Purification of Human Mini-TyrRS Protein

Human TyrRS was purified as previously described.^{7,36,37 (link)} The plasmid of mini-TyrRS (amino acids 1–341) as a gift from Paul Schimmel. Briefly, this plasmid was transformed into E. coli Arctic Express (DE3) cells. The cells were cultured at 37 °C in fresh LB liquid medium (5 g of NaCl, 10 g of bactotrypton, and 10 g of yeast extract per litre). Isopropyl-β-d-1-thiogalactopyranoside (IPTG, 0.5 mM) was used to induce expression when attenuance D_{600 nm} was 1, and the culture was grown for 20 h at 16 °C. Cells were collected by centrifugation at 4000 rpm min⁻¹ for 15 min at 4 °C and then re-suspended in lysis buffer (50 mM Hepes-Na, pH 7.5, 5 mM imidazole pH 7.5, 400 mM NaCl, 1× PMSF, and 2 mM BME). Cells were lysed using a cell disrupter. After centrifugation at 14 000 rpm min⁻¹ for 25 min at 4 °C, the supernatant was loaded onto a nickel-affinity chromatography column (GE-Healthcare) pre-equilibrated with lysis buffer. The protein was eluted with a linear gradient of imidazole (0–300 mM). The desired fractions were pooled, concentrated and subjected to gel-filtration (Superdex 200; GE-Healthcare) chromatography and the protein peak corresponding to homogenous protein in buffer (20 mM Hepes-Na, pH 7.5, 50 mM NaCl, and 2 mM BME) was collected (see Fig. S3†). The quality of the protein purification was validated by SDS-PAGE analysis (see Fig. S4†).