Metalation calculators for E. coli strain JM109 (DE3): Aerobic, anaerobic and hydrogen peroxide exposed cells cultured in LB media
Metalation calculators for E. coli strain JM109 (DE3): Aerobic, anaerobic and hydrogen peroxide exposed cells cultured in LB media
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大肠杆菌菌株 JM109 (DE3) 的金属化计算器：在 LB 培养基中培养的需氧、厌氧和过氧化氢暴露细胞
DOI：
10.1101/2022.05.06.490408
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发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Foster A
中科院分区：
文献类型：
--
作者：
Foster A
已经生成了三个基于网络的计算器和三个类似的电子表格，根据已知的金属亲和力来预测蛋白质的金属占比。计算利用了对电池内不同金属的不稳定缓冲池的可用性的估计。本文估计了一种大肠杆菌菌株的金属可利用性，这种菌株通常用于分子生物学和生物化学研究，例如重组蛋白的生产。通过定量聚合酶链反应（qPCR）监测一组金属反应转录本的丰度，研究了在Luria-Bertani （LB）培养基中生长的细胞在有氧、厌氧和h2o2条件下的金属可利用性。所选择的基因由dna结合金属传感器调节，这些传感器在相关细菌细胞中进行了热力学表征，使基因表达能够被解读为细胞内金属可用性的函数，表达为形成金属配合物的自由能。计算器将这些值与由金属亲和得到的与目标蛋白质形成络合物的自由能进行比较，以估计蛋白质在细胞内环境中与可交换结合位点竞争的效率。然后，计算器将不同的金属相互竞争，将场地的总占用率限制在最大化学计量1，以输出每种金属的占用率。除了提供这些新的条件计算器之外，本文的最初目的是提供一个教程，讨论这种方法的限制，并介绍在基础研究和应用研究以及下一代制造中利用这种计算器的方法。
Three Web-based calculators, and three analogous spreadsheets, have been generated that predictin vivometal occupancies of proteins based on known metal affinities. The calculations exploit estimates of the availabilities of the labile buffered pools of different metals inside a cell. Here, metal availabilities have been estimated for a strain ofEscherichia colithat is commonly used in molecular biology and biochemistry research, e.g. in the production of recombinant proteins. Metal availabilities have been examined for cells grown in Luria-Bertani (LB) medium aerobically, anaerobically, and in response to H2O2by monitoring the abundance of a selected set of metal-responsive transcripts by quantitative polymerase chain reaction (qPCR). The selected genes are regulated by DNA-binding metal sensors that have been thermodynamically characterized in related bacterial cells enabling gene expression to be read out as a function of intracellular metal availabilities expressed as free energies for forming metal complexes. The calculators compare these values with the free energies for forming complexes with the protein of interest, derived from metal affinities, to estimate how effectively the protein can compete with exchangeable binding sites in the intracellular milieu. The calculators then inter-compete the different metals, limiting total occupancy of the site to a maximum stoichiometry of 1, to output percentage occupancies with each metal. In addition to making these new and conditional calculators available, an original purpose of this article was to provide a tutorial that discusses constraints of this approach and presents ways in which such calculators might be exploited in basic and applied research, and in next-generation manufacturing.