His select ni2

Codon-optimized (DNA 2.0) DNA constructs corresponding to the mature form of human LRPPRC (amino acid 60–1394) or SLIRP (18–109) were cloned in a pJexpress 401 (DNA 2.0) vector and the LRPPRC–SLIRP complex in pCDFDuet-1 (Novagen). LRPPRC harbours a 6×His fusion tag at the N-terminus in both vectors whereas SLIRP was His-tagged in the pJexpress 401 vector and untagged in pCDFDuet-1. The proteins were expressed in Rosetta 2 cells (EMD chemicals) by induction with 0.5 mM isopropyl-1-thio-β-D-galactopyranoside (IPTG) at 30°C for 16 h in Enpresso B media (Biosilta). After lysis, the proteins were purified over a His-Select Ni²⁺ (Sigma-Aldrich) resin and dialysed against H-0.2 (25 mM Tris–HCl [pH 7.8], 0.5 mM ethylenediaminetetraacetic acid (EDTA), 10% glycerol, 1 mM dithiothreitol, 200 mM NaCl) after the addition of TEV protease at a 1:50 protease:protein ratio. Further purification was conducted over a heparin column equilibrated in H-0.2. After washing with H-0.2 the proteins were eluted with H-0.6 and purified to homogeneity over a HiLoad 16/60 Superdex 200 pg gel filtration column (GE Healthcare) in buffer H-0.2 lacking glycerol.

Codon-optimized (Genscript) DNA constructs corresponding to amino acids 39-216 of human REXO2 was cloned in a pETM-11 vector (EMBL). REXO2 was expressed in Rosetta 2 cells (EMD chemicals) by autoinduction in Magic media (Invitrogen) at 25°C for 16 hours. After lysis, the proteins were purified over a His-Select Ni²⁺ (Sigma-Aldrich) resin and dialyzed against H-0.2 (25 mM Tris-HCl [pH 7.8], 0.5 mM EDTA, 10% glycerol, 1 mM DTT, 0.2 M NaCl) after the addition of TEV protease at a 1:10 protease:protein ratio. Further purification was conducted over a HiLoad 16/60 Superdex 200 pg gel filtration column (GE Healthcare) in buffer H-0.2 lacking glycerol and concentrated using Vivaspin concentrators (10,000 Da MWCO, Sartorius). Dimerization mutants were dialyzed against H-0.1 after the Ni²⁺ purification step and further purified over a Hitrap-Q (1 ml) column equilibrated in H-0.1. After washing with H-0.1 the proteins were eluted with a 30 mL linear gradient between H-0.1 and H-1.0. The dimerization state was determined on a Superdex 75 increase (GE Healthcare) column equilibrated in H-0.5 lacking glycerol.