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 lg 10 À6 cells day À1 , 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-jB 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-jB activation, was examined. Suppression of NF-jB 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.Abbreviations: CHO -Chinese hamster ovary; dhfr -dihydrofolate reductase; DMEM -Dulbecco's modified Eagle's medium; ECL -enhanced chemiluminescence; ELISA -enzyme-linked immunosorbent assay; EMSA -electrophoretic mobility shift assay;

Dynamics of unfolded protein response in recombinant CHO cells

“…Another pathway that responds to protein accumulation in the ER membrane is the ER overload response (EOR), where NF-jB is activated on production of reactive oxygen intermediates and release of Ca 2? from the ER (Pahl and Baeuerle 1997;Teruya et al 2005).…”

Repressing expression of difficult‐to‐express recombinant proteins during the selection process increases productivity of CHO stable pools

“…Among the proteins tested, only two, IFN‐β and to a lesser extent IL7, did not appear to benefit from this approach. It is not clear whether these two proteins may be less apt to induce cellular stress during expression, or if other factors may limit their expression, such as nonoptimal nucleotide sequences, signal peptides, or maximal negative feedback already attained from the non‐induced (basal) level of the expressed r‐protein, which was previously shown for IL6 in CHO cells (Teruya et al, 2005).…”

Engineering of chaperone systems and of the unfolded protein response

“…Induction of BiP in several expression systems (Alete et al 2005;Dorner et al 1989;Downham et al 1996;Jones et al 2005;Miura et al 2001;Smales et al 2004;Watowich et al 1991), of NF-jB in human IL-6 expressing CHO cells (Teruya et al 2005), and of genes involved in the ER stress response including BiP, GRP94, calnexin, and HERP in human bone morphogenic protein 2 expressing CHO DUKX cells (Doolan et al 2008) demonstrates that the UPR is activated in many heterologous expression systems. Cooperativity of molecular chaperones and foldases may necessitate increasing expression of several chaperones simultaneously to reproducibly increase productivities.…”

“…ROS may damage the heterologous protein product. NF-jB, but not BiP, activation in CHO cells expressing human IL-6 suggests that these cells suffer from oxidative stress (Teruya et al 2005).…”