Hiprep 26 10 column

The soluble HLA-E/peptide complexes were produced as previously described^{23 (link),24 (link)}. Briefly, recombinant HLA-E*01:01 heavy chain and ß2microglobulin light chain were produced as inclusion bodies in bacterial strain BL21(DE3) and TG1, respectively, resuspended in 8 M urea buffer and stored at − 80 °C. The heavy and light chains were then refolded in vitro with the peptide of interest during 5 days at 4 °C with slow agitation. The solution was concentrated on a 10 kDa Amicon membrane (Millipore, Bedford, MA) and dialyzed overnight at 4 °C against 10 mM Tris pH 8 buffer. The monomers were then biotinylated with BirA enzyme (Avidity, Denvers CO) for 5 h at 30 °C and desalted on HiPrep 26/10 column (Cytiva) with 10 mM Tris–HCl pH 8, 150 mM NaCl buffer. Purification was completed with a size exclusion step on a Superdex 200 10/300 GL column (Cytiva). Biotinylation of the HLA-E/peptide complex was assessed by tetramerization assay with streptavidin (Sigma Aldrich) after one hour incubation at room temperature and injected onto the Superdex 200 10/300 GL column. Purified complexes were stored at − 80 °C until use.

The YSK₂ and YSK₂ constructs were expressed and purified using a slight modification of Livernois et al. [39 (link)]. Briefly, the cells were lysed by boiling for 20 min with agitation every 5 min and were subsequently cooled on ice. Sodium acetate (pH 5.0) was added to a final concentration of 20 mM and the supernatant was filtered before continuing with purification. The filtered supernatant was loaded onto a 5 mL HiTrap SP HP column (Cytiva, Vancouver, BC, Canada) equilibrated with 20 mM sodium acetate, pH 5.0 (buffer A). Elution occurred over a gradient of 0–1 M NaCl (Buffer B: 20 mM sodium acetate, pH 5.0, 1 M NaCl). Fractions containing the protein of interest were desalted into Milli-Q water using a HiPrep 26/10 column (Cytiva). After concentrating the eluted protein fraction, reversed-phase HPLC was performed. The protein sample was purified using a BioBasic C4 column (Thermo Scientific, Ottawa, ON, Canada), which was equilibrated with Buffer A (0.1% trifluoroacetic acid (TFA) (w/v)). The protein was eluted using a 1–100% Buffer B (0.1% TFA (w/v) in acetonitrile) gradient. Sample purity was determined using 12% SDS-PAGE and the fractions containing the protein of interest were pooled, flash frozen and lyophilized. The proteins were stored at −20 °C until use.

Recombinant SARS‐CoV‐2‐N protein was expressed in Escherichia coli BL21 (DE3) cells as described previously.^[19] His‐tagged SARS‐CoV‐2 N protein was purified using immobilized metal affinity chromatography with a 5 mL HisTrap excel column (Cytiva, Marlborough, MA). The column was equilibrated in 20 × 10⁻³m sodium phosphate pH 7.4 + 500 × 10⁻³m NaCl. The harvested supernatant was loaded onto the column and then washed with equilibration buffer containing 20 × 10⁻³m of imidazole. N protein was then eluted with 20 × 10⁻³m of sodium phosphate pH 7.4 + 500 × 10⁻³m NaCl + 500 × 10⁻³m imidazole. Eluted proteins were desalted into phosphate‐buffered saline (PBS) using a HiPrep 26/10 column (Cytiva, Marlborough, MA) and then filtered using a 0.22 m filter. The positive fractions of N protein was then pooled, and after aliquoting the products, it was stored at −80 °C until used. By using SDS‐PAGE and western blot with anti‐His tag antibodies, confirmed the purity of the N protein produced.