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Protein Expr Purif. 2016 Nov 4. pii: S1046-5928(16)30368-0. doi: 10.1016/j.pep.2016.11.001. [Epub ahead of print]
Expression and purification of native and functional influenza A virus matrix 2 proton selective ion channel.
Desuzinges Mandon E1, Traversier A2, Champagne A3, Benier L1, Audebert S4, Balme S5, Dejean E1, Rosa Calatrava M6, Jawhari A7.
Author information
Abstract
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.
Copyright ? 2016. Published by Elsevier Inc.
KEYWORDS:
Current/voltage; Influenza infection; Lipid bilayer; MDCK expression; Matrix 2 influenza A; Native solubilization; Oligomeric state; Proton selective ion channel; Purification
PMID: 27825980 DOI: 10.1016/j.pep.2016.11.001
[PubMed - as supplied by publisher]
Expression and purification of native and functional influenza A virus matrix 2 proton selective ion channel.
Desuzinges Mandon E1, Traversier A2, Champagne A3, Benier L1, Audebert S4, Balme S5, Dejean E1, Rosa Calatrava M6, Jawhari A7.
Author information
Abstract
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.
Copyright ? 2016. Published by Elsevier Inc.
KEYWORDS:
Current/voltage; Influenza infection; Lipid bilayer; MDCK expression; Matrix 2 influenza A; Native solubilization; Oligomeric state; Proton selective ion channel; Purification
PMID: 27825980 DOI: 10.1016/j.pep.2016.11.001
[PubMed - as supplied by publisher]