Hiload superdex 200

Plasmid DNA of pET15b-His-3C-Dot1 (158-582) was generated using Gibson Assembly (NEB). Yeast Dot1 plasmid DNA was transformed into ONESHOT™BL21(DE3) (ThermoFisher) competent cells and grown in 2xYT-Amp media. Yeast Dot1 was expressed as soluble protein by inducing with 0.1mM IPTG for 16 hours at 18°C upon the culture reaching OD600 = 0.4-0.6. Cells were harvested (Sorvall LYNX6000) and lysed (AvestinEmulsiflexC3), and the protein was purified through Ni-NTA agarose beads (Qiagen) (Lysis Buffer: 500 mM NaCl, 20 mM HEPES pH 7.5, 20 mM Imidazole, 1 mM BME, 1× Protease Inhibitor / Elution Buffer: 500 mM NaCl, 20 mM HEPES pH 7.5, 300 mM Imidazole, 1 mM BME). Eluted yeast Dot1 protein was digested with 3C PreScission Protease (GE Healthcare) while dialyzed in digestion buffer (Digestion Buffer: 100 mM NaCl, 20 mM HEPES pH 7.5, 1 mM DTT) overnight at 4°. After which, sample was purified through HiTrap SP HP (GE Healthcare) liquid chromatography column (Buffer A: 100 mM NaCl, 20 mM HEPES pH 7.5, 1 mM DTT / Buffer B: 1 M NaCl, 20 mM HEPES pH 7.5, 1 mM DTT). Selected fractions were further purified through HiLoad Superdex 200 (GE Healthcare) size-exclusion liquid chromatography column (S200 Buffer: 150 mM NaCl, 10 mM Tris pH 7.5, 5 mM DTT). Purified yeast Dot1 protein was then concentrated, flash frozen in liquid nitrogen and stored in −80°C for future use.

Medaka TRBD (residues 318–579) was expressed in Escherichia coli strain ScarabXpress T7lac cells (Scarab Genomics) with a modified pMAL-2CX vector with an MBP protein fused at its N terminus. After induction for 16 h with 0.1 mM IPTG at 20 °C, the cells were harvested by centrifugation, and the pellets were resuspended in lysis buffer (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 1 mM MgCl₂, 10% glycerol, 1 mM Tris(2-carboxyethyl)phosphine (TCEP), 1 mM PMSF, 5 mM benzamidine, 1 μg/ml leupeptin and 1 μg/ml pepstatin). The cells were then lysed by sonication, and the debris was removed by ultracentrifugation. The supernatant was subjected to 55% ammonium sulfate precipitation. The precipitated proteins were resuspended in lysis buffer and were mixed with amylose resin (New England BioLabs) for 2 h, and then the MBP-TRBD proteins were eluted with 10 mM maltose. After purification by gel-filtration chromatography on HiLoad Superdex200 (GE Healthcare), the MBP-TRBD proteins were treated with protease 3C at 4 °C overnight and were then rebound to the amylose resin to remove the MBP proteins. After a final step of gel-filtration chromatography on HiLoad Superdex200, the purified proteins were concentrated to 10 mg/ml and stored at −80 °C. Wild-type and mutant TRBD proteins used for ITC measurements were purified similarly.