Streptrap hp affinity column

Protein sequence of prefusion RSVF corresponds to the sc9-10 DS-Cav1 A149C Y458C S46G E92D S215P K465Q variant designed by Joyce and colleagues [42 (link)], which we refer to as RSVF DS2. RSVF DS2 was codon optimized for mammalian expression and cloned into the pHCMV-1 vector together with two C-terminal Strep-Tag II and one 8x His tag. Plasmids were transfected in HEK293-F cells and cultured in FreeStyle medium. Supernatants were harvested 1 week after transfection and purified via Ni-NTA affinity chromatography. Bound protein was eluted using buffer containing 10 mM Tris, 500 mM NaCl, and 300 mM Imidazole (pH 7.5), and eluate was further purified on a StrepTrap HP affinity column (GE Healthcare). Bound protein was eluted in 10 mM Tris, 150 mM NaCl and 20 mM desthiobiotin (pH 8) (Sigma) and size excluded in PBS (pH 7.4) on a Superdex 200 Increase 10/300 GL column (GE Healthcare) to obtain trimeric RSVF.

The sequence of the extracellular region of G proteins from HeV and NiV (HNV-G) were codon-optimized, the tPA signal peptide and Strep-II tag were added at the N-terminus. Gene was synthesized (General Biosystems Co., Ltd.) and inserted into pcDNA3.1 (+).
The plasmid was transfected into Expi-293F cells for expression. After transfection, cells were placed on a shaker at 120 rpm and 37°C. After 72 hours of cell culture, the supernatant was centrifuged at 3,000 g for 15 min and filtered through a 0.45 μm filter (Thermo Fisher Scientific, USA). The protein was purified using the StrepTrap HP affinity column (GE Health Care, USA). Purified proteins were concentrated using 30 K ultrafiltration tubes (Merck Millipore, Germany) and permuted using PBS buffer at pH 7.4. Finally, the obtained proteins were characterized using SDS-PAGE and quantified with a BCA Protein Assay Kit (Thermo Fisher Scientific, USA).