Assessing viability and cell-associated product of recombinant protein producing Pichia pastoris with flow cytometry
Assessing viability and cell-associated product of recombinant protein producing Pichia pastoris with flow cytometry
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DOI：
10.1016/s0168-1656(03)00049-x
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发表时间：
2003-05-08
影响因子：
4.1
通讯作者：
Mattanovich, D
中科院分区：
文献类型：
--
作者：
Hohenblum, H;Borth, N;Mattanovich, D
本文介绍了重组巴斯德毕赤酵母的流式细胞仪检测方法的建立及其在中试规模补料分批发酵中的应用。以一株能分泌人胰酶原的菌株为研究对象，分别用碘化丙啶和免疫荧光染色检测细胞活力和结合产物。在甲醇补料分批阶段，活力显著下降到70%以下，这表明发酵条件引发了一种应激状态。细胞相关产物在甲醇诱导后比分泌产物积累得更早。这些数据表明，流式细胞术是分析和优化重组蛋白生产过程的有力工具，也表明需要进一步改进广泛应用的巴斯德毕赤酵母发酵方案。(C)2003 Elsevier Science B.V.保留所有权利。
This paper describes the establishment of flow cytometric methods for recombinant Pichia pastoris strains, and their application to a lab scale fed batch fermentation. Using a strain which secretes human trypsinogen, the viability and the product which remained associated to the cell were measured with propidium iodide and immunofluorescent staining, respectively. Viability decreases significantly below 70% during the methanol fed batch phase, indicating a stress situation triggered by the fermentation conditions. Cell associated product is accumulated earlier after methanol induction than secreted product. These data demonstrate that flow cytometry is a powerful tool for the analysis and optimization of recombinant protein production processes, and they indicate the need to further improve a widely used fermentation protocol for P. pastoris. (C) 2003 Elsevier Science B.V. All rights reserved.