A modification of the split-tobacco etch virus method for monitoring interactions between membrane proteins in mammalian cells

Anal Biochem. 2012 Apr 1;423(1):109-18. doi: 10.1016/j.ab.2012.01.022. Epub 2012 Jan 31.

Abstract

Despite progress in the development of methods to monitor protein interactions, studies of interactions between membrane proteins in mammalian cells remain challenging. Protein complementation assays (PCAs) are commonly used to study interactions between proteins due to their simplicity. They are based on interaction-mediated reconstitution of a reporter protein, which can be easily monitored. Recently, a protein complementation method named split-TEV (tobacco etch virus) has been developed and is based on the functional reconstitution of TEV protease and subsequent proteolytic-mediated activation of reporters. In this work, we have developed a modification of the split-TEV method to study the interactions between membrane proteins with increased specificity. This assay was validated by addressing the interactions between different membrane proteins, including G protein-coupled receptors (GPCRs) and ion channels. By comparing it with another PCA, we found that this new method showed a higher sensitivity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biological Assay / methods*
  • Endopeptidases / metabolism*
  • HeLa Cells
  • Humans
  • Luminescent Measurements*
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Potyvirus / enzymology
  • Protein Interaction Mapping
  • Receptors, Dopamine D2 / genetics
  • Receptors, Dopamine D2 / metabolism
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism

Substances

  • Membrane Proteins
  • Receptors, Dopamine D2
  • Recombinant Fusion Proteins
  • Endopeptidases
  • TEV protease