The isolation of peroxisomes

Abstract

Peroxisomes are subcellular organelles bound by a single membrane. They are involved in a variety of metabolic processes such as ß-oxidation of very long chain fatty acids. Isolated and purified peroxisomes are necessary to obtain a detailed understanding of their structure and function. Here, our three protocols for the isolation of peroxisomes from rat liver, rat hepatoma H4IIE cells and yeast Komagataella phaffii (previously called Pichia pastoris) are presented. Highly pure peroxisomes can be prepared from rat liver by differential centrifugation followed by Nycodenz gradient centrifugation. It is difficult to prepare highly purified peroxisomes from cultured mammalian cells, but the subcellular fractionation of peroxisomes is still a powerful tool to analyze whether a certain protein is localized to peroxisomes. Peroxisomes are potently induced in methylotrophic yeast such as K. phaffii. A large amount of mammalian peroxisomal proteins can be expressed in cells under the control of the alcohol oxidase gene promoter in a medium containing methanol as the only carbon source. Therefore, purified peroxisomes are useful for characterizing mammalian peroxisomal proteins. The protocols for the isolation of peroxisomes by immuno-beads and peroxisomal membranes by a sodium carbonate procedure are also presented.