His select ni2 resin

A codon-optimized (Genscript) DNA construct corresponding to the mature form of human C6orf203 (amino acids 85–240) was cloned into a Fh8-Pet24d vector. Human C6orf203 was expressed in Arctic express cells (Agilent) at 16°C for 48 h in Magic Media (Thermo Fisher Scientific) with an approximate yield of 5 mg/l, assessed by Bradford protein assay. After lysis, the proteins were purified over a His-Select Ni²⁺ resin (Sigma-Aldrich) and dialyzed against H-0.2 (25 mM Tris-HCl (pH 7.4), 0.5 mM EDTA, 10% glycerol, 3 mM β-mercaptoethanol and 200 mM NaCl) after the addition of TEV protease at a 1:25 protease:protein ratio. The dialyzed protein was loaded on a second Ni²⁺ resin for TEV protease removal and the flow-through was collected. Further purification was conducted over a HiLoad 16/60 Superdex 200 pg gel filtration column (GE Healthcare) in buffer H-0.2 lacking glycerol with the addition of 2 mM dithiothreitol instead of β-mercaptoethanol. The purity was estimated at ∼95% following SDS-PAGE gel electrophoresis and staining with Coomassie (Supplementary Figure S1A). RNA EMSA were performed as previously described (19 (link)).

A codon-optimized (Genscript) DNA construct corresponding to the mature form of human mtIF2 (amino acids 38–727) or human mtIF3 (amino acids 32–278) was cloned into a pET-24b vector (Novagen). Both constructs were expressed in Rosetta 2 cells (EMD chemicals) at 25 °C for 16 h in Magic Media (Thermo Fisher Scientific). After lysis, the proteins were purified over a His-Select Ni²⁺ resin (Sigma–Aldrich) and dialyzed against H-0.2 (25 mM Tris-HCl pH 7.4, 0.5 mM EDTA, 10% glycerol, 1 mM DTT, 200 mM NaCl). Further purification was conducted over a HiLoad 16/60 Superdex 200 pg gel filtration column (GE Healthcare) in buffer H-0.2 lacking glycerol.
The first 30 or 51 nucleotides of mitochondrially encoded cytochrome C oxidase 2 (MT-CO2) mRNA was purchased from Sigma–Aldrich or Eurofins, respectively.
The unlabeled E. coli, fMet-tRNA^Met_i was generously provided by Prof. Marina Rodnina, and the fMet-tRNA^Met_i labeled at the dihydrouridine residues with Cy3 by Prof. Barry S. Cooperman¹⁷ .

A codon-optimised (Genscript) DNA construct corresponding to DmANGEL (amino acids 26–354) was cloned into a Pet24b vector and HsANGEL2 (amino acids 165–544) or MmANGEL2 (amino acids 164–544) was cloned into a Fh8-Pet24d vector. HsANGEL2 and MmANGEL2 were expressed in Arctic express cells (Agilent) at 16 °C for 48 h in Magic Media (Thermo Fisher Scientific). After lysis, the proteins were purified over a His-Select Ni2+ resin (Sigma-Aldrich) and dialysed against H-0.5 (25 mM Tris pH 7.4, 0.5 mM EDTA, 10% glycerol, 3 mM β-mercaptoethanol, 500 mM NaCl) after the addition of TEV protease at a 1:50 protease:protein ratio. The dialysed proteins were loaded on a second Ni2+ resin for TEV protease removal and the flow through was collected. Further purification was conducted over a HiLoad 16/60 Superdex 200 pg gel filtration column (GE Healthcare) in buffer H-0.5 lacking glycerol with the addition of 2 mM dithiothreitol instead of β-mercaptoethanol. DmANGEL was expressed and purified analogous to HsANGEL2 except that no His tag removal was performed.