Phenyl sepharose 6ff column

M2e5x protein was expressed from yeast. In brief, DNA fragment of M2e5x construct was cloned into the pPIC9 vector (Life technology, NY, USA) for secretory expression in the yeast. The recombinant plasmid was transformed into P. pastoris strain GS115, phenotype Mut+ (methanol utilization plus) by electroporation (Life Technologies). P. pastoris transformants were inoculated into BMGY medium (1% yeast extract, 2% peptone, 1.34% YNB, 1%glycerol, 100 mM potassium phosphate, pH 6.0) and incubated at 30°C for 48 h under vigorous agitation (240 rpm). For the induction of the M2e5x protein, the yeast transformants were transferred to BMMY medium (the same components as those of BMGY with glycerol replaced by 0.5% methanol). Methanol was added to a final concentration of 1% (v/v) on the second day and increased to 1.5% (v/v) on the third and fourth days. The culture was kept at 30°C with agitation for 72 h. Then the supernatants were recovered. M2e5x proteins were purified by ion exchange chromatography on Q-Sepharose (GE Healthcare, PA) followed by hydrophobic interaction chromatography on phenyl-Sepharose 6FF column (GE Healthcare, PA).

The gene construct for encoding M2e5x was genetically designed to contain five copies of influenza virus M2e sequences (a2-20) from human influenza virus-SLLTEVETPIRNEWGSRSN (2x), swine influenza virus -SLLTEVETPTRSEWESRSS (1x), avian influenza virus type I-SLLTEVETPTRNEWESRSS (1x), and avian influenza virus type II -SLLTEVETLTRNGWGCRCS (1x), and a tetramerizing leucine zipper domain of GCN4 (De Filette et al., 2008 (link); Kim et al., 2013b (link)). The M2e5x gene was cloned into yeast-expression shuttle vector pPIC9 and expressed in a secreted form through the use of the α-factor mating secretion signal using the yeast P. pastoris system by following the manufacturer’s instructions. Using ion exchange chromatography on Q-Sepharose FF (GE Healthcare, Pittsburgh, PA) followed by hydrophobic interaction chromatography on phenyl-Sepharose 6FF column (GE Healthcare), M2e5x proteins secreted into culture supernatants were purified. The yield of soluble M2e5x proteins purified from 1 L of yeast culture supernatant was approximately 15 mg.