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Expression, purification and functional characterization of influenza hemagglutinin II (HA2) protein containing transmembrane (TM) domain and ectodomain (781.1)
Author(s) -
Ratnayake Punsisi Upeka,
Weliky David
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.781.1
Subject(s) - ectodomain , lipid bilayer fusion , hemagglutinin (influenza) , transmembrane domain , fusion protein , circular dichroism , transmembrane protein , endosome , chemistry , lipid bilayer , biophysics , protein folding , biology , microbiology and biotechnology , biochemistry , virus , membrane , virology , recombinant dna , receptor , gene
Developing better vaccines against influenza requires investigating the viral infection process. Influenza hemagglutinin protein consists of two polypeptide chains HA1 and HA2. Conformational changes occurring in HA2 subunit initiate the fusion of viral membrane to endosomal membrane. The objective of this project was to assess the structure and function of the HA2 transmembrane domain while it is embedded in the lipid bilayer, during viral fusion process. Hydrophobicity and limited membrane space are two factors that limit membrane protein expression in cells. Therefore, expression of nonnative proteins in E‐coli causes formation of inclusion bodies. HA2 consisting of ectodomain and different lengths of TM without fusion peptide (FP) were expressed. Next, the proteins were purified from inclusion bodies followed by refolding. Also, HA2 ectodomain with and without FP were expressed and purified. Folding of all proteins was verified using circular dichroism spectroscopy. The activities of various proteins were analyzed using a lipid mixing assays at pH 5.0 and pH 7.0. No significance difference in the fusion activity was observed at pH 5.0 between the constructs with different lengths of TM domain. However, in the presence of FP, a significant increase of fusion activity was observed leading to conclude that fusion peptide is the main contributor in the fusion process. Grant Funding Source : Supported by National Institute Health (NIH) research grant 5R01AI047153‐13