Department of Neurobiology, University of Utah, Salt Lake City, UT, USA.
Methods Mol Biol. 2022;2303:251-257. doi: 10.1007/978-1-0716-1398-6_20.
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.
糖基磷脂酰肌醇（GPI）锚修饰将脂质锚定在蛋白质的 C 末端，将蛋白质固定在细胞膜上。从这种膜结合状态，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 脂酶活性。由于核心 GPI 结构通过 GPI 脂酶活性与蛋白质一起释放，而通过蛋白水解切割从蛋白质中去除，因此通过与 GPI 锚的核心结构域结合的α毒素（αToxin）进行亲和纯化，可以从培养基中分离出含有 GPI 的蛋白质。以下方法详细介绍了使用 His 标记的α毒素进行 GPI-AP 的亲和纯化的技术，用于鉴定 GPI 锚定蛋白、分析感兴趣的蛋白质的 GPI 锚定状态，或进行后续生化分析的纯化。
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