Expicho expression system kit

SARS-CoV-2 convalescent patients and immunized healthy adults were enrolled and peripheral blood was collected. Peripheral blood mononuclear cells (PBMCs) were prepared from peripheral blood using Ficoll-Paque (Sigma-Aldrich, USA). Single B cells were isolated with or without using biotinylated RBD (Beta variant) into the 96-well PCR plate containing lysis buffer as previously described^{6 (link)}. After performing reverse transcription to obtain cellular Ig cDNA, variable domain-encoding genes for heavy, kappa and lambda chains were amplified and inserted into human IgG1 expression vectors. For antibody expression, heavy and light chain expression vectors were transiently transfected into ExpiCHO cells (Thermo Fisher Scientific, A29133) using the ExpiCHO expression system kit. Human IgG1 monoclonal antibody-containing supernatant was harvested and purified by rProtein A Sepharose (GE healthcare), with the resulting monoclonal antibodies collected for further analysis.
To determine the individual gene segments employed by VDJ and VJ rearrangements and the number of nucleotide mutations and amino acid replacements, the variable domain sequences were aligned with germline gene segments using the international ImMunoGeneTics (IMGT) alignment tool (http://www.imgt.org/IMGT_vquest/input)^{34 (link)}.

For cryo-EM studies, a construct containing an HA secretion signal (MKTIIALSYIFCLVFA), a FLAG peptide (DYKDDDD), linker and 3 C cleavage site (KGSLEVLFQGPG), GCN4 homodimeric zipper (RMKQLEDKVEELLSKNYHLENEVARLKKLVGER), human GC-C regions corresponding to the small extracellular linker region, TM, and intracellular domains (residues 399–1,053), a second linker and 3 C cleavage site (AAALEVLFQGPGAA), a Protein C epitope tag (EDQVDPRLIDGK), and an 8 x His tag were cloned into a pD649 mammalian expression vector. This construct contains all domains of the native GC-C, with the exception of the ECD (Supplementary file 1). Protein was expressed using ExpiCHO Expression System Kit (Thermo Fisher). Briefly, ExpiCHO cells were maintained in ExpiCHO Expression Media at 37 °C with 5% CO₂ and gentle agitation, and transiently transfected by the expression construct and cultured according to the manufacturer’s protocol. Cells were pelleted and stored at –80 °C.

To produce secreted SARS-CoV-2 S proteins with a prefusion form, a DNA fragment encoding the Wuhan strain S protein was designed to contain a nonfunctional furin cleavage site (R682G, R683S, R685S) and two stabilizing prolines (K986P, V987P) in the hinge loop. Additionally, the transmembrane domain and the C-terminal intracellular tail were removed (S-2P) or replaced by a trimerization domain IZN4 (S-Trimer), followed by histidine (8-mer) at the carboxy terminus for purification. Both S variant DNA constructs were codon-optimized for Homo sapiens, synthesized, and cloned into the pcDNA3.1(+) plasmid vector by GenScript (Piscataway, NJ, USA). The production of the S-Trimer was compiled using the ExpiCHO™ Expression System Kit (ThermoFisher Scientific, Carlsbad, CA, USA). Briefly, the S-Trimer (or S-2P) was transiently expressed by ExpiCHO cells with serum-free medium according to the manufacturer’s instructions. The culture medium containing S-Trimer was centrifuged at 15,000 rpm for 30 min at 4 °C; later, the supernatant was dialyzed to equilibration buffer (50 mM Tris-HCl, 150 mM NaCl, and 20 mM imidazole; pH 8.9). S-Trimer was purified through an equilibrated Ni²⁺-NTA agarose column (GE). Finally, the S-Trimer was eluted by equilibration buffer with 0.5 M imidazole and dialyzed against buffer without imidazole (20 mM sodium phosphate; pH = 8.0).

HEK-293T (ATCC^® CRL-3216^TM) and African Green monkey kidney (Vero, ATCC, Old Town Manassas, VA, USA) cells were obtained from ATCC (Manassas, VA, USA). HEK-293T and Vero cell lines were cultured in DMEM (Corning, NY, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) and penicillin–streptomycin (Gibco, USA). ExpiCHO cell lines were utilized for recombinant protein expression according to the manufacturer’s protocol (ExpiCHO™ Expression System Kit, Thermo, Waltham, MA, USA). DNA transfection into HEK-293T cells in vitro utilized jetPRIME^® (Polyplus-transfection, Illkirch-Graffenstaden, France).

Both VH-IgG1 and VL-CK chain of the anti c-myc-ChBD expression vectors were co-transfected in CHO-S (Chinese hamster ovary) cells using the ExpiCHO Expression system kit (Thermo A29133). The antibody was expressed at 32 °C and 5% CO₂ for 10 days attending to the manufacturer’s instructions. Cells were isolated by centrifugation and the supernatant was measured by mouse IgG sandwich ELISA and purified by affinity chromatography with protein G column (HiTrap Protein G, GE Healthcare 29–0485-81). Pure antibody concentration was measured by absorbance at 280 nm and purity grade was analyzed by SDS-PAGE.

NN2101, a fully human monoclonal IgG1, was prepared as described previously [43 (link)]. Briefly, DNA sequences encoding the light and heavy chains were subcloned into the pCHO 1.0 vector (Thermo Fisher Scientific, Waltham, MA, USA). The recombinant vector was transiently transfected into Chinese hamster ovary cells using the ExpiCHO™ expression system kit (Thermo Fisher Scientific), and the cells were cultured for 14 days. NN2101 was purified using AKTA Avant 150 chromatography system (Merck Millipore, Burlington, MA, USA) equipped with MabSelect SuRe™ (Merck Millipore) and HiTrap^® Capto™ SP ImpRes (Merck Millipore). The samples were dialyzed against storage buffer (10 mm sodium phosphate, 40 mm NaCl, 0.03% polysorbate 20, and 5% sucrose; pH 6.2). We have previously reported the c‐Kit binding affinity, epitope map, c‐Kit‐positive cell‐binding ability, effector function, immunogenicity, and crystal structure of NN2101 [41 (link)].