Hitrap capto sp impres column

Plasmids encoding prefusion RSV F (DS-Cav1), L141W, and D489Y variants and post-fusion (F ΔFP) proteins based on strain A2 were transfected into FreeStyle 293 F and Expi293 cells, respectively (Invitrogen, Carlsbad, CA, USA)^{32 (link), 34 (link)}. Proteins were expressed in the presence of kifunensine (5 μM) and were purified over Strep-Tactin resin (IBA, Goettingen, Germany). Prefusion RSV F (PRDM) protein^{36 (link)} for DSF studies was purified from transiently transfected Expi293 cells using a two-step purification protocol including cation-exchange chromatography at pH 5.0 (HiTrap Capto SP ImpRes column; GE Healthcare Biosciences, Pittsburgh, PA, USA) and size-exclusion chromatography using a Superdex 200 column (GE Healthcare). For crystallization studies, purification tags were removed by overnight digestion with thrombin followed by gel filtration using a Superose 6 column (GE Healthcare Biosciences) with a running buffer of 2 mM TRIS pH 8.0, 200 mM NaCl. For ITC studies, tags were not removed and proteins were purified by gel filtration using a Superdex 200 column (GE Healthcare) with a running buffer of PBS.

The HiTrap Capto SP ImpRes column (1 mL, GE) was washed with 5 mL of ddH₂O and equilibrated with 10 mL of the starting buffer (20 mM Tris-HCl, 100 mM NaCl, pH 7.1). Since we used HiLoad Pump P-50 (GE), pH and conductivity of flowthrough (FT), eluates or eluents were determined by pH papers (Sigma) and the EC/salinity meter. The clarified 293F cell lysate were adjusted to a volume of 5 mL with the starting buffer and loaded on to the column at a flow rate of 1 mL/min. After washing with 10 mL of the starting buffer, the CEC-captured L1:P18I10 VLPs were one-step eluted with 5 mL of elution buffers (20 mM Tris-HCl, 1 M NaCl, pH 7.1) at a flow rate of 1 mL/min. The column was regenerated by washing with 5 mL of 2 M NaCl in 20 mM Tris-HCl buffer at pH 7.1 to remove remaining ionically bound proteins.