The evaluation of the factors that cause aggregation during recombinant expression in E. coli is simplified by the employment of an aggregation-sensitive reporter.
The evaluation of the factors that cause aggregation during recombinant expression in E. coli is simplified by the employment of an aggregation-sensitive reporter.
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DOI：
10.1186/1475-2859-5-28
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发表时间：
2006-09-01
影响因子：
6.4
通讯作者：
de Marco, Ario
中科院分区：
文献类型：
--
作者：
Schultz, Tina;Martinez, Lucia;de Marco, Ario
在细菌中表达的可溶性重组蛋白的产率通常很低，这是由于异源蛋白倾向于形成聚集体。因此，已经设想了聚集报告基因以简化不同表达条件之间的比较，并加速鉴定改善溶解度的合适方案。我们使用的探针由IbpAB启动子组成，该启动子被融合到编码β-半乳糖苷酶的序列的蛋白质聚集体特异性激活，因此，其活性成为聚集程度的指示。所收集的数据表明，该探针在实验条件范围内的重现性方面是可靠的，并且比基于SDS-PAGE和连续western blot的分析方法更快和更灵敏。β-半乳糖苷酶探针可用于确定哪些参数会影响模型蛋白的聚集，并建立优化的表达方案。根据β-半乳糖苷酶活性评估生长温度、诱导方式、与分子伴侣共表达以及添加渗透调节剂对聚集体积累的影响。有趣的是，在估计的聚集减少和可溶性蛋白质的较高产量之间观察到显著的相关性。我们还比较了一组具有各种调控功能的表达载体，发现单个特征，如启动子、拷贝数或聚合酶，与控制重组蛋白聚集无关，而导致的关键因素是系统的总表达率。在我们的实验中使用的聚集报告代表了一个有用的工具，以评估不同的因素，可以调节优化重组表达方案。此外，聚集度的快速估计使得能够将其与导致稀少的重组产量的其他原因区分开。
The yields of soluble recombinant proteins expressed in bacteria are often low due to the tendency of the heterologous proteins to form aggregates. Therefore, aggregation reporters have been envisaged to simplify the comparison among different expression conditions and to speed up the identification of suitable protocols that improve the solubility. The probe we used is composed by an IbpAB promoter specifically activated by protein aggregates fused to a sequence coding the β-galactosidase, the activity of which becomes, therefore, indicative of the aggregation degree. The collected data show that the probe is reliable in terms of reproducibility inside a range of experimental conditions and faster and more sensitive than the analysis methods based on SDS-PAGE and successive western blot. The β-galactosidase probe was useful to identify which parameters could influence the aggregation of the model proteins and to set up an optimized expression protocol. The effect of growth temperature, induction modality, co-expression with molecular chaperones and addition of osmolytes on the accumulation of aggregates were evaluated following the β-galactosidase activity. Interestingly, a significant correlation was observed between estimated decreased aggregation and higher yields of soluble protein. We also compared a set of expression vectors with various regulative features and found that the single characteristics, like promoter, copy number or polymerase, were not relevant for controlling the recombinant protein aggregation whilst the crucial factor resulted being the total expression rate of the system. The aggregation reporter used in our experiments represents a useful tool to evaluate the different factors that can be modulated to optimize a recombinant expression protocol. Furthermore, the rapid estimation of the aggregation degree enables to discriminate this from other causes responsible for scarce recombinant yields.
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影响因子：
4.1
作者：
de Marco, A;De Marco, V
通讯作者：
De Marco, V
影响因子：
3.5
作者：
Fox, JD;Routzahn, KM;Waugh, DS
通讯作者：
Waugh, DS
影响因子：
2.9
作者：
Vincentelli, R;Canaan, S;Bignon, C
通讯作者：
Bignon, C
影响因子：
8
作者：
Hammarström, M;Hellgren, N;Härd, T
通讯作者：
Härd, T
影响因子：
3.8
作者：
de Marco, A;Vigh, L;Goloubinoff, P
通讯作者：
Goloubinoff, P