Strep tactin xt resin

Protein expression and purification were conducted as described previously (Dejnirattisai et al., 2021a (link); Zhou et al., 2020 ). Briefly, plasmids encoding proteins were transiently expressed in HEK293T (ATCC CRL-11268) cells. The conditioned medium was concentrated using a QuixStand benchtop system. His-tagged Omicron RBD were purified with a 5 mL HisTrap nickel column (GE Healthcare) and further polished using a Superdex 75 HiLoad 16/60 gel filtration column (GE Healthcare). Twin-strep tagged Omicron spike was purified with Strep-Tactin XT resin (IBA lifesciences).
~4mg of ACE2 was mixed with homemade His-tagged 3C protease and DTT (final concentration 1mM). After incubated at 4 °C for one day, the sample was flown through a 5 mL HisTrap nickel column (GE Healthcare). His-tagged proteins were removed by the nickel column and purified ACE2 was harvested and concentrated.

The plasmid for expression of the SARS‐CoV‐2 prefusion‐stabilized spike ectodomain with a C‐terminal T4 fibritin trimerization motif was obtained from Wrapp et al.¹⁷ The plasmid was used to transiently transfect FreeStyle 293F cells using FreeStyle MAX reagent (Thermo Fisher Scientific). The ectodomain was purified from filtered supernatant on Streptactin XT resin (IBA Lifesciences), followed by size‐exclusion chromatography on a Superdex 200 in 5 mM Tris pH 8, 200 mM NaCl.
The RBD domain (RVQ – QFG) of SARS‐CoV‐2 was cloned upstream of a Sortase A recognition site (LPETG) and a 6xHIS tag, and expressed in 293F cells as described above. RBD‐HIS was purified from filtered supernatant on His‐Pur Ni‐NTA resin (Thermo Fisher Scientific), followed by size‐exclusion chromatography on a Superdex 200. The nucleocapsid was purchased from Sino Biological and was not used beyond assay development.

Protein expression and purification were conducted largely as described previously (Dejnirattisai et al., 2021a (link); Zhou et al., 2021 (link)). Twin-strep tagged Omicron spike was transiently expressed in HEK293T cells and purified with Strep-Tactin XT resin (IBA lifesciences). Plasmids encoding BA.1 RBD (319–541), BA.1 RBD (330–532) and BA.2 RBD (330–532) were transiently expressed in Expi293F™ Cells (ThermoFisher), cultured in FreeStyle™ 293 Expression Medium (ThermoFisher) at 30°C with 8% CO2 for 4 days. BA.1 RBD (330–532) was expressed in the presence of 1 μg/mL kifunensine. The harvested medium was concentrated using a QuixStand benchtop system. His-tagged ACE2 and RBDs were purified with a 5 mL HisTrap nickel column (GE Healthcare), followed by a Superdex 75 10/300 GL gel filtration column (GE Healthcare).

The DNA fragment encoding M1 protein (GenBank: CY147535.1) was cloned into the pEXPR-IBA 103 vector (IBA Lifesciences, Göttingen, Germany), and the plasmid DNA was transfected into ExpiCHO cells. Five days later, the cells were harvested and lysed by sonication (30% amplitude, 10 s on/10 s off, total 10 min). After filtration with a 0.45 μm filter (Corning), the lysate supernatant was bound to Strep-Tactin XT resin (IBA Lifesciences) using a gravity flow column. After washing, the bound protein was collected by treating the column with an elution buffer (IBA Lifesciences). The endotoxin level in the purified M1 was measured using the endotoxin test kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA). The protein was stored at –80 °C until further use.

To purify full length IgG mAbs, supernatants of mAb expression were collected and filtered by a vacuum filter system and loaded on protein A/G beads over night at 4 °C. Beads were washed with PBS three times and 0.1 M glycine pH 2.7 was used to elute IgG. The eluate was neutralized with Tris-HCl pH 8 buffer to make the final pH=7. The IgG concentration was determined by spectro-photometry and buffered exchanged into PBS.
To express and purify Fabs 158 and EY6A, heavy chain and light chain expression plasmids of Fab were co-transfected into HEK293T cells by PEI. After cells cultured for 5 days at 37°C with 5% CO2, culture supernatant was harvested and filtered using a 0.22 mm polyethersulfone filter. Fab 158 was purified using Strep-Tactin XT resin (IBA lifesciences) and Fab EY6A was purified with Ni-NTA column (GE HealthCare) and a Superdex 75 HiLoad 16/60 gel filtration column (GE Healthcare).
AstraZeneca and Regeneron antibodies were provided by AstraZeneca, Vir, Lilly and Adagio antibodies were provided by Adagio. For the antibodies heavy and light chains of the indicated antibodies were transiently transfected into 293Y cells and antibody purified from supernatant on protein A. Fab fragments of 58 and beta-55 were digested from purified IgGs with papain using a Pierce Fab Preparation Kit (Thermo Fisher), following the manufacturer’s protocol.