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首页> 外文期刊>Biotechnology Progress >Application of a Nonradioactive Method of Measuring Protein Synthesis in Industrially Relevant Chinese Hamster Ovary Cells
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Application of a Nonradioactive Method of Measuring Protein Synthesis in Industrially Relevant Chinese Hamster Ovary Cells

机译：非放射性方法在工业相关的中国仓鼠卵巢细胞中蛋白质合成测定的应用

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Due to the high medical and commercial value of recombinant proteins for clinical and diagnostic purposes, the protein synthesis machinery of mammalian host cells is the subject of extensive research by the biopharmaceutical industry. RNA translation and protein synthesis are steps that may determine the extent of growth and productivity of host cells. To address the problems of utilization of current radioisotope methods with proprietary media, we have focused on the application of an alternative method of measuring protein synthesis in recombinant Chinese hamster ovary (CHO) cells. This method employs puromy-cin as a nonradioactive label which incorporates into nascent polypeptide chains and is detectable by western blotting. This method, which is referred to as SUnSET, successfully demonstrated the expected changes in protein synthesis in conditions that inhibit and restore translation activity and was reproducibly quantifiable. The study of the effects of feed and sodium butyrate addition on protein synthesis by SUnSET revealed an increase following 1 h feed supplementation while a high concentration of sodium butyrate was able to decrease translation during the same treatment period. Finally, SUnSET was used to compare protein synthesis activity during batch culture of the CHO cell line in relation to growth. The results indicate that as the cells approached the end of batch culture, the global rate of protein synthesis declined in parallel with the decreasing growth rate. In conclusion, this method can be used as a "snapshot" to directly monitor the effects of different culture conditions and treatments on translation in recombinant host cells.
机译：由于重组蛋白对于临床和诊断目的具有很高的医学和商业价值，因此哺乳动物宿主细胞的蛋白合成机制已成为生物制药行业广泛研究的主题。 RNA翻译和蛋白质合成是可以确定宿主细胞生长程度和生产力的步骤。为了解决当前的放射性同位素方法在专有介质中的利用问题，我们集中于另一种在重组中国仓鼠卵巢（CHO）细胞中测量蛋白质合成的方法的应用。该方法采用嘌呤霉素作为非放射性标记，该标记掺入新生的多肽链中，并且可以通过蛋白质印迹法检测到。此方法称为SUnSET，成功地证明了在抑制和恢复翻译活性的条件下蛋白质合成的预期变化，并且可重复性地定量。饲料和丁酸钠的添加对SUnSET蛋白质合成的影响的研究表明，添加1 h饲料后增加，而高浓度的丁酸钠在相同的处理期内能够减少翻译。最后，使用SUnSET比较CHO细胞株分批培养过程中蛋白质合成活性与生长的关系。结果表明，随着细胞接近分批培养的结束，蛋白质合成的总体速率随着生长速率的降低而下降。总之，该方法可用作“快照”，以直接监测不同培养条件和处理对重组宿主细胞中翻译的影响。

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