eIF2α phosphorylation, stress perception, and the shutdown of global protein synthesis in cultured CHO cells
✍ Scribed by Michèle F. Underhill; John R. Birch; C. Mark Smales; Louise H. Naylor
- Publisher
- John Wiley and Sons
- Year
- 2005
- Tongue
- English
- Weight
- 648 KB
- Volume
- 89
- Category
- Article
- ISSN
- 0006-3592
No coin nor oath required. For personal study only.
✦ Synopsis
Abstract
The perception of environmental stress in animal cells engineered to produce heterologous protein leads to the induction of stress signaling pathways and ultimately apoptosis and cell death. Protein synthesis is regulated in response to various environmental stresses by phosphorylation of the α subunit of the eukaryotic initiation factor 2 (eIF2). In this study we have utilized a model system of Chinese hamster ovary cells engineered to secrete recombinant TIMP‐1 protein to investigate the relationship between the cellular rate of protein synthesis, eIF2α phosphorylation, cellular stress perception, and the rate of cell specific recombinant protein synthesis. The rate of total protein synthesis was maximal after 48 hours of culture, remaining relatively high until 96 hours of culture, after which a decline was observed. Towards the end of culture a marked increase in labeled secreted protein was observed. Total eIF2α expression levels were high during the exponential growth phase and decreased slightly towards the end of culture. On the other hand, the relative expression of phosphorylated eIF2α showed a bi‐phasic response with a small increase in phosphorylated eIF2α observed at 48 hours of culture, and a significant increase at 120 hours post‐inoculation. The large increase in phosphorylated eIF2α coincided with the observed increase in labeled secreted protein and the decline in total cellular protein synthesis. A marked increase in ubiquitination was also observed at 120 hours post‐inoculation that coincided with reduced rates of cellular protein synthesis and mRNA translation attenuation. We suggest that eIF2α phosphorylation is an indicator of cellular stress perception, which could be exploited in recombinant protein manufacturing to commence feeding and engineering strategies. © 2005 Wiley Periodicals, Inc.