Abstract

Protein aggregation and amyloid formation are associated with multiple human diseases, but are also a problem in protein production. Understanding how aggregation can be modulated is therefore of importance in both medical and industrial contexts. We have used bovine insulin as a model protein to explore how amyloid formation is affected by buffer pH and by the addition of short-chain alcohols. We find that bovine insulin forms amyloid fibrils, albeit with different rates and resulting fibril morphologies, across a wide pH range (2–7). At pH 4.0, bovine insulin displayed relatively low aggregation propensity in combination with high solubility; this condition was therefore chosen as basis for further exploration of how bovine insulin’s native state can be stabilized in the presence of short-chain alcohols that are relevant because of their common use as eluents in industrial-scale chromatography purification. We found that ethanol and isopropanol are efficient modulators of bovine insulin aggregation, providing a three to four times retardation of the aggregation kinetics at 30–35% (vol/vol) concentration; we attribute this to the formation of oligomers, which we detected by AFM. We discuss this effect in terms of reduced solvent polarity and show, by circular dichroism recordings, that a concomitant change in α-helical packing of the insulin monomer occurs in ethanol. Our results extend current knowledge of how insulin aggregates, and may, although bovine insulin serves as a simplistic model, provide insights into how buffers and additives can be fine-tuned in industrial production of proteins in general and pharmaceutical insulin in particular.

中文翻译：

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牛胰岛素的淀粉样蛋白形成在中等酸性的pH和通过添加短链醇被延迟

摘要

蛋白质聚集和淀粉样蛋白形成与多种人类疾病有关，但也是蛋白质生产中的问题。因此，在医学和工业环境中，了解如何调节聚集都很重要。我们已使用牛胰岛素作为模型蛋白来研究淀粉样蛋白的形成如何受到缓冲液pH和添加短链醇的影响。我们发现，在宽的pH范围内（2-7），牛胰岛素会形成淀粉样蛋白原纤维，尽管速率不同，从而形成原纤维形态。在pH 4.0时，牛胰岛素显示出较低的聚集倾向以及高溶解度。因此，选择该条件作为进一步探索在存在短链醇的情况下如何稳定牛胰岛素天然状态的基础，而短链醇由于在工业规模色谱纯化中通常用作洗脱剂而具有重要意义。我们发现乙醇和异丙醇是牛胰岛素聚集的有效调节剂，在30-35％（vol / vol）浓度下提供了三到四倍的聚集动力学迟滞。我们将其归因于由AFM检测到的低聚物形成。我们根据降低的溶剂极性来讨论这种效果，并通过圆二色性记录显示，在乙醇中发生了胰岛素单体的α螺旋堆积的伴随变化。我们的结果扩展了有关胰岛素如何聚集的当前知识，并且可能，