Amicon ultra 10 kda molecular weight cutoff concentrator

An uncleaved version of the ectodomain (residues 1–1208) of SARS-CoV-2 spike protein, namely SARS-CoV-2 S-2P was constructed as sorting probe that contains the following modifications - D614G mutaiotn, ₆₈₂RRAR₆₈₅ →SGAG substitution at the furin cleavage site, and two proline substitutions ₉₈₆ KV₉₈₇→PP. Additionally in the S-2P construct, the C terminus of the S ectodomain is appended with a GSG peptide linker, a foldon trimerization motif, an HRV3C protease cleavage site, an 8 × His Tag, and a TwinStrep Tag (SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK) as previously described.^{75 (link),76 (link)} The S-2P protein encoding sequence were codon optimized for human cell expression (GenScript, NJ), cloned into the mammalian cell expression vector pcDNA3.1(−), and confirmed by sequencing prior to transient transfection in FreeStyle 293-F cells with 293fectin transfection reagent (Thermo Fisher Scientific). Culture supernatants were harvested at 5 days post transfection, filtered, and purified by StrepTactin resin (IBA Lifesciences). Elutes were subjected to size exclusion chromatography using a Superpose 6 10/300 GL column (Sigma-Aldrich, MO) to collect trimer fraction. Elutes from both purification procedures were concentrated and buffer exchanged with phosphate-buffered saline (PBS) with an Amicon Ultra 10 kDa molecular weight cutoff concentrator (Millipore).