Hiload 26 60 superdex 200 column

DNA fragments encoding VH or VL of D27LEY were synthesized (IDT) and cloned into a vector derived from the pCEP4 vector (Invitrogen) which contains the CH1-CH2-CH3 sequence of IgG or the CL sequence of the kappa light chain. The resulting vectors, pCEP4(heavy) and pCEP4(k-light), were introduced into CHO-S cells (Gibco) at a density of 6x10⁶ cells/ml using ExpiFectamine (Gibco) to express D27LEY. The cells were grown in the ExpiCHO expression medium (Gibco) for 10 days. The culture supernatant was collected by centrifugation at 12,000g for 1 h at 4 ℃, diluted by half with Protein A binding buffer (PBS, pH 8.0), loaded onto an open column containing Protein A resin (Genscript), and eluted with Protein A elution buffer (0.1 M glycine, pH 3.0). The eluent was then immediately neutralized with Protein A neutralizing buffer (1M Tris-HCl, pH 8.5), and further purified using a HiLoad 26/60 Superdex 200 column (Cytiva) equilibrated with buffer A composed of 150 mM NaCl and 20 mM Tris-HCl (pH 8.0).

Purified RBD(N501Y) and D27LEY-Fab were mixed in a 1.2:1 molar ratio. The complex between the two was purified by size-exclusion chromatography using a Hiload 26/60 Superdex 200 column (Cytiva) equilibrated with buffer A. The complex was concentrated at 11.7 mg/ml, and an initial crystallization screening was performed in 96-well plates by the sitting-drop vapor diffusion method using Mosquito (Spt Labtech). Finally, crystals were grown in a solution containing 200 mM ammonium citrate tribasic (pH 7.0) and 20% (v/v) polyethyleneglycol 3350. For cryoprotection, the crystal was briefly immersed in a mother liquor containing an additional 10% (v/v) ethylene glycol. X-ray diffraction data were collected on the beamline 5C at the Pohang Accelerator Laboratory, Korea, and processed with HKL2000 (24 (link)). Model building and structure refinement were carried out using COOT and PHENIX (25 (link), 26 (link)).

For in vitro assays, human hemoglobin powder was purchased from Sigma Aldrich. A total of 50 mg were dissolved in PBS buffer and purified by gel filtration using a Hiload 26/60 Superdex-200 column (Cytiva). The concentration of human hemoglobin was determined using PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific) following the manufacturer’s instructions.