Akta pure 25 m1 system

All recombinant viral proteins were cloned into a mammalian expression vector with an N-terminal signal peptide and a C-terminal AviTag followed by a polyhistidine tag. Constructs were transfected in Expi293F cells and expressed for four days at 37°C. For biotinylated constructs, cells were co-transfected with a plasmid encoding for the Escherichia coli biotin ligase (BirA) obtained from the laboratory of Dr. Kai Zinn (California Institute of Technology). Recombinant proteins were isolated from cellular supernatants and purified via affinity chromatography using a HisTrap HP column (Cytiva) and size-exclusion chromatography on either HiLoad 16/600 Superdex 200 pg (Cytiva) or Superdex 200 Increase 10/300 GL (Cytiva) columns using an AKTA pure 25 M1 system (Cytiva). All proteins were stored in 1x TBS-Az at 4°C. Detailed sequence and design information for all recombinant viral protein constructs used in this study can be found in Data Table 2. pcDNA3-sACE2(WT)-8his encoding soluble human ACE2 (UniProt Q9BYF1, residues 1–732) was a gift from Erik Procko (Addgene plasmid #149268) and was expressed and purified as described above for the other recombinant proteins.

Sequences for S309, C118, C135, C952, C1520, C1533, and C1596 anti-SARS-CoV-2 IgG antibodies were previously published (20 (link), 23 (link), 43 (link), 73 (link)). Heavy IgG and light chains for each antibody were cloned into the AbVec2.1 expression plasmid (gift from Dr. Pamela Bjorkman, California Institute of Technology). Fab constructs were obtained by subcloning the variable heavy (V_H) and constant heavy chain 1 (CH₁) domain from the IgG vectors into an AbVec2.1 expression plasmid with a C-terminal 6x histidine tag. For IgG and Fab expression, heavy and light chains were co-transfected in Expi293F cells using the ExpiFectamine transfection kit (ThermoFisher Scientific, A14525) in a 1:1 ratio; ExpiFectamine transfection enhancer was added 20 h post-transfection. IgGs and Fabs were isolated and purified from filtered cell supernatants via affinity chromatography using HiTrap MabSelect SuRe (Cytiva) and HisTrap HP (Cytiva) columns, respectively. Elutions were concentrated using Amicon^™ spin concentrators and further purified by size-exclusion chromatography on a HiLoad 16/600 Superdex 200 pg (Cytiva) and Superdex 200 Increase 10/300 GL (Cytiva) columns using an AKTA pure 25 M1 system (Cytiva). Proteins were concentrated via Amicon^™ spin concentrators and stored in Tris-Buffered Saline with azide (1x TBS-Az; 20 mM Tris pH 8.0, 150 mM NaCl, 0.02% Sodium Azide) at 4°C.