The Effect of Temperature on the Proteome of Recombinant Pichia pastoris
The Effect of Temperature on the Proteome of Recombinant Pichia pastoris
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DOI：
10.1021/pr8007623
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发表时间：
2009-03-01
影响因子：
4.4
通讯作者：
Mattanovich, Diethard
中科院分区：
文献类型：
--
作者：
Dragosits, Martin;Stadlmann, Johannes;Mattanovich, Diethard
到目前为止，关于环境因素对酵母细胞产量的影响的研究还很少。因此，提高对包括巴斯德毕赤酵母在内的微生物蛋白生产宿主的细胞生理学的了解是一个主要关注的问题。采用双向荧光差示凝胶电泳法和蛋白质质谱鉴定技术，分析了培养温度对一株异源蛋白分泌型巴斯德毕赤酵母生理特性的影响。此外，还监测了比生产力，并计算了通过中心碳代谢的通量。恒化器培养条件用于评估在稳态条件下对不同生长温度(20、25、30摄氏度)的适应性。许多重要的细胞过程，包括中央碳代谢、应激反应和蛋白质折叠，都受到生长温度的影响。在较低的培养温度下，抗体Fab片段的比生产率提高了3倍，同时TCA循环的通量减少，参与氧化应激反应的蛋白质水平减少，细胞内分子伴侣水平降低。这些数据表明，在较低的培养温度下，折叠应力通常会降低，从而使巴斯德毕赤酵母宿主细胞能够更有效地分泌异源蛋白质。
The impact of environmental factors on the productivity of yeast cells is poorly investigated so far. Therefore, it is a major concern to improve the understanding of cellular physiology of microbial protein production hosts, including the methylotrophic yeast Pichia pastoris. Two-Dimensional Fluorescence Difference Gel electrophoresis and protein identification via mass spectrometry were applied to analyze the impact of cultivation temperature on the physiology of a heterologous protein secreting P. pastoris strain. Furthermore, specific productivity was monitored and fluxes through the central carbon metabolism were calculated. Chemostat culture conditions were applied to assess the adaption to different growth temperatures (20, 25, 30 degrees C) at steady-state conditions. Many important cellular processes, including the central carbon metabolism, stress response and protein folding are affected by changing the growth temperature. A 3-fold increased specific productivity at lower cultivation temperature for an antibody Fab fragment was accompanied by a reduced flux through the TCA-cycle, reduced levels of proteins involved in oxidative stress response and lower cellular levels of molecular chaperones. These data indicate that folding stress is generally decreased at lower cultivation temperatures, enabling more efficient heterologous protein secretion in P. pastoris host cells.