Expi293 cell

VLPs were produced in adherent HEK-293T [293T] (ATCC-CRL-3216) or the suspension Expi293 cells (Gibco, ThermoFischer Scientific, Waltham, MA, USA). HEK-293T cells were maintained in 10% fetal bovine serum (Atlanta Biologicals. Flowery Branch, GA, USA) in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, ThermoFischer Scientific, Waltham, MA, USA) with 1% penicillin–streptomycin (Gibco, ThermoFischer Scientific, Waltham, MA, USA) and 1% non-essential amino acids (neAA; Gibco, ThermoFischer Scientific, Waltham, MA, USA) at 37 °C in a humidified incubator with 5% CO₂. Expi293 cells were maintained in Expi293 Expression Medium (Gibco, ThermoFischer Scientific, Waltham, MA, USA) at 37 °C in a humidified incubator with 8% CO₂ shaking at 130 revolutions per minute.

pcDNA3.1 SARS-CoV-2 S D614G was a gift from Jeremy Luban (Addgene plasmid # 158075; http://n2t.net/addgene:158075; RRID: Addgene_158075). SARS-CoV-2 S D614G protein, RBD_wt, RBD_δ, and RBD_ο were produced in Expi293 cells (Thermo Fisher Scientific) and were purified using Ni-NTA agarose resin (Thermo Fisher Scientific) affinity chromatography, as described previously (24 (link), 25 (link)).
Briefly, Expi293 cells were cultured at 37 °C with 5% CO₂ for five days after transfection of each plasmid encoding SARS-CoV-2 S D614G protein, RBD_wt, RBD_δ, or RBD_ο (BA1). The supernatant was collected and passed over the Ni-NTA agarose resin column three times. After washing with 100 mL of phosphate-buffered saline (PBS), the his-tagged protein was eluted by elution buffer (pH8.0, 50 mM sodium phosphate, 300 mM NaCl, and 250 mM imidazole). Finally, samples were buffer-exchanged into pH 7.4 PBS using Amicon Ultra-4 (Merck Millipore, Burlington, MA, USA) spin columns with a 10 kDa cutoff. The purity of purified samples was assessed by 14% SDS-PAGE gel.

DS-Cav1 produced in CHO cells was purified by affinity and ion exchange chromatography. PreF mutants were cloned on DS-Cav1 with a C-terminal thrombin-cleavable double Strep tag II followed by a His-tag as a template. Single or multiple point mutations were generated using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs, Ipswich, MA). Proteins were transiently expressed in Expi293 cells (Thermo Fisher Scientific, Carlsbad, CA), and purified using affinity chromatography followed by removal of Strep/His tags using Thrombin protease, and a final size exclusion chromatography polishing step, as previously described [17 (link)].
RSB1 wildtype and mutant IgG was recombinantly expressed in Expi293 cells and purified using Protein A and size exclusion chromatography, as previously described [17 (link)]. Mutations in RSB1 sequence were generated using site directed mutagenesis using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs, Ipswich, MA). RSB1 Fab was expressed with a Strep Tag II at the heavy chain C terminus and purified using a StrepTrap HP column (GE Healthcare). The tag was proteolytically cleaved using TEV protease (AcTEV protease, Thermo Fisher Scientific) prior to size exclusion chromatography. DS-Cav1 was incubated with a 1:1.5 molar excess of RSB1 Fab prior to size exclusion chromatography to prepare the complex for crystallization.