Decreased Protein Expression and Intermittent Recoveries in BiP Levels Result from Cellular Stress during Heterologous Protein Expression in Saccharomyces cerevisiae
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- 作者：Kenneth J. Kauffman ; Eric M. Pridgen ; Francis J. Doyle ; III ; Prasad S. Dhurjati ; and Anne Skaja Robinson
- 刊名：Biotechnology Progress
- 出版年：2002
- 出版时间：October 2002
- 年：2002
- 卷：18
- 期：5
- 页码：942 - 950
- 全文大小：139K
- 年卷期：v.18,no.5(October 2002)
- ISSN：1520-6033
文摘
Cells are inherently robust to environmental perturbations and have evolved to recoverreadily from short-term exposure to heat, pH changes, and nutrient deprivation duringtimes of stress. The stress of unfolded protein accumulation has been implicatedpreviously in low protein yields during heterologous protein expression. Here wedescribe the dynamics of the response to this stress, termed the unfolded proteinresponse (UPR), during the expression of the single chain antibody 4-4-20 (scFv) inSaccharomyces cerevisiae. Expression of scFv decreased the growth rate of yeast cellswhether the scFv was expressed from single-copy plasmids or integrated into thechromosome. However, the growth rates recovered at longer expression times, andsurprisingly, the recovery occurred more quickly in the high-copy integration strains.The presence of a functional UPR pathway was necessary for a recovery of normalgrowth rates. During the growth inhibition, the UPR pathway appeared to be activated,resulting in decreased intracellular scFv levels and intermittent recovery of thechaperone BiP within the endoplasmic reticulum. Intracellular scFv was observedprimarily in the endoplasmic reticulum, consistent with activation of the UPR pathway.Although the intracellular scFv levels dropped over the course of the expression, thiswas not a result of scFv secretion. A functional UPR pathway was necessary for thedrop in intracellular scFv, suggesting that the decrease was a direct response of UPRactivation. Taken together, these results suggest that control of heterologous geneexpression to avoid UPR activation will result in higher production levels.