Flow cytometric analysis of bacterial physiology during induction of foreign protein synthesis in recombinant Escherichia coli cells
Flow cytometric analysis of bacterial physiology during induction of foreign protein synthesis in recombinant Escherichia coli cells
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DOI：
10.1002/(sici)1097-0320(19980201)31:2
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发表时间：
1998-02-01
期刊：
影响因子：
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通讯作者：
Katinger, H
中科院分区：
文献类型：
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作者：
Borth, N;Mitterbauer, R;Katinger, H
在许多情况下，诱导蛋白质合成会导致细胞能量和代谢前体的迅速耗竭，从而导致细胞死亡。因此，持续生产外源蛋白质需要对特定的生产速率进行一些微调，以满足细胞的能力。这促使我们用流式细胞术分析重组大肠杆菌细胞产生人超氧化物歧化酶（SOD）的生理行为。我们用以下参数比较了在T7/lac组合启动子或phi 10启动子控制下产生SOD的两株菌株：(a)总DNA含量作为细胞分裂的指标，(b)总RNA含量作为蛋白质合成活性的指标，(c)代表细胞大小的总蛋白质含量，以及(d)细胞内SOD含量作为生产力的指标。结果表明，诱导外源蛋白合成后生物量继续增加的细胞，其比产率也最高；这些结果证实了微调表达系统对于延长诱导后细胞的寿命的重要性。这将导致产量的增加。(C) 1998 Wiley-Liss, Inc。
The production of foreign proteins at high yields represents a severe metabolic stress for Escherichia coli cells, In many cases, induction of protein synthesis results in rapid exhaustion of the cellular energy and metabolic precursors and thus in cell death, Therefore, sustained production of foreign proteins requires some fine tuning of the specific production rate to meet the capabilities of the cell. This has stimulated us to analyze by flow cytometry the physiological behaviour of recombinant E. coli cells producing human superoxide dismutase (SOD),Two strains that produce SOD under the control of either a combined T7/lac promoter or the phi 10 promoter were compared by using the following parameters: (a) total DNA content as an indicator of cell division, (b) total RNA content as a measure for protein synthesis activity, (c) total protein content representing cell size, and (d) intracellular SOD content as a measure for productivity. Results show that those cells that continue to increase their biomass after induction of foreign protein synthesis also have the highest specific production rate, Cells, however; do not divide to a measureable degree but rather increase their size, The results confirm the importance of fine-tuning expression systems to prolong the lifetime of cells after induction. This will result in an increased yield. (C) 1998 Wiley-Liss, Inc.