Exchange of single amino acids at different positions of a recombinant protein affects metabolic burden in Escherichia coli.
Exchange of single amino acids at different positions of a recombinant protein affects metabolic burden in Escherichia coli.
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DOI：
10.1186/s12934-015-0191-y
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发表时间：
2015-01-23
影响因子：
6.4
通讯作者：
Büchs J
中科院分区：
文献类型：
--
作者：
Rahmen N;Fulton A;Ihling N;Magni M;Jaeger KE;Büchs J
大肠杆菌通常在学术界和工业界用于表达重组蛋白，因为其良好表征的分子遗传学和许多表达载体和菌株的可用性。重组蛋白生产过程中的一个重要问题是所谓的“代谢负荷”：通常为微生物代谢保留的物质和能量，其从细菌中消耗以产生重组蛋白。这种物质和能量的流失损害生物质的形成并改变呼吸作用。据我们所知，迄今为止还没有研究调查重组蛋白中的单个氨基酸交换是否影响代谢负荷现象。因此，在本研究中，15 E.研究了表达融合标签、重组野生型脂肪酶或13种不同脂肪酶变体的coliBL 21（DE 3）克隆，以定量分析在不同位置处的单个氨基酸交换对每个克隆的呼吸、生物量和蛋白质产量的相应影响。因此，应用了两种小规模在线监测系统，即呼吸活动监测系统（拉莫斯）和基于微量滴定板的培养系统（BioLector）。在所有酶变体的表达后，在各个E. coli克隆。观察到两种不同的呼吸行为模式，因此，克隆可分为两组（A型和B型）。评价了解释这些模式的潜在因素（例如质粒拷贝数、包涵体形成）。然而，目前还无法确定任何决定性因素。从OTR曲线可以确定五个不同的培养阶段，该曲线提供关于碳源消耗、生物量和蛋白质产量的实时信息。一般来说，发现产物的量随着活跃呼吸的持续时间而增加。这项工作表明，重组蛋白中的单个氨基酸交换影响蛋白质生产过程中的代谢负荷。小规模在线监测设备拉莫斯和BioLector能够实时检测E.大肠杆菌克隆研究。因此，本研究强调了并行在线监测系统的重要性，以揭示重组蛋白生产的单个氨基酸交换的相关性。本文的在线版本（doi：10.1186/s12934-015-0191-y）包含补充材料，可供授权用户使用。
Escherichia coli is commonly used in academia and industry for expressing recombinant proteins because of its well-characterized molecular genetics and the availability of numerous expression vectors and strains. One important issue during recombinant protein production is the so-called ‘metabolic burden’: the material and energy normally reserved for microbial metabolism which is sapped from the bacterium to produce the recombinant protein. This material and energy drain harms biomass formation and modifies respiration. To the best of our knowledge, no research has investigated so far whether a single amino acid exchange in a recombinant protein affects the metabolic burden phenomenon. Thus, in this study, 15 E. coli BL21(DE3) clones expressing either the fusion tags, a recombinant wild type lipase, or 13 different lipase variants are investigated to quantitatively analyze the respective effects of single amino acid exchanges at different positions on respiration, biomass and protein production of each clone. Therefore, two small-scale online monitoring systems, namely a Respiration Activity MOnitoring System (RAMOS) and a microtiter plate based cultivation system (BioLector) are applied. Upon expression of all enzyme variants, strong variations were found in the Oxygen Transfer Rate (OTR), biomass and protein (lipase) production of the respective E. coli clones. Two distinct patterns of respiration behavior were observed and, so, the clones could be classified into two groups (Type A and B). Potential factors to explain these patterns were evaluated (e.g. plasmid copy number, inclusion body formation). However, no decisive factor could yet be identified. Five distinct cultivation phases could be determined from OTR curves which give real-time information about carbon source consumption, biomass and protein production. In general, it was found that the quantity of product increased with the duration of active respiration. This work demonstrates that single amino acid exchanges in a recombinant protein influence the metabolic burden during protein production. The small-scale online monitoring devices RAMOS and BioLector enable the real-time detection of even smallest differences in respiration behavior, biomass and protein production in the E. coli clones investigated. Hence, this study underscores the importance of parallel online monitoring systems to unveil the relevance of single amino acid exchanges for the recombinant protein production. The online version of this article (doi:10.1186/s12934-015-0191-y) contains supplementary material, which is available to authorized users.
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影响因子：
3.8
作者：
Flores, S;de Anda-Herrera, R;Bolívar, FG
通讯作者：
Bolívar, FG
影响因子：
3.8
作者：
Hoffmann, F;Rinas, U
通讯作者：
Rinas, U
影响因子：
17.3
作者：
Hannig, G;Makrides, SC
通讯作者：
Makrides, SC
影响因子：
3.9
作者：
Anderlei, T;Zang, W;Büchs, J
通讯作者：
Büchs, J
影响因子：
4.1
作者：
Harcum, SW
通讯作者：
Harcum, SW