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

STAR Protoc. 2021 Dec 29;3(1):101068. doi: 10.1016/j.xpro.2021.101068. eCollection 2022 Mar 18.

Abstract

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).

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

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Bacterial Toxins
  • Brain / metabolism
  • Cholesterol* / metabolism
  • Hemolysin Proteins* / genetics
  • Humans
  • Liver / metabolism
  • Mice

Substances

  • Bacterial Toxins
  • Hemolysin Proteins
  • Clostridium perfringens theta-toxin
  • Cholesterol