Imidazole

The human FTO gene was cloned into a pET28a vector and transformed into BL21(DE3) cells (NEB). Successfully transformed bacteria were cultured at 37°C in 2xYT broth Teknova) to an O.D. of 0.8–1.0. The culture was cooled to 16°C and supplemented with 0.1 mM IPTG (Sigma), 10 μM ZnSO₄ (sigma), and 2 μM (NH₄)₂Fe(SO₄)₂ (sigma). Bacteria were cultured overnight at 16°C following induction.
Bacteria were collected via centrifugation and lysed in buffer D (300 mM NaCl (Fisher), 50 mM imidazole (Fisher), and 50 mM of Na₂HPO₄ (Sigma); pH 8.0). The lysate was clarified by centrifugation and loaded onto a nickel column (Ni Sepharose 6 FF, Cytiva), washed with buffer E (150 mM NaCl, 25 mM imidazole, and 10 mM Tris-HCl (Invitrogen); pH 7.5), and eluted with buffer F(150 mM NaCl, 250 mM imidazole, and 10 mM Tris-HCl; pH 7.5). The eluate was loaded onto an anion-exchange column (SOURCE 15Q, Cytiva) and fractionated with 0–50% of Buffer G (1.5 M NaCl, 20 mM Tris-HCl; pH 7.5) over 30 min. The resulting protein was concentrated and buffer exchanged using a 10 kD MWCO filter (Cat. No. 28932296, Cytiva) before being flash frozen in 30% glycerol for future use.

HEK293T cells were seeded at 15 × 10⁶ cells/25 mL/flask in T-150 flasks and transfected via the linear PEI method. Culture media were changed to serum-free DMEM 16 hours post-transfection, and supernatants were harvested at 48 and 72 hours post-media change. Supernatants from different collection times were pooled, and His-tagged proteins were batch-bound to Ni-NTA resin (Life Technologies) in binding buffer (500 mM NaCl, 20 mM Tris, pH 8.0) for 1 hour prior to being washed three times with binding buffer supplemented with 20 mM imidazole (Fisher Scientific, Hampton, NH), and then eluted with binding buffer supplemented with 500 mM imidazole through a chromatography column. Eluted proteins were buffer-exchanged into storage buffer (50 nM NaCl, 20 mM Tris pH7.4, 10% glycerol) by successive concentration and resuspension steps in Amicon centrifugal columns (10 kDa; EMD Millipore, Billierica, MA) following manufacturer’s recommendations. Purified proteins were then aliquoted and frozen at −20°C.