Large-scale Production of Bio-inactive N-SLIT2ΔD2

Large-scale production of bio-inactive human N-SLIT2ΔD2 was performed by transfecting FreeStyle 293-F cells with human N-SLIT2ΔD2 cDNA (1 ug/mL) using PEI reagent (Polyethyleneimine, linear, M.W. 25,000, Thermo Fisher Scientific) (Patel et al., 2012 (link)). After 5 days, the culture medium was loaded onto HisPur Ni-NTA resin (Thermo Fisher Scientific), washed with imidazole (35 mM), eluted with imidazole (250 mM), and desalted using a Pierce Zeba desalting column (7 K MWCO; Thermo Fisher Scientific). Protein molecular weight was determined by immunoblotting using anti-6XHIS-HRP conjugated antibody. Protein activity was assayed by using a spreading assay in RAW264.7 cells, as described previously (Bhosle et al., 2020 (link)). Recombinant human N-SLIT2 was purchased from PeproTech (Cranbury, NJ, USA). Endotoxin levels in all preparations were measured using ToxinSensor Chromogenic LAL Endotoxin Assay Kit (GenScript, Piscataway, NJ, USA) and were less than 0.05 EU/ml.