Expression and purification of native and functional influenza A virus matrix 2 proton selective ion channel

Protein Expr Purif. 2017 Mar;131:42-50. doi: 10.1016/j.pep.2016.11.001. Epub 2016 Nov 5.


Influenza A virus displays one of the highest infection rates of all human viruses and therefore represents a severe human health threat associated with an important economical challenge. Influenza matrix protein 2 (M2) is a membrane protein of the viral envelope that forms a proton selective ion channel. Here we report the expression and native isolation of full length active M2 without mutations or fusions. The ability of the influenza virus to efficiently infect MDCK cells was used to express native M2 protein. Using a Calixarene detergents/surfactants based approach; we were able to solubilize most of M2 from the plasma membrane and purify it. The tetrameric form of native M2 was maintained during the protein preparation. Mass spectrometry shows that M2 was phosphorylated in its cytoplasmic tail (serine 64) and newly identifies an acetylation of the highly conserved Lysine 60. ELISA shows that solubilized and purified M2 was specifically recognized by M2 antibody MAB65 and was able to displace the antibody from M2 MDCK membranes. Using a bilayer voltage clamp measurement assay, we demonstrate a pH dependent proton selective ion channel activity. The addition of the M2 ion channel blocker amantadine allows a total inhibition of the channel activity, illustrating therefore the specificity of purified M2 activity. Taken together, this work shows the production and isolation of a tetrameric and functional native M2 ion channel that will pave the way to structural and functional characterization of native M2, conformational antibody development, small molecules compounds screening towards vaccine treatment.

Keywords: Current/voltage; Influenza infection; Lipid bilayer; MDCK expression; Matrix 2 influenza A; Native solubilization; Oligomeric state; Proton selective ion channel; Purification.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Membrane / chemistry
  • Cell Membrane / genetics
  • Cell Membrane / metabolism
  • Dogs
  • Gene Expression*
  • Humans
  • Influenza A Virus, H1N1 Subtype* / chemistry
  • Influenza A Virus, H1N1 Subtype* / genetics
  • Ion Channels* / biosynthesis
  • Ion Channels* / chemistry
  • Ion Channels* / genetics
  • Ion Channels* / isolation & purification
  • Madin Darby Canine Kidney Cells
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Viral Matrix Proteins* / biosynthesis
  • Viral Matrix Proteins* / chemistry
  • Viral Matrix Proteins* / genetics
  • Viral Matrix Proteins* / isolation & purification


  • Ion Channels
  • M2 protein, Influenza A virus
  • Recombinant Proteins
  • Viral Matrix Proteins