Superdex 200 hr column

Purification of Pa-30S and Pa-aIF1A were performed as described in (24 (link)). The untagged version of Pa-aIF2 was purified as follows. Cultures (250 ml) of cells overproducing each of the three subunits of Pa-aIF2 (32 (link)) were harvested, mixed in 40 ml of buffer B (500 mM NaCl, 10 mM HEPES pH 7.5, 10 mM 2-mercaptoethanol, 0.1 mM EDTA, 0.1 mM PMSF, 0.1 mM benzamidine) and disrupted by sonication. After centrifugation, the supernatant was heated for 10 min at 80°C. After pelleting, the supernatant was loaded onto a Q-Hiload column (10 mm × 4 cm; GE Healthcare) equilibrated in buffer B. The flow through was recovered and diluted two fold with buffer A. The sample was then loaded onto an S-Hiload column (10 mm × 4 cm; GE Healthcare) equilibrated in buffer A plus 250 mM NaCl. The assembled heterotrimer was eluted by applying a step of buffer A plus 700 mM NaCl. The recovered sample was then concentrated to 1 ml. The final heterotrimer preparation was obtained after purification by molecular sieving on a Superdex 200 HR column (10 mm × 30 cm, GE Healthcare) equilibrated in buffer B. Finally, glycerol (55% final concentration) and 1 mM Gpp(NH)p-Mg²⁺ were added. The protein was stored at –20°C for further use in toeprinting experiments. Routinely, 6 mg of purified protein were obtained from the three mixed 250 ml cultures.

Refolding was performed as previously described [4 (link)]. Briefly, for a 1 L MEL5 TCR refold, 30 mg of MEL5 α-chain was incubated at 37°C for 30 min with 10 mM DTT and added to refold buffer at 4°C (50 mM TRIS pH 8.1, 2 mM EDTA, 2.5 M urea, 6 mM cysteamine hydrochloride, and 4 mM cystamine). After 30 min, 30 mg MEL5 β-chain, also incubated at 37°C for 30 min with 10 mM DTT, was added. For a 1 L pMHCI refold, 30 mg HLA A*0201 α-chain was mixed with 30 mg β2m and 4 mg EAAGIGILTV peptide at 37°C for 30 min with 10 mM DTT. This mixture was then added to refold buffer at 4°C (50 mM TRIS pH 8.1, 2 mM EDTA, 400 mM L-arginine, 6 mM cysteamine hydrochloride, and 4 mM cystamine). The MEL5 TCR and A2-EAA refolds were mixed at 4°C for 1–3 h and dialyzed against 10 mM TRIS pH 8.1 until the conductivity of the refolds was <2 mS/cm. Refolded proteins were purified by ion exchange using a Poros50HQ™ column (GE Healthcare, Buckinghamshire, UK) and gel filtered into crystallization buffer (10 mM TRIS pH 8.1 and 10 mM NaCl) or BIAcore buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.005% v/v surfactant P20) using a Superdex200HR™ column (GE Healthcare, Buckinghamshire, UK).