Graduate School of Genetic Resources Technology, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka, 812-8581, Japan.
Cytotechnology. 2001 Jan;35(1):9-16. doi: 10.1023/A:1008179919857.
We previously established a ras-oncogene amplified Chinesehamster ovary (CHO) cell line, named ras clone I, as anuniversal host cell line for oncogene activated production(OAP) system to mass-produce recombinant protein by activationof the cytomegalovirus immediate early (CMV) promoter with ras protein. The lambda light chain(C5lambda) of human monoclonal antibody HB4C5 is expected tobe potentially useful for lung cancer targeting. We generated aC5lambda hyper-producing cell line by transfecting ras cloneI with the C5lambda gene expression plasmid regulated by theCMV promoter, of which productivity was 5.3 times greater thanthe hyper productive CHO cell line generated by using conventional CHO cells. Introduction of the adenovirus E1A geneinto the hyper-producing cell line derived from ras clone I resulted in further 9.5 times enhancement of the productivity,suggesting the synergistic effect of E1A and ras oncogenes on the recombinant protein production driven by the CMV promoter. In addition, intracellular accumulation of C5lambda andupregulation of BiP was found in hyper-producing cell lineswhich were introduced E1A and ras oncogene. This resultsuggests that excessive intracellular accumulation ofC5lambda protein, which might be caused by that the amount of produced C5lambda in ER is beyond the ability of CHO cells to secrete, might signal the BiP promoter. Our data imply that ras clone I is available as a general host cell for establishing the recombinant protein hyper-producing CHOcells by the OAP system, and suggest that further mass production of recombinant proteins in the OAP system can be possible by clarifying the accurate role of upregulated BiP protein.
我们之前建立了一个 ras-oncogene 扩增的中国仓鼠卵巢（CHO）细胞系，命名为 ras 克隆 I，作为一个通用的宿主细胞系，用于 oncogene 激活生产（OAP）系统，通过 ras 蛋白激活巨细胞病毒立即早期（CMV）启动子大量生产重组蛋白。人类单克隆抗体 HB4C5 的 lambda 轻链（C5lambda）有望成为潜在的肺癌靶向药物。我们通过将 C5lambda 基因表达质粒转染 ras 克隆 I，生成了一个 C5lambda 高表达细胞系，该质粒由 CMV 启动子调控，其产物比使用传统 CHO 细胞生成的高表达 CHO 细胞系高出 5.3 倍。将腺病毒 E1A 基因引入源自 ras 克隆 I 的高表达细胞系，导致产物进一步提高了 9.5 倍，表明 E1A 和 ras 癌基因对 CMV 启动子驱动的重组蛋白生产具有协同作用。此外，在引入 E1A 和 ras 癌基因的高表达细胞系中发现了 C5lambda 的细胞内积累和 BiP 的上调。这一结果表明，C5lambda 蛋白的细胞内积累过多，可能是由于内质网中产生的 C5lambda 蛋白量超过了 CHO 细胞分泌的能力，可能会引发 BiP 启动子。我们的数据表明，ras 克隆 I 可作为通过 OAP 系统建立重组蛋白高表达 CHO 细胞的通用宿主细胞，并且通过阐明上调的 BiP 蛋白的准确作用，提示在 OAP 系统中进一步大规模生产重组蛋白是可能的。
