Superdex 200 increase 10 300 gel filtration chromatography

All proteins were expressed in Escherichia coli Rosetta (DE3) strains that provide additional tRNAs. For purification of individual full-length dyskerin or dyskerin_ΔC protein, cells were resuspended in 50 mM Tris-Cl pH 7.3, 400 mM NaCl, 5% glycerol, and 2 mM DTT and lysed by a microfluidizer. Proteins were purified through HisTrap-HP column (GE Healthcare) and Superdex 200 increase 10/300 gel filtration chromatography (GE Healthcare). For reassembly and purification of dyskerin_ΔC-NOP10- GAL1_53-145, the heterotrimer was co-expressed from a pETDuet-1 plasmid expressing dyskerin_ΔC and Nop10 and a pET28a plasmid expressing GAL1_53-145. The complex was purified through a HisTrap-HP column (GE Healthcare) and Superdex 200 increase 10/300 gel filtration chromatography (GE Healthcare) in buffer (50 mM Tris-Cl pH 7.3, 350 mM KCl and 2 mM DTT). An additional 5 mM DTT was provided before storage at -80°C.