Imidazole

The CM from transfected 293F cells were collected, centrifuged at 800g for 5 minutes, filtered with a 0.22 μm filter (Thermo Fisher Scientific), and stored at –80°C before processing. Affinity purification was performed using an Akta Pure fast protein liquid chromatography system (Cytiva, formerly GE Healthcare). The S-RBD-His product (S-RBD1) purification was performed using nickel-nitrilotriacetic acid resin, where samples were supplemented to a final concentration of 30 mM imidazole (MilliporeSigma) and 1× EDTA-free protease inhibitor cocktail (Roche) before loading onto a HisTrap FF 1 mL column (Cytiva, formerly GE Healthcare), equilibrated in wash buffer (PBS 300 mM NaCl, 40 mM imidazole, pH 8.0), and eluted with a linear gradient from 40 to 500 mM imidazole. Eluted fractions were pooled, concentrated with 3000 Da (MW cutoff) ultrafiltration membrane spin column (Amicon) per the manufacturer’s instructions, dialyzed into PBS, and stored at –80°C. The purified S-RBD1 was quantified using NanoDrop OneC (280 nm absorbance, Thermo Fisher Scientific), BCA assay (Thermo Fisher Scientific) relative to bovine serum albumin (BSA) standard, and/or Qubit 3 Fluorometer (Qubit Protein Assay, Thermo Fisher Scientific) according to the manufacturer’s instructions. The reagents for the protein purifications are listed in Supplemental Table 1.

LALF_32-51-E7 crude extracts were prepared from 50 g FLW using the enhanced extraction strategy. Imidazole (Merck Millipore) and NaCl were added to a final concentration of 20 mM and 500 mM respectively. A 5 ml Ni²⁺ affinity column (HisTrap^™ HP, GE Healthcare) was equilibrated with 5 column volumes (CV) of equilibration buffer [500 mM NaCl, 8 M urea, 20 mM Imidazole in 50 mM Na-PO₄ pH 8.0]. The crude extract was loaded onto the column using a 150 ml SuperLoop. Thereafter the column was washed with 10 CV of equilibration buffer or wash buffer [equilibration buffer at pH 6.5]. LALF_32-51-E7 was eluted over 20 CV of a linear gradient (from 0% to 100%) of elution buffer [equilibration buffer with 500 mM Imidazole] and an additional 5 CV step of 100% elution buffer. The ÄKTA Explorer^™ system and the UNICORN 4.11 software (GE Healthcare) were used. All fractions were collected in 5 ml aliquots and stored at -20°C. Relevant fractions were analysed by western blots and SDS-PAGE gels.
Relevant elution fractions were pooled and extensively dialysed against 130 v/v renaturing buffer [10 mM Tris, pH 8.0]. The dialysis was carried over 4 h, followed by an overnight round and a final round of 4 h. Thereafter, dialysed samples were filter-sterilized through a 0.2 μm Corning^® syringe filters (Sigma-Aldrich) and stored at 4°C.

Human FcγRI, FcγRIIa_R131 and FcγRIIIa_V158 receptors used in this work were expressed in-house in HEK293F for FcγRI, FcγRIIa_R131 (transient expression) and in CHO cells for FcγRIIIa_V158, respectively. The CHO DG44 cell line was stably transfected [36 (link)]. Purification of the receptor was achieved by affinity chromatography using Ni Sepharose High Performance material (GE Healthcare, Munich, Germany) followed by elution with 300 mM imidazole (Sigma, Munich, Germany) an case of FcγRI, FcγRIIa_R131 and 100 mM imidazole in case of FcγRIIIa_V158, and a size exclusion chromatography on Superdex 200 26/60 column (GE Healthcare, Munich, Germany) with PBS pH7.4 (FcγRI, FcγRIIa_R131) or 2 mM MOPS, 150 mM NaCl, 0.02% Tween 20 pH 7.0 buffer (FcγRIIIa_V158). Fcγ receptors were biotinylated using the biotinylation kit from Avidity according to the manufacturer instructions (Bulk BIRA, Avidity LLC, Denver, CO, USA) and dialyzed at 4°C over night to remove excess of biotin. The product quality was characterized by standard methods. The glycosylation pattern of FcγRI and FcγRIIa is not relevant for the antibody interaction, and was not addressed in detail. Analysis of the glycosylation pattern of FcγRIIIa was described previously [36 (link)].