Kta start fplc system

The expression and purification procedure of recombinant PPX_Δ12 was performed as described previously (21 (link)). In brief, DH5α competent E. coli cells were cultured at 37°C on an orbital shaker after transformation with plasmid DNA (pTrcHisB backbone) coding for PPX_Δ12. Induction of protein expression through the pTrc promoter was achieved by addition of 0.5 mM isopropylthio-β-D-galactoside (Sigma-Aldrich). After lysis of cells by sonication, protein purification was performed using an ÄKTA™ start FPLC system (GE Healthcare) connected to a 1 ml HisTrap FF crude column (GE Healthcare). Histidine-tagged proteins were eluted with a buffer containing 20 mM NaH₂PO₄, 500 mM NaCl and 500 mM imidazole. The buffer of the elution fractions was changed to PBS, pH 7.4.

The PEG-precipitated RSV resuspended in NT was filtered through an ÄKTA Start FPLC system (GE Healthcare Life Sciences, Mississauga, ON, Canada) stored within a biosafety cabinet. The instrument was equipped with a HiTrap Capto Core 700 column (Cat. No. 17-5481-51 GE Healthcare Life Sciences) with a 1 mL bed volume. The matrix beads of the column bind small impurities while allowing large molecules to pass through the column (MW > 700,000). ÄKTA Running Buffer A was comprised of NT buffer +1% sucrose, pH 7.4, 0.2 μm filtered, and ÄKTA Elution Buffer B was 1N sodium hydroxide in 30% isopropanol, 0.2 μm filtered. The system and column were washed with sterile filtered distilled water at 5 mL/min. The column was primed and equilibrated with 5 mL of Buffer A running at 1 mL/min. The 2-mL injection loop was flushed with buffer A and then loaded with the PEG-precipitated virus resuspended in NT buffer. Fractionation was set to collect 1 mL fractions as soon as the sample was injected onto the column at 0.5 mL/min, collecting 5 mL total throughout the absorbance peak at 250 nm. Isocratic elution was set at 100% Buffer B to remove the impurities bound to the column to allow for re-use. The column was stored at 4 °C in 20% ethanol.