Strep tactin resin

Given the oxidative instability of Fe²⁺ bound to HsARD, it is necessary to purify the enzyme under anaerobic conditions, with buffers degassed and argon saturated prior to use, and all steps after lysis performed in an anaerobic chamber (Coy Mfg., Ann Arbor, MI). After cell lysis, cell extracts were collected anaerobically in a sealed centrifuge tube to maintain an anaerobic head space during centrifugation. Supernatants collected after centrifugation were passed through Strep-Tactin resin (IBA Life Sciences) pre-equilibrated with wash buffer. The bound protein was washed with five column volumes (CV) of wash buffer and eluted with two CV of elution buffer (200 mM HEPES, pH 7.0, 10 mM D-desthiobiotin). Eluted Fe-HsARD was then concentrated and exchanged by microcentrifugation with Amicon filter concentrators into storage buffer (degassed and Ar-saturated 50 mM HEPES, pH 7.0, 100 mM NaCl), and stored at −80° C.

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).