Production of 13C, 1H Ile-δ1–labeled TR

Plasmids encoding His₆-SUMO-TR and corresponding point mutants were transformed into E. coli C41 (DE3) cells. Each Ile in TR was mutated one at a time to Leu (except I232 that was mutated to Ala, and I522 that was mutated to Ser) by site-directed mutagenesis using the Phusion Master Mix with HF Buffer and Q5 High-Fidelity 2× Master Mix (New England BioLabs). Fresh C41 (DE3) colonies were inoculated into starter LB cultures supplemented with ampicillin (100 mg/L), grown at 37 °C until the A₆₀₀ reached 0.6, centrifuged (4000 × g for 15 minutes at 4 °C). The cell pellet was re-suspended in a starter M9 medium prepared in D₂O and grown overnight. The overnight culture was inoculated into a 0.5 L D₂O-based M9 medium supplemented with 1 g/L ¹⁵NH₄Cl and 2 g/L ² H-glucose and grown at 37 °C. When the A₆₀₀ reached 0.5, 50 mg/L (methyl-¹³C, 3,3-d₂) α-ketobutyric acid (Cambridge Isotope Laboratories) was added to the growth that was incubated for 1 hour, followed by induction with 0.5 mM IPTG at 18 °C for 22 hours. Cells were harvested by centrifugation (2820 × g for 30 minutes at 4 °C) and re-suspended in lysis buffer (50 mM Tris at pH 7.8, 500 mM NaCl, 150 mM KCl, 5 mM β-mercaptoethanol, 20 mM imidazole, and 1 mM CaCl₂). After lysis by sonication, the lysate was centrifuged (15,000 × g for 30 minutes at 4 °C). The supernatant was extracted and incubated with Ni-NTA beads (Qiagen) for 1 hour at 4 °C. The beads were poured into an Econo-Column (Bio-Rad), washed with 10 column volumes of lysis buffer, and eluted with elution buffer (lysis buffer containing 250 mM imidazole). ULP1 protease (1:1000 ULP1 to fusion mass ratio) and λ-phosphatase (0.25 nanomoles) were added into the eluate, which was then dialyzed against 4 L dialysis buffer (20 mM Tris at pH 7.5, 150 mM NaCl, 5 mM β-mercaptoethanol, and 1 mM MnCl₂) at 4 °C for 16 hours, using 12–14 kDa MWCO dialysis bag (Spectra/Por). Dialyzed samples were centrifuged (15,000 × g for 30 minutes at 4 °C), filtered (0.22 μm), and purified over a 5 mL HiTrap Q HP (GE Healthcare Biosciences) column using a linear gradient of 0.2 – 0.8 M NaCl (20 mM Tris at pH 7.5, 5 mM β-mercaptoethanol). The TR sample was further purified using a gel filtration column (Superdex 200 10/300 GL, GE Heathlcare Biosciences) pre-equilibrated with NMR buffer (20 mM BisTris at pH 6.8, 150 mM KCl, 10 mM CaCl₂, 5 mM β-mercaptoethanol, and 0.1 % NaN₃).