Yano T, Teruya K, Shirahata S, Watanabe J, Osada K, Tachibana H, Ohashi H, Kim E H, Murakami H
Laboratory of Cellular Regulation Technology, Graduate School of Genetic Resources Technology, Kyushu University, Fukuoka, Japan.
Cytotechnology. 1994;16(3):167-78. doi: 10.1007/BF00749904.
In order to enhance recombinant protein productivity in animal cells, we developed the oncogene activated production (OAP) system. The OAP system is based on the premise that oncogenes are able to enhance promoter activity. To this end, we constructed reported plasmids by fusing various promoters to the human interleukin-6 (hIL-6) cDNA, and the effector plasmids by inserting individual oncogenes, for example c-myc, c-fos, v-jun, v-myb and c-Ha-ras, downstream from the human cytomegalovirus immediate early (CMV) promoter. Results of transient expression experiments with BHK-21 cells suggest that the CMV promoter is the most potent promoter examined and that the ras product is able to transactivate the beta-actin, CMV and SR alpha promoters. Recombinant BHK-21 cells producing hIL-6 under the control of the CMV promoter were contransfected with the ras oncogene and dihydrofolate reductase gene, then selected with 50 nM methotrexate to coamplify the ras oncogene. We were able to rapidly establish a stable and highly productive clone which exhibited a 35-times higher production rate as compared to the control value.
为了提高动物细胞中重组蛋白的产量，我们开发了癌基因激活生产（OAP）系统。OAP系统基于癌基因能够增强启动子活性这一前提。为此，我们通过将各种启动子与人白细胞介素-6（hIL-6）cDNA融合构建了报告质粒，并通过在人巨细胞病毒立即早期（CMV）启动子下游插入单个癌基因（例如c-myc、c-fos、v-jun、v-myb和c-Ha-ras）构建了效应质粒。用BHK-21细胞进行的瞬时表达实验结果表明，CMV启动子是所检测的最有效的启动子，并且ras产物能够反式激活β-肌动蛋白、CMV和SRα启动子。在CMV启动子控制下产生hIL-6的重组BHK-21细胞与ras癌基因和二氢叶酸还原酶基因共转染，然后用50 nM甲氨蝶呤进行选择以共扩增ras癌基因。我们能够快速建立一个稳定且高产的克隆，其产量比对照值高35倍。
