Ni2 imac sepharose 6 fast flow

Recombinant proteins were purified as reported previously with small modifications.⁹¹ (link)^,92 (link) Mammalian expression vectors containing secreted, codon-optimized CoV2 S (pHL-Sec⁹³ (link); aa. 1-1208, C-terminal 8His-Twin-Strep) and RBD (pOPINTTGNeo; aa. 330-532, C-terminal 6His) were transiently transfected with linear PEI into Expi239^TM cells cultured in roller bottles in FreeStyle 293 media. Cell culture media was harvested after 3 days at 37°C for RBD or 3 days at 30°C for Spike and then buffered to 1X PBS. Proteins were first pulled down on Ni²⁺ IMAC Sepharose® 6 Fast Flow (GE) with stringent washing (>50 CV with 40 mM imidazole). RBD was polished on a Superdex 75 16/600 column (GE) equilibrated with 1X PBS, while Spike was directly dialyzed into 1X PBS using SnakeSkin^TM 3,500 MWCO dialysis tubing. Proteins were concentrated with VivaSpin® centrifugal concentrators, centrifuged at 21,000 × g for 30 min to remove precipitates, and flash frozen at 1 mg/mL.

The receptor-binding domain (RBD; aa 330–532) of SARS-CoV-2 Spike protein (Genbank MN908947) was inserted into the pOPINTTGneo expression vector fused to an N-terminal signal peptide and a C-terminal 6xHis tag²² . RBD protein was produced by transient transfection in Expi293F™ cells (ThermoFisher Scientific, UK) using purified DNA (1.0 mg/L cells), a 1:3 DNA:L-PEI ratio, and sodium butyrate as an additive. Cells were grown in suspension in FreeStyle293™ expression medium at 37 °C with 8% CO₂ and 75% humidity. Supernatants from transfected cells were harvested three days post transfection and the supernatant was collected by centrifugation. Clarified supernatant was incubated with 5 mL of Ni²⁺ IMAC Sepharose^®6 Fast Flow (GE) at room temperature for 2 h. Using gravity flow, resin was washed with 50 CV of base buffer (1X PBS) and 50 CV of WB (1X PBS + 25 mM imidazole) before elution with EB (0.5 M imidazole in 1X PBS). Protein was concentrated using a 10,000-MWCO Amicon Ultra-15 before application to a Superdex 75 16/600 column pre-equilibrated with 1X PBS pH 7.4. Peak monomeric fractions were pooled and concentrated to 2 mg/mL, flash-frozen in LN₂, and stored at −80 °C until use. The final purified yield was >15 mg of RBD protein per L of transfected cells.