Streptactin superflow plus columns

The purification strategy was the same for both reconstituted 20S proteasome and 20S-PA200 complexes. Briefly, thawed cells were resuspended in 3-4 times volume of buffer W (50 mM Tris pH 7.5, 150 mM NaCl, 5% (v/v) glycerol, 1 mM DTT, 1 mM EDTA), lysed by sonication and the lysate cleared by centrifugation at 48,400 g for 30 minutes. The clear lysate was then passed through a 5 μm filter and loaded onto tandem Streptactin Superflow Plus columns (QIAGEN), equilibrated in buffer W, and eluted in buffer E (50 mM Tris pH7.5, 150 mM NaCl, 5% (v/v) glycerol, 1 mM DTT, 1mM EDTA, 2.5 mM d-desthiobiotin). Proteasome containing fractions were pooled together and the TwinStrep-tag cleaved by overnight dialysis against buffer W containing TEV protease, at a ratio of 1:50 (TEV to protein). The sample was filtered again with a 0.4 μm filter and loaded onto Streptactin Superflow Plus columns, where the flow-through was collected. The protein was concentrated using 30 kDa cut-off Vivaspin 20 concentrators (Sartorius) and loaded onto a Superose 6 Increase 10/300 gel filtration column (GE Healthcare) equilibrated with 50 mM Tris pH 7.4, 100 mM NaCl, 1 mM EDTA (Figure S1B). Typical yields for the purification of reconstituted 20S proteasomes and 20S-PA200 complexes are 2-3 mg and 1 mg of protein purified from 1 L of Sf9 cultures, respectively.