Protein expression and purification

The C-terminal domain of RCD1 for NMR study was expressed as GST-fusion protein in E.coli BL21 (DE3) Codon Plus strain and purified using GSH-Sepharose beads (GE Healthcare) according to the manufacturer’s instruction. Cleavage of GST tag was performed with thrombin (GE Healthcare, 80 units per mL of beads) for 4 hr at room temperature and the C-terminal domain of RCD1 was eluted from the beads with PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.4). The protein was further purified by gel filtration with HiLoad 16/600 Superdex 75 column (GE Healthcare) equilibrated with 20 mM sodium phosphate buffer (pH 6.4), 50 mM NaCl at 4°C.