The expression and purification of DICER and DICER-TRBP

The pXG-DICER and pXGR-TRBP plasmids were gifts from Dr. Narry Kim (Seoul National University, Korea). To express DICER (or DICER-TRBP), pXG-DICER (or pXG-DICER and pXGR-TRBP) was transfected into 100 of 100 mm dishes of the HEK293E cells, and the transfected cells were collected after 3 days of transfection. The cell pellets were dissolved in T500 buffer containing 20 mM Tris-HCl [pH 7.5], 500 mM NaCl, 4 mM β-mercaptoethanol (Thermo Fisher Scientific), 2 μg/ml RNase A (Thermo Fisher Scientific), together with a protease inhibitor cocktail (Thermo Fisher Scientific). After sonication and high-speed centrifugation, the 45 mL of clear cell lysate were obtained and then mixed with 2 mL of Ni-NTA resin (Bio-Rad). The protein-bound resin was sequentially washed with three buffers containing 20 mM Tris-HCl [pH 7.5], 4 mM β-mercaptoethanol, and 2000 mM NaCl (T2000), 0 mM NaCl (T0), or 150 mM NaCl (T150). The resin-bound proteins were eluted from Ni-NTA resin with T150 (20 mM Tris-HCl [pH 7.5], 4 mM β-mercaptoethanol, and 150 mM NaCl) plus 250 mM imidazole. Next, the eluted proteins were loaded on Q Sepharose Fast Flow resin (GE Healthcare). The Q Sepharose beads were washed with T150, and the proteins were finally eluted from Q Sepharose beads by T500-plus buffer containing 20 mM Tris-HCl [pH 7.5], 500 mM NaCl, 10% glycerol, and 2 mM dithiothreitol (DTT) (Sigma-Aldrich).