Rapid purification and analysis of α-synuclein proteins:: C-terminal truncation promotes the conversion of α-synuclein into a protease-sensitive form in Escherichia coli
Rapid purification and analysis of α-synuclein proteins:: C-terminal truncation promotes the conversion of α-synuclein into a protease-sensitive form in Escherichia coli
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DOI：
10.1042/ba20020004
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发表时间：
2002-08-01
影响因子：
2.8
通讯作者：
Rhim, H
中科院分区：
文献类型：
--
作者：
Choi, JY;Sung, YM;Rhim, H
帕金森病（PD）是最常见的神经退行性运动障碍，其特征是多巴胺能神经元的丧失和嗜酸性细胞浆内包涵体的形成，即路易小体。虽然已知a-synuclein是PD发病机制中的关键因素，但其功能仍有待阐明。我们利用pGEX表达系统开发了一种简单快速的纯化a-突触核蛋白的方法，以获得合适形式的a-突触核蛋白，用于其功能的生化研究。野生型α -突触核蛋白在大肠杆菌中过表达，纯化到约为。纯度为80%，收率较高。我们还利用该表达系统研究了α -突触核蛋白不同结构域的表达模式。除了m-synuclein蛋白在氨基酸残基95处被截断外，α -synuclein的所有结构域都以相似的水平纯化，且产量相对较高。出乎意料的是，在大肠杆菌的表达和纯化过程中，α -突触核蛋白c端酸性区氨基酸残基96-140的去除促进了其向蛋白酶敏感形式的转化。我们的研究为研究α -突触核蛋白调控PD发病机制的分子机制提供了一种有用的试剂。
Parkinson's disease (PD) is the most common neurodegenerative movement disorder and is characterized by the loss of dopaminergic neurons and the formation of eosinophilic intracytoplasmic inclusion bodies known as Lewy bodies. Although a-synuclein is known to be a pivotal factor implicated in the pathogenesis of PD, its function remains to be elucidated. We used the pGEX expression system to develop a simple and rapid method for purifying a-synuclein proteins in suitable forms for biochemical studies of their functions. The wild-type alpha-synuclein protein was overexpressed in Escherichia coli and purified to approx. 80% purity with relatively high yields. We also used this expression system to investigate the expression pattern of the various domains of alpha-synuclein. With the exception of the m-synuclein protein that was truncated at amino acid residue 95, all domain constructs of alpha-synuclein were purified at similar levels with relatively high yields. Unexpectedly, removal of amino acid residues 96-140 in the C-terminal acidic region of alpha-synuclein promotes its conversion to a protease-sensitive form during expression and purification in E. coli. Our study suggests a method for generating useful reagents to investigate the molecular mechanism by which alpha-synuclein regulates the pathogenesis of PD.