Extracellular vesicles as a platform to study cell-surface membrane proteins

Methods. 2020 Aug 1;180:35-44. doi: 10.1016/j.ymeth.2020.03.004. Epub 2020 Mar 7.

Abstract

Producing intact recombinant membrane proteins for structural studies is an inherently challenging task due to their requirement for a cell-lipid environment. Most of the procedures developed involve isolating the protein by solubilization with detergent and further reconstitutions into artificial membranes. These procedures are highly time consuming and suffer from further drawbacks, including low yields and high cost. We describe here an alternative method for rapidly obtaining recombinant cell-surface membrane proteins displayed on extracellular vesicles (EVs) derived from cells in culture. Interaction between these membrane proteins and ligands can be analyzed directly on EVs. Moreover, EVs can also be used for protein structure determination or immunization purposes.

Keywords: Antibody generation; Extracellular vesicle; Immunization; Membrane protein; Protein-ligand interaction.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 5'-Nucleotidase / immunology
  • Cloning, Molecular
  • Cryoelectron Microscopy
  • Detergents / chemistry
  • Dynamic Light Scattering
  • Extracellular Vesicles / immunology
  • Extracellular Vesicles / metabolism*
  • Extracellular Vesicles / ultrastructure
  • GPI-Linked Proteins / immunology
  • HEK293 Cells
  • Humans
  • Ligands
  • Mass Spectrometry
  • Membrane Proteins / genetics
  • Membrane Proteins / isolation & purification*
  • Membrane Proteins / metabolism
  • Microscopy, Electron
  • Plasmids / genetics
  • Recombinant Proteins / isolation & purification*

Substances

  • Detergents
  • GPI-Linked Proteins
  • Ligands
  • Membrane Proteins
  • Recombinant Proteins
  • 5'-Nucleotidase
  • NT5E protein, human