Kamboj Sumaer, Harms Chase, Kumar Lokender, Creamer Daniel, West Colista, Klein-Seetharaman Judith, Sarkar Susanta K
Department of Physics, Colorado School of Mines, Golden, CO 80401, USA.
Department of Chemistry, Colorado School of Mines, Golden, CO 80401, USA.
Heliyon. 2021 Jan 11;7(1):e05874. doi: 10.1016/j.heliyon.2020.e05874. eCollection 2021 Jan.
Research has implicated alpha-synuclein (aSyn) in pathological protein aggregation observed in almost all patients with Parkinson's disease and more than 50% of patients with Alzheimer's disease. An easy and inexpensive method of purifying aSyn and developing an model system of Lewy body formation would enhance basic biomedical research. We report aSyn purification technique that leverages the amyloidogenic property of aSyn suitable for purifying monomeric aSyn without chromatography and denaturing agents. We expressed full-length and untagged aSyn in Rosetta(DE3) pLysS and purified ~60 μg of aSyn from 500 mL culture within 24 h. After IPTG-induced expression of aSyn in , we disrupted the cells with a sonicator. We centrifuged the cell lysate in a 15 mL tube, which leads to aSyn-induced aggregation of native proteins. After removing aggregates, centrifugation in a 30 kDa cut-off filter followed by a 10 kDa cut-off filter led to purified water-soluble aSyn. The identity of aSyn was confirmed by Western blot using anti-aSyn antibody and Edman sequencing. Its mass was determined to be 14.6 kDa using a MALDI TOF-MS mass spectrometer. The majority of aSyn led to water-suspended (as opposed to precipitated) aggregation of proteins with visible fibrous structures. The broad-spectrum binding and amyloidogenic property of aSyn is thus not only useful for inexpensive aSyn production for diverse applications, but it also expands studying its possible roles in human physiology. The aggregate of proteins induced by aSyn during the purification process may serve as a Lewy body model.
研究表明，α-突触核蛋白（aSyn）与几乎所有帕金森病患者以及超过50%的阿尔茨海默病患者中观察到的病理性蛋白质聚集有关。一种简单且廉价的纯化aSyn并建立路易小体形成模型系统的方法将加强基础生物医学研究。我们报告了一种aSyn纯化技术，该技术利用了aSyn的淀粉样蛋白生成特性，适用于在不使用色谱法和变性剂的情况下纯化单体aSyn。我们在Rosetta(DE3) pLysS中表达全长且无标签的aSyn，并在24小时内从500毫升培养物中纯化出约60微克的aSyn。在用异丙基-β-D-硫代半乳糖苷（IPTG）诱导aSyn表达后，我们用超声破碎仪裂解细胞。我们将细胞裂解物在一个15毫升的试管中离心，这导致了aSyn诱导的天然蛋白质聚集。去除聚集体后，在截留分子量为30 kDa的滤器中离心，然后在截留分子量为10 kDa的滤器中离心，得到纯化的水溶性aSyn。使用抗aSyn抗体的蛋白质免疫印迹法和埃德曼测序法确认了aSyn的身份。使用基质辅助激光解吸电离飞行时间质谱仪（MALDI TOF-MS）测定其质量为14.6 kDa。大多数aSyn导致具有可见纤维结构的蛋白质形成水悬浮（而非沉淀）聚集。因此，aSyn的广谱结合和淀粉样蛋白生成特性不仅有助于以低成本生产用于各种应用的aSyn，还扩展了对其在人类生理学中可能作用的研究。在纯化过程中由aSyn诱导的蛋白质聚集体可作为路易小体模型。
