Expifectamine 293 kit

The sequence for the WT CSF3R 5FnIII domain (residues 530–626) with the CSF3R signal peptide (residues 1–24) and a C-terminal 6xHis tag was cloned into a pAdd2 mammalian expression vector at the EcoRI and XhoI sites by Gibson assembly, as described above. Expi293F cells were transfected with the pAdd2 5FnIII construct using the ExpiFectamine 293 kit (Thermo Fisher Scientific A14524) according to the manufacturer’s protocols. Six days after transfection, the supernatant was centrifuged at 300g for 5 min, adjusted to a pH of 8 with NaOH, centrifuged again at 3700g for 20 min, and filtered with a 0.22 μm bottle-top filter (EMD Millipore S2GPT02RE). His-tagged CSF3R protein was enriched by nickel-nitrilotriacetic acid agarose (Qiagen 30210) and further purified by size-exclusion chromatography on a Superdex 75 10/300 Gl column (Cytiva 17517401) with an AKTA Pure system (Cytiva).

The RBD antigen was prepared as described [8 ]. Briefly, The RBD sequence is based on the genomic sequence of the first virus isolated Wuhan-Hu-1 and optimized for mammalian cell expression. The natural Spike N terminal signal peptide (amino acids 1–14) was fused to the RBD amino acids 319–541 and also fused with a C-terminal hexahistidine tag to allow isolation. The recombinant protein was produced in Expi293F cells (Thermo fisher) by transfecting these cells with purified DNA using Expifectamine 293 kit (Thermo fisher). Three days post-transfection the cells were harvested and the RBD protein was isolated from the supernatant by 2 hours incubation at RT with HisPure Ni-NTA resin (Thermo). The RBD was eluted and dialyzed against PBS.