Schultz Tina, Martinez Lucia, de Marco Ario
EMBL Scientific Core Facilities, Meyerhofstr, 1, D-69117, Heidelberg, Germany.
Microb Cell Fact. 2006 Sep 1;5:28. doi: 10.1186/1475-2859-5-28.
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 beta-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 beta-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 beta-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.
由于异源蛋白易于形成聚集体，细菌中表达的可溶性重组蛋白产量往往较低。因此，设想使用聚集报告基因来简化不同表达条件之间的比较，并加速鉴定可提高溶解度的合适方案。我们使用的探针由一个IbpAB启动子组成，该启动子由与编码β-半乳糖苷酶的序列融合的蛋白聚集体特异性激活，因此其活性可指示聚集程度。
收集的数据表明，该探针在一系列实验条件下具有良好的可重复性，并且比基于SDS-PAGE和后续蛋白质印迹的分析方法更快、更灵敏。β-半乳糖苷酶探针有助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。通过β-半乳糖苷酶活性评估了生长温度、诱导方式、与分子伴侣共表达以及添加渗透剂对聚集体积累的影响。有趣的是，观察到估计的聚集减少与可溶性蛋白产量增加之间存在显著相关性。我们还比较了一组具有不同调控特征的表达载体，发现启动子、拷贝数或聚合酶等单一特征与控制重组蛋白聚集无关，而关键因素是系统的总表达率。
我们实验中使用的聚集报告基因是评估可调节以优化重组表达方案的不同因素的有用工具。此外，聚集程度的快速估计能够将其与导致重组产量低的其他原因区分开来。
