流式细胞仪检测重组毕赤酵母发酵过程中的细胞活性
Assessing Viability of Recombinant Pichia pastoris in Fermentation Process with Flow Cytometry
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摘要: 建立了流式细胞仪检测重组巴斯德毕赤酵母(P.p astoris)细胞活性的方法,并在高密度发酵过程中得到应用。使用碘化丙锭染色法测定细胞活性,发现甲醇诱导时酵母细胞活性开始下降,随着细胞密度的增加,由于细胞胁迫和甲醇的影响,细胞活性明显下降,活细胞率最大下降了14%。与此同时,细胞生长减缓,目的蛋白发生降解,表明细胞活性与发酵过程中细胞的生长和目的蛋白生成等参数密切相关。实验证明:流式细胞仪可作为检测毕赤酵母发酵过程中细胞活性参数的一个便捷精确的有力工具。Abstract: This paper describes the establishment of flow cytometric methods to assess the viability for recombinant methylotrophic P.pastoris strains and its application to a lab scale high cell density fermentation.Using a recombinant strain expressing human reteplase(hrPA),the cell viabilities were measured with propidium iodide staining.The cell viability decreases significantly by 14% during the methanol(induction) phase,indicating there was a stress situation triggered by the fermentation conditions.These(data) demonstrate that cell viability is an important physiological parameter and flow cytometry is a power-(ful) tool for the analysis and optimization of recombinant protein production processes expressed with P.(pastoris.)