamster ovary (CHO) cells. Technological advances have rendered the selection procedure for higher producers a robust protocol. However, information on molecular mechanisms that impart the property of hyper-productivity in the final selected clones is currently lacking. In this study, an IgG-producing industrial cell line and its methotrexate (MTX)-amplified progeny cell line were analyzed using transcriptomic, proteomic, phosphoproteomic, and chromatin immunoprecipitation (ChIP) techniques. Computational prediction of transcription factor binding to the transgene cytomegalovirus (CMV) promoter by the Transcription Element Search System and upstream regulator analysis of the differential transcriptomic data suggested increased in vivo CMV promoter-cAMP response element binding protein (CREB1) interaction in the higher producing cell line. Differential nuclear proteomic analysis detected 1.3-fold less CREB1 in the nucleus of the high productivity cell line compared with the parental cell line. However, the differential abundance of multiple CREB1 phosphopeptides suggested an increase in CREB1 activity in the higher producing cell line, which was confirmed by increased association of the CMV promotor with CREB1 in the high producer cell line. Thus, we show here that the nuclear proteome and phosphoproteome have an important role in regulating final productivity of recombinant proteins from CHO cells, and that CREB1 may play a role in transcriptional enhancement. Moreover, CREB1 phosphosites may be potential targets for cell engineering for increased productivity.
生物制药过程中大多数治疗性单克隆抗体是在中国仓鼠卵巢（CHO）细胞中生产的。技术进步使筛选高产细胞的程序变得更加可靠。然而，目前尚缺乏关于最终选定克隆中赋予高产特性的分子机制的信息。在本研究中，使用转录组学、蛋白质组学、磷酸蛋白质组学和染色质免疫沉淀（ChIP）技术分析了一种产生IgG的工业细胞系及其甲氨蝶呤（MTX）扩增的子代细胞系。通过转录元件搜索系统对转录因子与转基因巨细胞病毒（CMV）启动子结合的计算预测以及差异转录组数据的上游调节因子分析表明，高产细胞系中体内CMV启动子与环磷酸腺苷反应元件结合蛋白（CREB1）的相互作用增加。差异核蛋白质组分析检测到，与亲代细胞系相比，高产细胞系细胞核中的CREB1减少了1.3倍。然而，多种CREB1磷酸化肽段的丰度差异表明，高产细胞系中CREB1的活性增加，这在高产细胞系中通过CMV启动子与CREB1的结合增加得到了证实。因此，我们在此表明，核蛋白质组和磷酸蛋白质组在调节CHO细胞中重组蛋白的最终产量方面具有重要作用，并且CREB1可能在转录增强中发挥作用。此外，CREB1磷酸化位点可能是提高产量的细胞工程的潜在靶点。
