Superdex 75 10 300 gl gel filtration column

Protein expression and purification were largely the same as described previously^{6 (link),34 (link)}. Twin-strep tagged Wuhan, BA.2.12.1, BA.4, XBB.1.5, and BA.2.86 spikes were transiently expressed in HEK293T cells and purified with Strep-Tactin XT resin (IBA Lifesciences). Plasmids encoding BA.4 RBD, NTD, and SD1 with a 6*His-tag were separately transiently expressed in Expi293F™ Cells (Thermo Fisher), cultured in FreeStyle™ 293 Expression Medium (Thermo Fisher) at 30 °C with 8% CO₂ for 4 days. The harvested medium was concentrated and buffer-exchanged using a QuixStand benchtop system. His-tagged proteins were purified with a 5 mL HisTrap nickel column (Cytiva), followed by a Superdex 75 10/300 GL gel filtration column (Cytiva).

The DNA sequence of lyophyllin was synthesized by Integrated DNA technologies, IDT. The sequence was cloned to pET-28a vector with 6X His tag. Constructed plasmid was transformed to OverExpress C43(DE3) cell (Cat. No. CMC0019, Sigma, St Louis, MO, USA) and grown until OD600 0.8, then induced by 0.1 mM IPTG at 25 °C overnight. The yield was about 50 μg lyophyllin/L cells. Corresponding variants were made by site-direct mutagenesis (Table S1).
To express the recombinant protein, E. coli cells were harvested by centrifugation at 11,655× g (Rotor JA-14, Beckman Coulter, Indianapolis, IN, USA) for 4 min, and then lysed by flow cell disrupter JN-Mini (JNBIO, Guangdong, China) at 1200 bar and 4 °C in buffer A (20 mM Tris pH 7.5, 100 mM NaCl, 50 mM Imidazole, 5% glycerol). The nickel NTA beads (QIAGEN, Venlo, The Netherlands) were equilibrated in the same buffer before loaded with the cell lysate. Beads were washed by buffer A for 10X column volume (CV) and then buffer B (20 mM Tris pH 7.5, 100 mM NaCl, 100 mM Imidazole, 5% glycerol) for another 10 CV. Finally, protein was eluted by buffer C (20 mM Tris pH 7.5, 100 mM NaCl, 300 mM Imidazole, 5% glycerol). It was then concentrated to suitable volume and injected to AKTA Prime (Cytiva, Marlborough, MA, USA) with Superdex 75 10/300 GL gel filtration column (Cytiva, Marlborough, MA, USA).