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Analysis of Golgi pH in Chinese hamster ovary cells using ratiometric pH-sensitive fluorescent proteins

机译：使用比率pH敏感荧光蛋白的中国仓鼠卵巢细胞Golgi pH分析

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摘要

Acidic Golgi pH plays an important role in protein glycosylation, one of the critical quality attributes of therapeutic proteins. To determine the intracellular Golgi pH during culture, stable Chinese hamster ovary (CHO) cell clones expressing pHluorin2, a ratiometric pH-sensitive fluorescent protein (FP), in the cis- and trans-Golgi, were constructed by fusing pHluorin2 with specific targeting proteins, acetylglucosaminyltransferase, and a galactosyltransferase, respectively. Stable CHO cell clones expressing pHluorin2 in the cytoplasm were also constructed. The subcellular localization of FPs was confirmed by immunofluorescence analysis. Live-cell imaging revealed that the intracellular pH (pHi) of clones expressing the ratiometric pH-sensitive FPs converged to a specific pH range (cis-Golgi: 6.4-6.5; trans-Golgi: 5.9-6.0; and cytoplasm: 7.1-7.2). The pHi was successfully evaluated in various culture conditions. Although culture pH was maintained at 7.2 in a bioreactor, the Golgi pH increased with culture time. Elevated ammonia concentration and osmolality were partially responsible for the increased Golgi pH during bioreactor cultures. Taken together, the application of ratiometric pH-sensitive FPs in monitoring the Golgi pH of CHO cells during culture provides a new perspective to improve protein glycosylation through pHi control.
机译：酸性高尔基pH在蛋白质糖基化中起重要作用，治疗蛋白的临界品质属性之一。为了确定培养过程中的细胞内Golgi pH，通过融合氟丙胺2与特异性靶向蛋白质构建CIS-and-GOLGI中表达氟胺2的稳定的中国仓鼠卵巢（CHO）细胞克隆，在CIS-和Trans-roolgi中表达鉴定荧光蛋白2。 ，乙酰甘氨酸氨基氨基转移酶和半乳糖基转移酶。还构建了在细胞质中表达普洛林2的稳定CHO细胞克隆。通过免疫荧光分析证实FPS的亚细胞定位。活细胞成像显示，表达克隆的细胞内pH（phi）表达收敛于特定pH范围（Cis-golgi：6.4-6.5; Trans-golgi：5.9-6.0;和细胞质：7.1-7.2 ）。在各种培养条件下成功评估了PHI。尽管在生物反应器中保持培养物pH值为7.2，但GOLGI pH随着培养时间而增加。氨浓度和渗透压升高是在生物反应器培养过程中增加的高尔基pH值的部分原因。在一起，在培养过程中，在监测CHO细胞的GOLGI pH监测中的应用提供了一种通过PHI对照改善蛋白质糖基化的新视角。

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