Increasing Yeast Secretion of Heterologous Proteins by Regulating Expression Rates and Post-Secretory Loss
Increasing Yeast Secretion of Heterologous Proteins by Regulating Expression Rates and Post-Secretory Loss
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DOI：
10.1002/bit.22019
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发表时间：
2008-12-15
影响因子：
3.8
通讯作者：
Shusta, Eric V.
中科院分区：
文献类型：
--
作者：
Huang, Dagang;Gore, Patrick R.;Shusta, Eric V.
异源蛋白质分泌涉及蛋白质合成、蛋白质折叠和分泌运输的耦合过程。对这些过程如何相互关联的更完整的理解可以帮助直接优化分泌系统。在这里，我们提供了一个详细的研究，从酵母细胞内蛋白水平，分泌蛋白水平，和未折叠蛋白反应（UPR）的异源蛋白分泌的动力学。研究了三种不同的蛋白质表达诱导温度（20、30和37 ° C）作为调节表达速率并因此调节细胞应答的手段。在20 ℃下诱导产生最慢的初始分泌速率，但产物的绝对水平最高。相应地，细胞内蛋白质积累和未折叠蛋白反应（UPR）激活的水平和速率也在20 ℃时最低。此外，分别在30和37摄氏度下约22小时后分泌停止，而在20摄氏度下分泌持续到营养耗尽。观察到分泌水平的最大值是分泌停止和分泌后蛋白质损失的累加效应的结果。分泌后蛋白质的损失似乎不是由于溶液相蛋白水解或聚集，但需要酵母细胞的存在。酵母氮碱和酪蛋白氨基酸的再补料成功地防止了在高温（37摄氏度）和低温（20摄氏度）下蛋白质的分泌后损失，并在20摄氏度下进一步增加了分泌水平1.5倍，其中分泌途径仍然起作用。总之，这些发现表明，存在一个适当的平衡之间的蛋白质合成，加工和分泌优化所需的分泌速率。
Heterologous protein secretion involves the coupled processes of protein synthesis, protein folding, and secretory trafficking. A more complete understanding of how these processes interrelate could help direct optimization of secretion systems. Here we provide a detailed study regarding the dynamics of heterologous protein secretion from yeast in terms of intracellular protein levels, secreted protein levels, and unfolded protein response (UPR). Three different protein expression induction temperatures (20, 30, and 37 degrees C) were investigated as a means to modulate expression rates and thus cellular responses. Inducing at 20 degrees C yielded the slowest initial secretion rate, but the highest absolute level of product. Correspondingly, the level and the rate of both intracellular protein accumulation and unfolded protein response (UPR) activation were also the lowest at 20 degrees C. In addition, secretion ceased after approximately 22 h at 30 and 37 degrees C, respectively, while it was continuous until nutrient depletion at 20 degrees C. Maxima in secretion levels were observed that were a result of the additive effects of secretion cessation and post-secretory protein loss. The post-secretory loss of protein did not appear to result from solution phase proteolysis or aggregation, but required the presence of yeast cells. Refeeding of both yeast nitrogen base and casamino acids successfully prevented the post-secretory loss of protein at both high (37 degrees C) and low (20 degrees C) temperatures, and further increased secretion levels 1.5-fold at 20 degrees C where the secretory pathway was still functioning. Taken together, these findings suggest that there exists an appropriate balance between protein synthesis, processing and secretion rates required for secretion optimization.