CBD-SA protein was designed, produced, and purified similarly to previously reported CBD proteins (9). The sequences encoding for the fusion of human VWF A3 domain residues Cys1670-Gly1874 (907-1111 of mature VWF) and mouse SA without pro-peptide (25 to 608 amino acids of whole SA) were synthesized and subcloned into the mammalian expression vector pcDNA3.1(+) by GenScript. A sequence encoding for a His-tag (6-His) was inserted at the C terminus for further purification of the recombinant protein. Suspension-adapted HEK293F cells were routinely maintained in serum-free FreeStyle 293 Expression Medium (Gibco). On the day of transfection, cells were diluted into fresh medium at a density of 1 × 106 cells/ml. Plasmid DNA (2 μg/ml), linear 25-kDa polyethylenimine (2 μg/ml; Polysciences), and OptiPRO SFM medium (4% final concentration; Thermo fisher Scientific) were added. The culture flask was agitated by orbital shaking at 135 rpm at 37°C in the presence of 5% CO2. Seven days after transfection, the cell culture medium was collected by centrifugation and filtered through a 0.22-μm filter. Culture medium was loaded into a HisTrap HP 5-ml column (GE Healthcare), using an ÄKTA pure 25 (GE Healthcare). After washing of the column with wash buffer [20 mM imidazole, 20 mM NaH2PO4, and 0.5 M NaCl (pH 7.4)], protein was eluted with a gradient of 500 mM imidazole [in 20 mM NaH2PO4 and 0.5 M NaCl (pH 7.4)]. The eluent was further purified with size exclusion chromatography using a HiLoad Superdex 200PG column (GE Healthcare). All purification steps were carried out at 4°C. The protein was verified as >90% pure by SDS-PAGE.

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