SARS-CoV-2 RBD Protein Purification

A SARS-CoV-2 RBD construct containing a His-tag and Avi-tag was generated, as previously described (51 (link)). Residues 319–541 of the S protein were codon optimized with the N-terminal of signal peptide (MFVFLVLLPLVSSQ) and C-terminal of 6-His tag and Avi-tag (GLNDIFEAQKIEWHE). The DNA encoding sequence was cloned into the mammalian cell expression vector pCAGGS and confirmed by sequencing, before transient transfection in FreeStyle 293-F cells with 293fectin transfection reagent (Thermo Fisher). Culture supernatants were harvested at 5 d after transfection, filtered, and purified by in-house packed affinity purification column with cOmplete His-tag purification resin (Roche). Elutes were buffer exchanged with phosphate-buffered saline (PBS) and concentrated with an Amicon Ultra 10 kDa molecular weight cutoff concentrator (Millipore). Biotinylation was performed with a BirA biotin-protein ligase standard reaction kit (Avidity), according to the manufacturer’s instructions. Excess biotin was removed by five buffer exchanges with an Ultra 10K concentrator (Amicon).