Abstract
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.
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Acknowledgments
GFP-αToxin-His plasmid is a gift from Dr. Yeongjin Hong (Chonnam National University Medical Center, Gwangju, Korea). This work was supported by National Institute of Neurological Disorders and Stroke (R01NS102444) to S.P.
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Huang, K., Park, S. (2022). Affinity Purification of Glycosylphosphatidylinositol-anchored Proteins by Alpha-Toxin . In: Balagurunathan, K., Nakato, H., Desai, U., Saijoh, Y. (eds) Glycosaminoglycans. Methods in Molecular Biology, vol 2303. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1398-6_20
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DOI: https://doi.org/10.1007/978-1-0716-1398-6_20
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