Expifectamine 293 transfection kit

LayV F ectodomain constructs include codon-optimized LayV F (residue 1 to 482) fused to a C-terminal GCN4 followed by a linker (GSGGGS) and a hexa-histidine tag (HHHHHH). The GhV F ectodomain construct used for cryoEM includes residues 1 to 586 C-terminally fused to the I53-50A component via an intervening 16 GS linker (70 (link)) followed by a GSGGGS linker and a 6×His tag whereas the other constructs have a C-terminal GSGGGS linker followed by a 6×His tag. The LayV F wildtype, N95C/A114C, I167F/S186P, I167F/S186P/N95C/A114C and GhV F wildtype, S196C/A215C, I268F/Q287P, I268F/Q287P/S196C/A215C were synthesized by Twist Bioscience and cloned into pTwist CMV vector. LayV F and GhV F ectodomains were produced in 100 mL Expi293F cells grown in suspension using Expi293 Expression Medium at 37 °C in a humidified 8% CO₂ incubator rotating at 130 rpm. The cultures were transfected using ExpiFectamine™ 293 Transfection Kit (ThermoFisher Scientific) with cells grown to a density of 3 million cells per mL and cultivated for 4 d. The supernatants were harvested, and proteins were purified from clarified supernatants using a 1 mL HisTrap HP column (Cytiva), buffer exchanged, concentrated, and flash frozen in either TBS (50 mM Tris, 150 mM NaCl and 10 mM EDTA at pH 8.0). SDS-PAGE was run to check purity.

Antibodies were expressed transiently in Expi293F cells (A14527, ThermoFisher Scientific, Waltham, MA, USA) through cotransfection of heavy (γ1/μ/α1/α2) and light-chain expression plasmids (κ/λ) using ExpiFectamine 293 Transfection Kit (A14525, ThermoFisher Scientific) at a heavy chain (HC):light chain (LC) ratio of 1:1. In the case of IgM and dIgAs, an expression plasmid encoding the human J chain precursor (UniProtKB number:human J chain P01591, Geneva, Switzerland) [5 (link)] was also included in the cotransfections at HC:LC:J chain ratios of 5:5:1. Cells were maintained in Expi293 expression medium (A1435102, ThermoFisher Scientific) for four to five days at 37 °C, 8% CO₂ with continuous shaking at 135 rpm. The culture supernatants were harvested and clarified via centrifugation at 4000× g for 30 min and filtered through a 0.22 μm pore size hydrophilic polyethersulfone (PES) membrane.

Recombinant chimeric antibodies were produced using Expi293 Expression System (Thermo Fisher Scientific) following the manufacturer’s instructions. Briefly, Expi293F cells were transfected with the vectors encoding the heavy and light chains (30 μg each) per cell culture of 60 mL using the ExpiFectamine 293 Transfection Kit (Thermo Fisher Scientific). The cell culture was incubated on a shaker for 6–7 days post transfection, and the cells were removed by centrifugation at 6,000 × g for 20 min. The culture supernatant was filtered through a 0.20 μm filter, and the antibodies were captured in a 1 mL HiTrap Protein A HP Column (Cytiva). The column was washed with 20 mL phosphate-buffered saline (PBS), and the antibodies were subsequently eluted with 5 mL citrate buffer (100 mM, pH 3.0). The eluate was dialyzed in PBS and used without further purification.