Hiprep q hp 16 10

The DNA fragment encoding the RHA^N300 (RHA residues 1–300) [11 (link)] was cloned into a modified pMCSG7 vector that incorporates an N-terminal His₆-Mocr for enhanced protein expression and solubility [32 (link)]. The recombinant RHA^N300 protein was expressed in E.coli BL21-CodonPlus (DE3)-RIPL cells (Agilent Technologies) induced with 0.3 mM IPTG at 37°C for 5 h. The harvested bacteria were lysed in buffer A (40 mM NaH₂PO4, pH 7.5, 300 mM NaCl). Then the fusion protein was isolated on a Cobalt column (HisPur Cobalt Resin, Thermo Scientific) for affinity chromatography. RHA^N300 was eluted with Buffer A supplemented with 120 mM imidazole. Pooled fractions were dialyzed against dialysis buffer (30mM Tris, pH 7.5, 100 mM NaCl, 2 mM EDTA, 5 mM β-Mercaptoethanol) supplemented with TEV protease (1 mg protease/ 50 mg of RHA^N300) for His₆-Mocr tag cleavage at 4°C for 20 h. After dialysis, the preparation was loaded to an anion exchange column (Hiprep Q HP 16/10, GE Healthcare) for removing the His₆-Mocr tag and contamination of nucleic acids. The untagged proteins were purified by size exclusion chromatography using a Superdex 75 column (GE Healthcare) equilibrated with ITC Buffer (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 300 mM KCl, 1 mM MgCl₂, 1 mM β-Mercaptoethanol). RHA^N300 was concentrated to 6 mg/ml for storage at −80°C.