Recombinant Human Robo4 Extracellular Domain Purification

The recombinant extracellular domain of human Robo4 (Robo4-Ig), including Ig 1 and 2 domains, was expressed as we expressed human Robo1 (46). In brief, human Robo4 Ig1-2 cDNA was cloned into pGEn2 vector and transiently expressed in HEK293 cells^{34 (link)}. Robo4-Ig-GFP-His in conditioned media was purified using a Ni–NTA superflow column (Qiagen, Valencia, CA). Fractions containing fluorescence were pooled and concentrated to ∼ 1 mg/ml using an ultrafiltration pressure cell membrane (Millipore, Billerica, MA) with a 10 kDa molecular weight cut-off. Following, the GFP-His tag was cleaved from Robo4-Ig-GFP-His protein by TEV protease digestion. The GFP-His tag and Tev-protease were removed through the second Ni–NTA column. Flow-through from the second run was collected and concentrated to 2 mg/ml before loading onto Superdex 75 for further purification. Fractions of purified Robo4-Ig were pooled and concentrated to 2 mg/ml and stored at − 80 °C.