Superose 6

For large-scale purification both RSV full-length and truncated and HMPV full-length constructs utilized the same method. The Sf9 cell pellets were resuspended in lysis buffer (50 mM Tris pH 8.0, 200 mM NaCl, 1 mM TCEP, 10% glycerol) and were lysed by sonication. The lysate was then centrifuged, and protein complex captured with a prepacked Anti-FLAG M2 agarose column (Sigma) that had been equilibrated in lysis buffer. The protein complex was eluted with 100 µg/mL 3xFLAG peptide (Sigma) and subsequently loaded onto a Heparin HP column (Cytiva). The protein was gradient eluted (200 mM NaCl to 1 M NaCl) from the Heparin column. Additional purification utilized size-exclusion chromatography (Superose 6, Cytiva) using a buffer of 25 mM HEPES pH 7.4, 300 mM NaCl, 6 mM MgSO₄, 0.5 mM TCEP. Final protein preparations were concentrated to 2 mg/mL for RSV and HMPV full-length complexes and 1.2 mg/mL for the RSV truncated complexes. Protein preparations were flash-frozen and stored at −70 ˚C until use.

Prior to expression, all expression construct plasmids were sequence verified (ACGT). Subsequently, constructs were transiently transfected (SC and D1 variants) or co-transfected (Fcα and JC) into Expi293F (Gibco: A14527) cells using the ExpiFectamine 293 Transfection kit (Gibco: A14525) and following previously described methods (11 (link)). Four to five days after transfection, supernatants were harvested and filtered through 0.22μm PES bottletop filters (Millipore Sigma). Proteins and complexes were purified from the cell supernatants using Nickel-NTA affinity resin (Qiagen: 30210), followed by Superose 6 (Cytiva) or Superdex 200 (Cytiva) size exclusion chromatography (SEC) on an AKTA FPLC (Cytiva). During and after SEC, purified molecules were maintained in TRIS-buffered saline (20mM Tris+ 150mM NaCl, pH=7.4). The SEC-eluted protein peaks corresponding to the correct retention volume based on the molecular weight standards (BioRad: 1511901) were collected and concentrated using Amicon Ultra centrifugal filters (Millipore Sigma) to stock concentrations between 2–4mg/ml for SC, 1.5–2mg/ml for D1, and 0.4–0.7mg/ml for dFcα constructs, and were then filtered using 0.22μm spin filters (Millipore Sigma) and utilized for SPR experiments.

The ectodomains of EBOV GP_ΔTM (residues 1–636; Makona variant; GenBank: KM233070), BDBV GP_ΔTM (residues 1–643; strain200706291 Uganda; GenBank: NC_014373), SUDV GP_ΔTM (residues 1–637; Gulu variant; GenBank: NC_006432), and MARV GP_ΔTM (residues 1–648; Angola2005 variant; GenBank: DQ447653) were expressed transiently in Expi293F cells with a C-terminal strep II tag using the pcDNA3 plasmid vector. Secreted proteins were purified from filtered supernatant using 5 mL StrepTrap HP columns (Cytiva, Chicago, Il, USA) following the manufacturer’s protocol, and subjected to size exclusion chromatography using Superose 6 (Cytiva, Chicago, Il, USA) and buffer exchanged into PBS. Trimer fractions were pooled from SEC tested in ELISA through binding against control antibodies.