Superdex 200 size exclusion column

Wild-type (WT) and truncated His₆-tagged OA was expressed in a polycistronic pST39 vector in BL21 cells and induced with 0.4 mM IPTG for 16 hr at 18°C. Cells were lysed with a French press and OA was immobilized on a Ni-charged IMAC resin column (Bio-Rad) in 50 mM sodium phosphate buffer, pH 7.0 supplemented with 200 mM NaCl, protease inhibitors (Roche), 5 mM imidazole, and 1 mM PMSF, washed, and eluted in the same buffer with 300 mM imidazole. OA was further purified using a Superdex 200 size exclusion column (GE Healthcare) in SEC-OA buffer (50 mM sodium phosphate buffer, pH 7.0 supplemented with 200 mM NaCl.)
WT and truncated FLAG-tagged OA was expressed similarly to His₆-tagged OA except that it was immobilized on an anti-FLAG M2 affinity gel (A2220; Sigma). The affinity gel was washed, and protein was eluted with 0.1 mg·mL⁻¹ 3X FLAG Peptide (F4799; Sigma). OA was further purified using a Superdex 200 size exclusion column (GE Healthcare) in SEC-OA buffer.
After SEC, OA was concentrated with Amicon Ultra centrifugal filters (Millipore), stored in SEC-OA buffer with 5% glycerol, and immediately stored at −80°C.

All GST- and MBP-tagged constructs were expressed and purified using glutathione-conjugated sepharose (GE Healthcare) or Amylose sepharose (NEB), respectively. The GST- and MBP-tagged proteins were cleaved using GST-PreScission Protease (GE Healthcare) and TEV protease (Sigma), respectively. The cleaved proteins were applied to Superdex 75 or Superdex 200 size-exclusion columns (GE Healthcare) that were equilibrated using respective buffers. Ube1 was purified using Ni-NTA-conjugated sepharose (Roche). The purified protein was then applied to the Superdex 200 size-exclusion column.

Wild-type or P1482T mutant ProRS fragment of human EPRS1 (aa 930-1512) was subcloned in pTRC-HisB (Invitrogen) for N-terminal 6X-His tagging, and sequence verified. Recombinant protein was expressed in BL21 Codon-plus (DE3)RIPL (Agilent) strain as described^{21 (link),103 (link)}. Briefly, protein was induced with isopropyl β-D-1-thiogalactopyranoside (200 μM) at 37 °C, and cells harvested by centrifugation 4–6 h post-induction. The pellet was resuspended in purification buffer containing 50 mM Tris-HCl, pH 8.0, 100 mM NaCl,10% glycerol,1 mg/ml lysozyme, protease inhibitors, and 10 mM imidazole, and sonicated on ice for 20 min. Lysate was cleared by centrifugation at 26,000 × g for 45 min, and purified using HisTrap HP column (GE Life Sciences, Pittsburgh, PA). Protein oligomeric state was determined using a Superdex 200 size-exclusion column (GE Life Sciences) pre-calibrated with Bio-Rad gel-filtration standards (Bio-Rad) in purification buffer on an NGC Chromatography System with ChromLab v5.0.2.11 (Bio-Rad). ProRS aminoacylation activity was confirmed as described^{21 (link)}.

Genes encoding EsaG, EsaDc, NT_L and CT_L were cloned into a modified pRSF-Duet vector which is generated by inserting the sequence of SUMO-tag ahead the multiple cloning site, respectively. The proteins were over-expressed with a fused N-terminal His₆-SUMO tag in E. coli BL21(DE3) cells. Cells were induced with 0.4 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at an optical density at 600 nm of 0.8 and grown at 16 °C overnight. The fusion proteins were first purified using a nickel column. The His₆-SUMO tag was removed by ULP1. The tagless proteins were further purified using ion-exchange column (GE Healthcare) and the Superdex 200 size exclusion column (GE Healthcare). For NMR study, the ¹⁵N-labeled proteins were produced in M9 minimal media supplemented with ¹⁵NH₄Cl as sole nitrogen resource, and the ¹⁹F-labeled EsaDc was produced in M9 minimal media supplemented with 120 mg/L of 5-fluoro-D/L-tryptophan.

Csy genes and a synthetic crRNA were co-expressed on separate vectors in E. coli BL21 (DE3) cells as previously described (27 (link)). Expression was induced with 0.5 mM isopropyl-β-d-1-thiogalactopyranoside (IPTG) at OD₆₀₀ = 0.5 nm. Cells were incubated overnight at 16°C, then pelleted by centrifugation (5000 x g for 15 min at 4°C) and re-suspended in lysis buffer (50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH7.5, 300 mM potassium chloride, 5% glycerol, 1 mM Tris(2-carboxyethyl) phosphine hydrochloride (TCEP), 1x protease inhibitor cocktail (Thermo Scientific)). Pellets were sonicated on ice for 2 × 2.5 min (1 s on, 3 s off), then lysate was clarified by centrifugation at 22 000 x g for 30 min at 4°C. The Csy complex self-assembles in vivo and the intact complex was affinity purified on Ni-NTA resin (Qiagen) using hexa-histidine tags on either Csy3 or Csy1. Elution was performed using the lysis buffer supplemented with 300 mM imidazole. Tobacco etch virus (TEV) protease was added to remove the N-terminal His₆ tags and the sample was dialyzed at 4°C overnight in gel filtration buffer (20 mM HEPES pH 7.5, 100 mM KCl, 5% glycerol, 1 mM TCEP). Protein was concentrated (Corning Spin-X concentrators) at 4°C before further purification on a Superdex 200 size-exclusion column (GE Healthcare).