DNA fragment encoding ectodomain of S from SARS-CoV-2 with the disruption of S1/S2 furin cleavage site and the substitutions of six amino acids with proline (F817P, A892P, A899P, A942P, K986 P, V987P) was placed into the mammalian expression vector pCAGGS with N-terminal signal peptide (S-6P). The C terminus was engineered with a thrombin site (LVPRGS) linked by a folded trimerization motif (YIPEAPRDGQAYVRKDGEWVLLSTFL) and a 6×His Tag. The S-6P plasmid was transiently transfected into Expi293 cells using PEI reagent. The cell supernatants containing spike protein were harvested 108 h after transfection, clarified by centrifuge (20 min at 8000 × g, 4 °C), and filtered through a 0.45 μm filter. The soluble S proteins were recovered using 2 ml of High-Affinity Ni-charged Resin (GenScript), further purified by passage over SuperoseTM 6 Increase 10/300GL (Cytiva).
Variable regions of heavy and light chains were cloned into a Fab expression vector and sequence-corrected plasmids were transiently co-transfected into Expi293 cells with a ratio of 1:1. At 144 h post-transfection, cell supernatants were collected and secreted Fabs were purified with High-Affinity Ni-charged Resin (GenScript), following the purification by gel filtration of SuperoseTM 200 Increase.
Variable regions of heavy and light chains were cloned into a Fab expression vector and sequence-corrected plasmids were transiently co-transfected into Expi293 cells with a ratio of 1:1. At 144 h post-transfection, cell supernatants were collected and secreted Fabs were purified with High-Affinity Ni-charged Resin (GenScript), following the purification by gel filtration of SuperoseTM 200 Increase.