On-line estimation of the metabolic burden resulting from the synthesis of plasmid-encoded and heat-shock proteins by monitoring respiratory energy generation
On-line estimation of the metabolic burden resulting from the synthesis of plasmid-encoded and heat-shock proteins by monitoring respiratory energy generation
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DOI：
10.1002/bit.10098
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发表时间：
2001-11-01
影响因子：
3.8
通讯作者：
Rinas, U
中科院分区：
文献类型：
--
作者：
Hoffmann, F;Rinas, U
利用温度诱导表达系统，在高密度培养的重组大肠杆菌中生产人碱性成纤维细胞生长因子（hFGF-2）。蛋白质的合成速率随后通过S-35-甲硫氨酸标记的蛋白质组的二维凝胶电泳。在温度诱导hFGF-2合成后，单位生物质的总蛋白质合成速率增加了三倍，这主要是由于hFGF-2和热休克蛋白的额外合成。在没有hFGF-2基因的对照菌株中，热休克蛋白和组成型质粒编码蛋白的合成速率在温度升高后也增加，但遵循与生产菌株不同的时间曲线。额外的质粒编码的和热休克蛋白的合成的能量需求导致呼吸活性升高，因此，在减少的生长速率和生物质产量。开发了一种程序，将这些蛋白质的额外合成的能量需求与呼吸途径中能量的产生联系起来。比能源生产估计的基础上在线可测量的耗氧率，或二氧化碳的演变和增长，分别。以这种方式，从质粒编码的和热休克蛋白的合成所产生的代谢负荷从在线可访问的数据进行定量。(C)John Wiley & Sons，Inc.
Human basic fibroblast growth factor (hFGF-2) was produced in high-cell density cultures of recombinant Escherichia coli using a temperature-inducible expression system. The synthesis rates of proteins were followed by two-dimensional gel electrophoresis of the S-35-methionine-labeled proteom. After temperature induction of hFGF-2 synthesis, the rate of total protein synthesis per biomass increased by a factor of three, mainly as a result of the additional synthesis of hFGF-2 and heat-shock proteins. The synthesis rates of heat-shock proteins and constitutive plasmid-encoded proteins increased after the temperature upshift also in the control strain without hFGF-2 gene but followed time profiles different from the producing strain. The energy demand for the extra synthesis of plasmid-encoded and heat-shock proteins resulted in an elevated respiratory activity and, consequently, in a reduction of the growth rate and the biomass yield. A procedure was developed to relate the energy demand for the additional synthesis of these proteins to the generation of energy in the respiratory pathway. Specific energy production was estimated based on on-line measurable rates of oxygen consumption, or carbondioxide evolution and growth, respectively. In this way, the metabolic burden resulting from the synthesis of plasmid-encoded and heat-shock proteins was quantified from on-line accessible data. (C) 2001 John Wiley & Sons, Inc.