GST-Perfringolysin O production for the localization and quantification of membrane cholesterol in human and mouse brain and liver

Abnormal cholesterol metabolism is linked to many neurodegenerative disorders. Here, we present a protocol for the production of a recombinant protein consisting of a Glutathione-S-Transferase tag fused with the Perfringolysin O (PFO). The GST-PFO tag enables analysis of the localization of cholesterol in subcellular membranes of human and mice brain and liver tissues. We have used this approach for samples from Niemann-Pick type C disease and non-alcoholic steatohepatitis models. The construct may also have applications for the diagnosis of cholesterol-accumulating disorders. For complete details on the use and execution of this protocol, please refer to Kwiatkowska et al. (2014).

Biotechnology and bioengineering, Cell Biology, Cell Membrane, Health Sciences, Metabolism, Microscopy, Model Organisms, Molecular Biology, Molecular/Chemical Probes, Neuroscience, Protein expression and purification