A regulatable selective system facilitates isolation of heterologous protein hyper-producing mammalian cells without gene amplification.
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.
Teruya
Kiichiro
K
Department of Genetic Resources Technology, Faculty of Agriculture, Kyushu University, Hakozaki, Higashi-ku, Fukuoka, Japan, 92 642 3047), kteruya@grt.kyushu-u.ac.jp.
Zhang
Yingpei
Y
Katakura
Yoshinori
Y
Shirahata
Sanetaka
S