Purification of RGI3 Extracellular Domain

The ECD construct of RGI3 (RGI3^ECD) was previously described (Smakowska‐Luzan et al, 2018 (link)). RGI3^ECD was cloned into the baculovirus transfer vector pMeIBac B1 (Invitrogen) using RecA‐mediated sequence and ligation‐independent cloning strategy. A C‐terminal Strep II‐9x His tag was fused to RGI3^ECD. RGI3^ECD‐Strep II‐9x His was produced by secreted expression in baculovirus‐infected High Five insect cells and harvested 72 h postinfection. Subsequently, the protein was purified by nickel affinity chromatography (Ni Sepharose excel beads; GE Healthcare) and subjected to a size exclusion chromatography column (Superdex 200 10/300; GE Healthcare) pre‐equilibrated with 20 mM NaH₂PO₄/ Na₂HPO₄, pH 7.5, 200 mM NaCl, and 5% glycerol.