Ni sepharose chromatography

Plasmids encoding RSV F prefusion (DS-Cav1) and postfusion (F ΔFP) proteins based on strain A2 sequences with mammalian codon-optimization were used to transfect Expi 293F cells (Invitrogen) and proteins were purified as described previously [10 (link), 11 (link)]. Briefly, RSV F proteins were purified from cell culture supernatants using Ni-Sepharose chromatography (GE healthcare). Postfusion RSV F was further purified by Strep-Tactin chromatography (Strep-Tactin Superflow Plus, Qiagen). Tags were removed by digestion with thrombin overnight. Prefusion F was further purified by a second Ni-Sepahrose chromatography step to remove IMAC contaminants and uncleaved RSV F. Both forms of RSV F proteins were further purified by gel filtration chromatography (Superdex 200, GE Healthcare).

The recombinant mouse IL9 (rIL9) gene with a C-terminal hexahistidine and ALFA tag was cloned between the BamHI and NotI site in the pVL1393 baculovirus transfer vector. The recombinant baculovirus was generated by cotransfection of the BestBac 2.0v linearized baculovirus genome (Expression Systems) into Sf9 insect cells (Thermo Fisher Scientific). The rIL9 protein was produced by infecting High Five insect cells (Thermo Fisher Scientific) with the recombinant baculovirus. The secreted rIL9 protein was purified by Ni-Sepharose chromatography (GE Healthcare). After cleavage by thrombin to remove the hexahistidine and ALFA tag, the mIL9 protein was further purified by SP-Sepharose (GE Healthcare) cation-exchange chromatography and Superdex-200 gel filtration chromatography (GE Healthcare) equilibrated with a buffer containing 20 mmol/L Tris pH 8.0, 200 mmol/L NaCl, and 0.1 mmol/L phenylmethylsulfonyl fluoride. The protein sample buffer was changed into PBS (Gibco) using PD MiniTrap G-25 desalting columns (Cytiva). Endotoxin was removed from rIL9 using Pierce High-Capacity Endotoxin Removal Resin (Thermo Fisher Scientific), and the protein samples were sterilized by 0.2 μmol/L sterile membrane filtration. The protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific).

As previously described, plasmids encoding mammalian codon-optimized RSV F prefusion (DS-Cav1) and postfusion (FΔFP) proteins were used to transfect Expi 293F cells (Life Technologies), and proteins were purified from culture supernatants [13 (link), 21 (link)]. Briefly, cell culture supernatants were harvested day 3 (FΔFP) or 7 (DS-Cav1) post-plasmid transfection, and RSV F proteins were purified using Ni-Sepharose chromatography (GE Healthcare). FΔFP was further purified by Strep-Tactin chromatography (Strep-Tactin Superflow Plus, Qiagen). Tags were removed from DS-Cav1 and FΔFP by overnight digestion with thrombin. To remove IMAC contaminants and uncleaved F protein, DS-Cav1 was subjected to a second Ni-Sepharose chromatography step. Both DS-Cav1 and FΔFP were polished by gel filtration chromatography (Superdex 200, GE Healthcare) and were stored in a buffer of 50 mM HEPES pH 7.5, 300 mM NaCl.