Vera Andrea, González-Montalbán Nuria, Arís Anna, Villaverde Antonio
Institut de Biotecnologia i de Biomedicina, Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain.
Biotechnol Bioeng. 2007 Apr 15;96(6):1101-6. doi: 10.1002/bit.21218.
Protein aggregation is a major bottleneck during the bacterial production of recombinant proteins. In general, the induction of gene expression at sub-optimal growth temperatures improves the solubility of aggregation-prone polypeptides and minimizes inclusion body (IB) formation. However, the effect of low temperatures on the quality of the recombinant protein, especially within the insoluble cell fraction, has been hardly ever explored. In this work, we have examined the conformational status of a recombinant GFP protein when produced in Escherichia coli below 37 degrees C. As expected, the fraction of aggregated protein largely decreased at lower temperatures, while the conformational quality of both soluble and aggregated GFP, as reflected by its specific fluorescence emission, progressively improved. This observation indicates that physicochemical conditions governing protein folding affect concurrently the quality of the soluble and the aggregated forms of a misfolding-prone protein, and that protein misfolding and aggregation are clearly not coincident events.
蛋白质聚集是重组蛋白细菌生产过程中的一个主要瓶颈。一般来说，在次优生长温度下诱导基因表达可提高易聚集多肽的溶解度，并将包涵体（IB）形成降至最低。然而，低温对重组蛋白质量的影响，尤其是对不溶性细胞部分的影响，几乎从未被研究过。在这项工作中，我们研究了重组绿色荧光蛋白（GFP）在大肠杆菌中于37摄氏度以下生产时的构象状态。正如预期的那样，聚集蛋白的比例在较低温度下大幅下降，而可溶性和聚集态GFP的构象质量，由其特定荧光发射反映，逐渐提高。这一观察结果表明，控制蛋白质折叠的物理化学条件同时影响易错误折叠蛋白的可溶性和聚集态形式的质量，并且蛋白质错误折叠和聚集显然不是同时发生的事件。
