title: An Approach to Further Enhance the Cellular Productivity of Exogenous Protein Hyper-producing Chinese Hamster Ovary (CHO) Cells.
abstract: The cell line D29, which was easily and rapidly established by the promoter-activated production and glutamine synthetase hybrid system, secreted recombinant human interleukin-6 (hIL-6) at a productivity rate of 39.5 mug 10(-6) cells day(-1), one of the highest reported levels worldwide. The productivity rate was about 130-fold higher than that of the cell line A7, which was established without both promoter activation and gene amplification. Although D29 cells had a high copy number and high mRNA level of the hIL-6 gene as well as a high secretion rate of hIL-6, large amounts of intracellular hIL-6 protein accumulated in D29 cells compared to A7 cells. Northern blotting analysis showed no change in the GRP78/BiP expression level in D29 cells. In contrast, an electrophoresis mobility shift assay revealed strong activation of NF-kappaB in D29 cells. These results suggest that large amounts of hIL-6 translated from large amounts of hIL-6 mRNA cause excess accumulation of intact hIL-6 in the endoplasmic reticulum (ER), and that subsequent negative feedback signals via the ER overload response inhibit hIL-6 protein secretion. To enhance the hIL-6 productivity rate of D29 cells by releasing the negative feedback signals, the effect of pyrrolidinedithiocarbamate, an inhibitor of NF-kappaB activation, was examined. Suppression of NF-kappaB activation in D29 cells produced a 25% augmentation of the hIL-6 productivity rate. Therefore, in highly productive cells like D29 cells, the release of negative feedback signals could increase the total amount of recombinant protein secretion.
authors.affiliation: Department of Genetic Resources Technology, Faculty of Agriculture, Kyushu University, Fukuoka, 812-8581, Japan, kteruya@grt.kyushu-u.ac.jp.
authors.forename: Kiichiro
authors.initials: K
authors.lastname: Teruya
authors.forename: Yoshihito
authors.initials: Y
authors.lastname: Daimon
authors.forename: Xiao-Yan
authors.initials: XY
authors.lastname: Dong
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Katakura
authors.forename: Takumi
authors.initials: T
authors.lastname: Miura
authors.forename: Akira
authors.initials: A
authors.lastname: Ichikawa
authors.forename: Tsukasa
authors.initials: T
authors.lastname: Fujiki
authors.forename: Makiko
authors.initials: M
authors.lastname: Yamashita
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Mori
authors.forename: Hideya
authors.initials: H
authors.lastname: Ohashi
authors.forename: Sanetaka
authors.initials: S
authors.lastname: Shirahata