Department of Genetic Resources Technology, Faculty of Agriculture, Kyushu University, Hakozaki, Higashi-ku, Fukuoka, Japan, 92 642 3047),
Cytotechnology. 2002 Nov;40(1-3):13-22. doi: 10.1023/A:1023945517446.
In this article, we describe a new method that facilitates to isolate mammalian cells inducible hyper-producing heterologous proteins. This method uses the tetracycline-inducible system to express both the selection marker and the heterologous gene, therefore, allows to increase the selection pressure by reducing the transcription of the selection maker gene. Using this method, we were able to isolate recombinant Chinese hamster ovary cells with a high efficiency. One of established clones produced the recombinant bovine beta-lactoglobulin as heterologous protein at a peak rate of 12 mug 10(-6) cells/day with an inducibility of about 100-fold. This clone was over expressed them RNA of beta-lactoglobulin and the drug resistant gene but did not amplify their genes. When cultured in a hollow fiber bioreactor, the cells were able to secrete beta-lactoglobulinover 300 mug ml(-1). This method is applicable to a broad range of eukaryotic systems and is of general value to technology for recombinant protein production.
在本文中，我们描述了一种新的方法，可用于分离哺乳动物细胞诱导产生异源蛋白。该方法使用四环素诱导系统表达选择标记和异源基因，从而通过降低选择标记基因的转录来增加选择压力。使用这种方法，我们能够高效地分离出重组中国仓鼠卵巢细胞。其中一个建立的克隆以约 100 倍的诱导率产生重组牛β-乳球蛋白作为异源蛋白，峰值速率为 12 微克/10(-6)细胞/天。该克隆过度表达了β-乳球蛋白和耐药基因的 RNA，但没有扩增它们的基因。当在中空纤维生物反应器中培养时，细胞能够分泌超过 300 微克/毫升的β-乳球蛋白。该方法适用于广泛的真核系统，对重组蛋白生产技术具有普遍价值。
