Expanded surface roller bottles

Human Notum protein was produced in mammalian cells. HEK293S GNTI‐ cells35 were cultured in DMEM (high glucose, Sigma) with 1 mmol/L glutamine, 1× nonessential amino acids and 10% FBS (Invitrogen) at 37°C with 5% CO₂. For large scale production, cells were grown in expanded surface roller bottles (Greiner).
We cloned the human Notum enzyme core sequence comprising S81‐T451 with a C330S mutation (Notum_core)29 into a stable cell line vector pNeo_sec.36 HEK293S GNTI‐ cells35 were co‐transfected with pNeo‐Notum_core and a PhiC31 integrase expression vector (pgk‐phiC31).37 The polyclonal cell population resulting from G418 (1 mg/mL) selection was cultured to produce protein. For protein purification, the dialysed conditioned media were incubated with talon beads for 1 hour at 16°C. The beads were collected and washed with 10 mmol/L imidazole PBS and eluted with 200 mmol/L imidazole PBS. To remove flexible glycans, which can prevent crystal growth, the protein was deglycosylated with endo‐β‐N‐acetylglucosaminidase F1 (37°C, 1 hour) and further purified by size‐exclusion chromatography (Superdex 200 16/60 column, GE Healthcare) in 10 mmol/L Hepes, pH 7.4, 150 mmol/L NaCl buffer.

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.