Kauffman Kenneth J, Pridgen Eric M, Doyle Francis J, Dhurjati Prasad S, Robinson Anne Skaja
Department of Chemical Engineering, University of Delaware, Newark, Delaware 19716, USA.
Biotechnol Prog. 2002 Sep-Oct;18(5):942-50. doi: 10.1021/bp025518g.
Cells are inherently robust to environmental perturbations and have evolved to recover readily from short-term exposure to heat, pH changes, and nutrient deprivation during times of stress. The stress of unfolded protein accumulation has been implicated previously in low protein yields during heterologous protein expression. Here we describe the dynamics of the response to this stress, termed the unfolded protein response (UPR), during the expression of the single chain antibody 4-4-20 (scFv) in Saccharomyces cerevisiae. Expression of scFv decreased the growth rate of yeast cells whether the scFv was expressed from single-copy plasmids or integrated into the chromosome. However, the growth rates recovered at longer expression times, and surprisingly, the recovery occurred more quickly in the high-copy integration strains. The presence of a functional UPR pathway was necessary for a recovery of normal growth rates. During the growth inhibition, the UPR pathway appeared to be activated, resulting in decreased intracellular scFv levels and intermittent recovery of the chaperone BiP within the endoplasmic reticulum. Intracellular scFv was observed primarily in the endoplasmic reticulum, consistent with activation of the UPR pathway. Although the intracellular scFv levels dropped over the course of the expression, this was not a result of scFv secretion. A functional UPR pathway was necessary for the drop in intracellular scFv, suggesting that the decrease was a direct response of UPR activation. Taken together, these results suggest that control of heterologous gene expression to avoid UPR activation will result in higher production levels.
细胞对环境扰动具有内在的稳健性，并且已经进化到能够在应激期间从短期暴露于热、pH变化和营养剥夺中迅速恢复。未折叠蛋白积累的应激先前被认为与异源蛋白表达过程中的低蛋白产量有关。在这里，我们描述了在酿酒酵母中表达单链抗体4-4-20（scFv）期间对这种应激（称为未折叠蛋白反应（UPR））的反应动态。无论scFv是从单拷贝质粒表达还是整合到染色体中，scFv的表达都会降低酵母细胞的生长速率。然而，在较长的表达时间后生长速率恢复，令人惊讶的是，在高拷贝整合菌株中恢复更快。功能性UPR途径的存在是正常生长速率恢复所必需的。在生长抑制期间，UPR途径似乎被激活，导致细胞内scFv水平降低以及内质网中伴侣蛋白BiP的间歇性恢复。细胞内scFv主要在内质网中观察到，这与UPR途径的激活一致。尽管在表达过程中细胞内scFv水平下降，但这不是scFv分泌的结果。功能性UPR途径是细胞内scFv下降所必需的，表明这种下降是UPR激活的直接反应。综上所述，这些结果表明，控制异源基因表达以避免UPR激活将导致更高的生产水平。
