Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain.
Microb Cell Fact. 2011 Oct 12;10:79. doi: 10.1186/1475-2859-10-79.
The effects and effectiveness of the chaperone pair GroELS on the yield and quality of recombinant polypeptides produced in Escherichia coli are matter of controversy, as the reported activities of this complex are not always consistent and eventually indicate undesired side effects. The divergence in the reported data could be due, at least partially, to different experimental conditions in independent research approaches.
We have then selected two structurally different model proteins (namely GFP and E. coli β-galactosidase) and two derived aggregation-prone fusions to explore, in a systematic way, the eventual effects of GroELS co-production on yield, solubility and conformational quality. Host cells were cultured at two alternative temperatures below the threshold at which thermal stress is expected to be triggered, to minimize the involvement of independent stress factors.
From the analysis of protein yield, solubility and biological activity of the four model proteins produced alone or along the chaperones, we conclude that GroELS impacts on yield and quality of aggregation-prone proteins with intrinsic determinants but not on thermally induced protein aggregation. No effective modifications of protein solubility have been observed, but significant stabilization of small (encapsulable) substrates and moderate chaperone-induced degradation of larger (excluded) polypeptides. These findings indicate that the activities of this chaperone pair in the context of actively producing recombinant bacteria discriminate between intrinsic and thermally-induced protein aggregation, and that the side effects of GroELS overproduction might be determined by substrate size.
伴侣蛋白 GroELS 对大肠杆菌中重组多肽产量和质量的影响和效果存在争议，因为该复合物的报道活性并不总是一致的，最终表明存在不良的副作用。报告数据的差异至少部分归因于独立研究方法中不同的实验条件。
我们选择了两种结构不同的模型蛋白（即 GFP 和大肠杆菌β-半乳糖苷酶）和两种衍生的易于聚集的融合蛋白，以系统地探索 GroELS 共表达对产量、可溶性和构象质量的潜在影响。在低于预计会引发热应激的温度下培养宿主细胞，以最小化独立应激因素的参与。
从单独或与伴侣蛋白一起生产的四种模型蛋白的蛋白产量、可溶性和生物活性分析中，我们得出结论，GroELS 对具有内在决定因素的易聚集蛋白的产量和质量有影响，但对热诱导的蛋白聚集没有影响。没有观察到蛋白质可溶性的有效修饰，但对较小（可封装）底物有显著的稳定作用，并且对较大（被排除）多肽有适度的伴侣诱导降解。这些发现表明，在积极生产重组细菌的情况下，这种伴侣蛋白对内在和热诱导的蛋白聚集具有区分能力，并且 GroELS 过表达的副作用可能由底物大小决定。
