Recombinant PvGAMA-Ecto Protein Expression

The recombinant protein PvGAMA-Ecto domain comprising amino acid 22–771 was expressed and purified as previously described [13 (link)]. Briefly, the PVX_088910 fragments encoding PvGAMA-ectodomain and a hexahistidine (His) tag at the C-terminus, were amplified using sense primers with XhoI sites and antisense primers with BamHI restriction sites. The amplified fragments were then restricted and ligated into the WGCF expression vector pEU-E01-His-TEV-MCS (CellFree Sciences, Matsuyama, Japan). The cloned inserts were sequenced using an ABI 3700 Genetic Analyzer (Genotech, Daejeon, Korea). The recombinant proteins with His tags were expressed using a WGCF system (CellFree Sciences) and purified using a Nickel-Sepharose column (GE Healthcare Life Sciences, Uppsala, Sweden).