Affinity Purification of Glycosylphosphatidylinositol-anchored Proteins by Alpha-Toxin.
Affinity Purification of Glycosylphosphatidylinositol-anchored Proteins by Alpha-Toxin.
复制标题
通过 Alpha-Toxin 亲和纯化糖基磷脂酰肌醇锚定蛋白。
DOI：
10.1007/978-1-0716-1398-6_20
复制
发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Park,Sungjin
中科院分区：
文献类型：
--
作者：
Huang,Kevin;Park,Sungjin
糖基磷脂酰肌醇（GPI）锚定修饰将脂质锚定在蛋白质的c端，将蛋白质拴在细胞膜表面。在这种膜结合状态下，GPI锚定蛋白（GPI- aps）可以通过多种机制释放到细胞外空间，包括蛋白水解脱落和GPI脂肪酶活性。由于核心GPI结构通过GPI脂肪酶活性与蛋白质共同释放，而通过蛋白水解裂解从蛋白质中去除，因此通过α毒素（α毒素）结合GPI锚定的核心结构域进行亲和纯化，从培养基中分离出含GPI的蛋白质。下面的方法详细介绍了使用his标记的α毒素对GP-APs进行亲和纯化的技术，用于鉴定gpi锚定蛋白，分析感兴趣的蛋白质的gpi锚定状态，或纯化用于随后的生化分析。
The glycosylphosphatidylinositol (GPI)-anchor modification attaches a lipid anchor to the C-terminus of a protein, tethering the protein to the cell surface membrane. From this membrane-bound state, GPI-anchored proteins (GPI-APs) can be released into the extracellular space by multiple mechanisms, including proteolytic shedding and GPI lipase activity. Since the core GPI structure is co-released with the protein by GPI lipase activity, while removed from the protein by proteolytic cleavage, affinity purification by alpha-toxin (αToxin), which binds to the core domain of the GPI-anchor, isolates GPI-containing proteins from the culture medium. The following method details a technique for affinity purification of GP-APs using His-tagged αToxin for identification of GPI-anchored proteins, analysis of the GPI-anchor status of a protein of interest, or purification for subsequent biochemical analysis.