Streptactin superflow column

To generate a stable Drosophila S2 cell line expressing recombinant MHV spike ectodomain, we used Effectene (Qiagen) and 2 μg of the plasmid encoding the MHV S protein ectodomain. A second plasmid, encoding blasticidin S deaminase was cotransfected as dominant selectable marker. Stable MHV S ectodomain expressing cell lines were selected by addition of 10 μg ml⁻¹ blasticidin S (Invivogen) to the culture medium 48 h after transfection.
For large-scale production of MHV S ectodomain the cells were cultured in spinner flasks and induced by 5 μM CdCl₂ at a density of approximately 10⁷ cells per ml. After a week at 28 °C, clarified cell supernatants were concentrated 40-fold using Vivaflow tangential filtration cassettes (Sartorius, 10-kDa cut-off) and adjusted to pH 8.0, before affinity purification using StrepTactin Superflow column (IBA) followed by gel filtration chromatography using Superose 6 10/300 GL column (GE Life Sciences) equilibrated in 20 mM Tris-HCl, pH 7.5, 100 mM NaCl. The purified protein was quantified using absorption at 280 nm and concentrated to approximately 4 mg ml⁻¹.

To generate a stable Drosophila S2 cell line expressing the recombinant HCoV-NL63 S ectodomain, we used Effectene (Qiagen) and 2 μg of plasmid. puromycin N-acetyltransferase was cotransfected and used as a dominant selectable marker. Stable HCoV-NL63 S–expressing cell lines were selected by addition of 7 μg/ml puromycin (Invivogen) to the culture medium 48 h after transfection. For large-scale production, the cells were cultured in spinner flasks and induced by 5 μM of CdCl₂ at a density of approximately 10⁷ cells/mL. After one week at 28 °C, clarified cell supernatants were concentrated 40-fold with Vivaflow tangential filtration cassettes (Sartorius, 10-kDa cutoff) and adjusted to pH 8.0, before affinity purification with a StrepTactin Superflow column (IBA) followed by gel-filtration chromatography with a Superose 6 10/300 GL column (GE Life Sciences) equilibrated in 20 mM Tris-HCl, pH 7.5, and 100 mM NaCl. The purified protein was quantified according to absorption at 280 nm and concentrated to approximately 3 mg/mL.