Histrap nickel 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 gene for nanobody C1 (NbC1) and F2 (NbF2) and were codon-optimized using the IDT Codon Optimization Tool, synthesized as a ready-to-clone gene fragment (Integrated DNA Technologies), and cloned into the phagemid vector pADL-23c. The nanobodies were produced as previously described (Huo et al., 2021 (link)). Briefly, the plasmid was transformed into the WK6 E. coli strain and protein expression induced by 1 mM IPTG grown overnight at 28°C. Periplasmic extract was prepared by osmotic shock, and the nanobody protein was purified with a 5 mL HisTrap nickel column (Cytiva), followed by size exclusion with a Hiload 16/60 Superdex 75 column.