Protein Purification and SDS-PAGE Analysis.

Suspension of bacterial cell pellets (15 mL) from the expressions were prepared for lysis by adding 2 Pierce EDTA free protease inhibitor mini tablets, 1.7 mL of 10× BugBuster protein extraction reagent, and 0.75 μL of Benzonase nuclease. The cells were lysed using a French pressure cell followed by centrifugation at 25 000g for 30 min at 4 °C to obtain soluble protein in the cleared lysate. The cleared lysate containing either Scc4-Scc1-His₆, His₆-Scc4-Scc1-FT, or His₆-Scc4 was immediately loaded onto 4 mL of Ni-IMAC resin (1 cm i.d. column) at a flow rate of 1 mL/min using a fast protein liquid chromatography system (FPLC). The captured proteins were washed with 20 mL of Tris wash buffer and eluted with 20 mL of Tris elution buffer (20 mM Tris, 300 mM NaCl, 500 mM imidazole, and 5% glycerol at pH 8.0) at a flow rate of 2 mL/min. The eluted proteins were detected using absorbance at 280 nm (A₂₈₀) and pooled from 1 mL fractions (typically 5–6 mL). The Tris elution buffer was exchanged to the desired buffer using 15 mL of P4 gel (polyacrylamide beads in a 1 cmi.d. column) at a flow rate of 0.5 mL/min with 1 mL fractions. The protein fractions were combined based on A₂₈₀ and analyzed using the Bradford protein assay following the manufacturer’s 96-well plate instructions (Pierce Comassie Plus Assay Kit, Thermo Fisher Scientific). The results from the Bradford protein assay are reported in the results as mg protein or complex per L culture. Samples taken during the purification of the proteins, including the purified proteins, were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Sample aliquots (10 μL) were mixed with 10 μL of 2X Laemmli buffer with 5% (by volume) of β-mercaptoethanol, heated at 95 °C for 5 min, and loaded into 4–20% Tris/glycine gels. The gels were run at 120 V for 45 min in running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3 buffer) followed by staining with brilliant blue R-250 solution (0.1% (m/V) brilliant blue R-250 dye, 50% methanol, 40% water, and 10% acetic acid). After destaining in 50% water, 40% methanol, 10% acetic acid, the gels were imaged using a Bio-Rad Gel Doc EZ System.