Removal of phospholipid contaminants through precipitation of glycosylphosphatidylinositols

Abstract

Parasitic glycosylphosphatidylinositols (GPIs) are thought to be involved in induced cell signaling that leads to proinflammatory responses. Increasing interest in elucidation of the mechanisms involved in signaling pathways drives the finding of rapid and reliable methods to purify GPIs. GPIs are usually extracted using mixtures of chloroform/methanol/water, followed by a phase partition between water and water-saturated n-butanol. GPIs recovered in the butanol phase are separated by thin-layer chromatography, scraped, eluted from the silica, and used for studying the structure–function relationship. The presence of phospholipid contaminants or other hydrophobic components in the samples cannot be excluded. Furthermore, the standard procedures to purify GPIs harbor several drawbacks, including the need to handle large amounts of culture, poor yields, time-consuming, and interfering contaminants. Here we report on the development of a simple and reliable method to isolate and purify both free and bound GPIs from one cell pellet. We exploited the low solubility of GPIs in water-saturated n-butanol to remove the phospholipid contaminants completely. After delipidation, GPI proteins were solubilized from the pellet using a mixture of organic solvent containing ethanol and water.