A method of purifying alpha-synuclein in E. coli without chromatography.
A method of purifying alpha-synuclein in E. coli without chromatography.
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DOI：
10.1016/j.heliyon.2020.e05874
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发表时间：
2021-01
期刊：
影响因子：
4
通讯作者：
Sarkar SK
中科院分区：
文献类型：
--
作者：
Kamboj S;Harms C;Kumar L;Creamer D;West C;Klein-Seetharaman J;Sarkar SK
研究表明，α-突触核蛋白(ASyn)参与了几乎所有帕金森病患者和超过50%的阿尔茨海默病患者的病理性蛋白质聚集。一种简单、廉价的纯化aSyn的方法和开发一种路易体形成的体外模型系统将加强基础生物医学研究。我们报道了一种Syn纯化技术，该技术利用了aSyn的淀粉样变性特性，适合于在没有层析和变性剂的情况下纯化单体aSyn。我们在Rosetta(DE3)pLysS中表达了全长的、未加标签的aSyn，并在24 h内从500毫升的培养物中纯化了约60μg的aSyn。我们将细胞裂解物放在15毫升的试管中离心，这导致了Syn诱导的天然大肠杆菌蛋白质的聚集。去除聚集体后，先用30 kDa截滤器离心，再用10 kDa截滤器离心，得到纯化的水溶性aSyn。用抗aSyn抗体进行Western印迹和Edman测序证实了aSyn的同源性。用MALDI TOF-MS质谱仪测定其质量为14.6 kDa。大多数aSyn导致具有可见纤维结构的大肠杆菌蛋白质的水悬浮(而不是沉淀)聚集。因此，aSyn的广谱结合和淀粉样变特性不仅有助于廉价生产各种应用的aSyn，而且还扩大了对其在人体生理学中可能作用的研究。在纯化过程中，aSyn诱导的大肠杆菌蛋白聚集体可作为路易体模型。重组人α-突触核蛋白，α-突触核蛋白诱导的聚集，路易体模型系统，蛋白质印迹。
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 in vitro 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 E. coli, 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 E. coli 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 E. coli 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 E. coli proteins induced by aSyn during the purification process may serve as a Lewy body model. Recombinant human alpha-synuclein, aggregation induced by alpha-synuclein, Lewy body model system, Western blot.
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影响因子：
82.9
作者：
Conway, KA;Harper, JD;Lansbury, PT
通讯作者：
Lansbury, PT
影响因子：
14
作者：
通讯作者：
--
影响因子：
2.8
作者：
Choi, JY;Sung, YM;Rhim, H
通讯作者：
Rhim, H
帕金森氏病的患病率在北美。
DOI：
10.1038/s41531-018-0058-0
发表时间：
2018
期刊：
NPJ Parkinson's disease
影响因子：
--
作者：
Marras C;Beck JC;Bower JH;Roberts E;Ritz B;Ross GW;Abbott RD;Savica R;Van Den Eeden SK;Willis AW;Tanner CM;Parkinson’s Foundation P4 Group
通讯作者：
Parkinson’s Foundation P4 Group
影响因子：
3.5
作者：
JAKES, R;SPILLANTINI, MG;GOEDERT, M
通讯作者：
GOEDERT, M