Western University of Health Sciences, Pomona, California, United States of America.
PLoS One. 2021 Nov 22;16(11):e0260143. doi: 10.1371/journal.pone.0260143. eCollection 2021.
The protein aggregation is one of the major challenges of the biotechnological industry, especially in the areas of development and commercialization of successful protein-based drug products. The inherent high aggregation tendency of proteins during various manufacturing processes, storage, and administration has significant impact upon the product quality, safety and efficacy. We have developed an interesting protein purification approach that separates the functionally active protein from inactive aggregates using a detergent concentration gradient. The C-terminally His tagged nucleocapsid protein of Crimean Congo Hemorrhagic fever virus (CCHFV) has high aggregation tendency and rapidly precipitates upon purification by NiNTA chromatography. Using the new purification approach reported here, the freshly purified protein by NiNTA chromatography was further processed using a detergent gradient. In this new purification approach the active protein is retained in the low detergent concentration zone while the inactive aggregates are promptly removed by their rapid migration to the high detergent concentration zone. The method prevented further aggregation and retained the RNA binding activity in the native protein despite numerous freeze thaw cycles. This simple approach prevents protein aggregation by rapidly separating the preformed early aggregates and creating the appropriate microenvironment for correctly folded proteins to retain their biological activity. It will be of potential importance to the biotechnological industry and other fields of protein biochemistry that routinely face the challenges of protein aggregation.
蛋白质聚集是生物技术行业面临的主要挑战之一，尤其是在成功的蛋白质类药物产品的开发和商业化领域。在各种制造过程、储存和给药过程中，蛋白质固有的高聚集倾向对产品质量、安全性和功效有重大影响。我们开发了一种有趣的蛋白质纯化方法，该方法使用去污剂浓度梯度将具有功能活性的蛋白质与无活性的聚集物分离。克里米亚-刚果出血热病毒（CCHFV）的 C 端组氨酸标记核衣壳蛋白（nucleocapsid protein）聚集倾向高，在用 NiNTA 色谱法纯化时会迅速沉淀。使用这里报道的新的纯化方法，用 NiNTA 色谱法刚纯化的蛋白质，进一步用去污剂梯度进行处理。在这种新的纯化方法中，活性蛋白质保留在低去污剂浓度区，而无活性的聚集物则迅速迁移到高去污剂浓度区，从而被迅速去除。该方法防止了进一步的聚集，并在多次冻融循环后保留了天然蛋白质中的 RNA 结合活性。尽管面临蛋白质聚集的挑战，但该简单方法通过快速分离早期形成的预聚集物并为正确折叠的蛋白质创造适当的微环境来保留其生物活性。它将对生物技术行业和其他蛋白质生物化学领域具有潜在的重要意义。
