Recombinant Notum Enzyme Production

The human Notum enzyme core comprising amino acids S81–T451 with a C330S mutation^{16 (link)} was cloned into a stable cell line vector pNeo_sec^{46 (link)}. A stable polyclonal cell line was obtained by G418 selection of HEK293S GNTI- (ATCC CRL-3022) cells^{47 (link)}. The stable cells were cultured in DMEM (high glucose, Sigma) supplemented with 1 mM glutamine, 1× non-essential amino acids and 10% foetal bovine serum (Invitrogen) at 37 °C with 5% CO₂. Large scale protein expression was performed by growing cells in expanded surface roller bottles (Greiner). For protein purification, the dialyzed conditioned media were passed through a 5 ml HisTrap Excel column (GE Healthcare) which was then washed with 20 mM imidazole PBS, and Notum protein was eluted with 300 mM imidazole PBS. To remove flexible glycans, the protein was deglycosylated with endo-β-N-acetylglucosaminidase F1 (37 °C, 1 h) and further purified by size-exclusion chromatography (Superdex 200 16/600 column, GE Healthcare) in 10 mM Hepes, pH 7.4, 150 mM NaCl buffer.