Isolation of Glycosomes from Trypanosoma brucei.

Glycosomes, belonging to the sub-class of peroxisomes, are single-membrane-bound organelles of trypanosomatid parasites. Glycosomes compartmentalize mainly glycolytic and other essential metabolic pathways such as gluconeogenesis, pentose phosphate pathway, sugar nucleotide biosynthesis, etc. Since glycosomes are parasite-specific and their biogenesis is essential for the parasite survival, they have attracted a lot of interest over the years. Understanding the glycosomal enzyme composition and machinery involved in the biogenesis of this organelle requires the knowledge of the glycosomal proteome. Here we describe a method to isolate highly purified glycosomes and further enrichment of the glycosomal membrane proteins from the pro-cyclic form of Trypanosoma brucei. The isolation method is based on the controlled rupture of the cells by silicon carbide, followed by the differential centrifugation, and density gradient centrifugation. Further, the glycosomal membrane proteins are enriched from the purified glycosomes by the successive treatments with low-salt, high-salt, and alkaline carbonate buffer extractions.