2.3. Protein Expression and Purification

The in vivo-associated complexes, Scc4:His₆-Scc1 and His₆-Scc4:Scc1-FLAG, were expressed and purified as described by Ukwaththage, et al. [22]. Briefly, Scc4 and His₆-Scc1 were co-expressed from the pET28Scc4 and pACYCHis₆-Scc1 vectors in E. coli BL21-Gold (DE3) cells, and His₆-Scc4 and Scc1-FLAG were co-expressed from the pCDFScc4 and pScc1-FT vectors in E. coli T7 Express cells. The cultures were first grown in LB medium to an optical density at 600 nm of 0.6, and the cells were transferred to M9 minimal medium (with ammonium chloride or ¹⁵N-ammonium chloride). The protein expression was induced with 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG), and the cultures were grown at 16 °C with 250 rpm shaking for 16 h. The cells were harvested by centrifugation (4000× g, 15 min, 4 °C) and lysed using a French pressure cell 3 times. The protein complexes were purified from the clarified lysate using Ni-IMAC with 4 mL of resin and Tris buffer followed by SEC with NMR buffer. The purified proteins were analyzed using SDS-PAGE and the Coomassie protein assay.

Co-expression cultures of the Scc4:His₆-Scc1 and His₆-Scc4:Scc1-FLAG complexes were used to purify Scc4 and Scc1-FLAG and prepare immobilized His₆-Scc1 as described by Ukwaththage, et al. [22]. Briefly, the complexes were immobilized on 4 mL of Ni-IMAC resin, and 0.5% sarkosyl in Tris buffer was used to dissociate the complexes and elute either Scc4 or Scc1-FLAG, leaving the His-tagged proteins (His₆-Scc1 or His₆-Scc4) on the resin. The immobilized His₆-Scc1 was washed with Tris buffer to remove residual sarkosyl. Sarkosyl was removed from the purified Scc4 and Scc1-FLAG proteins by diluting the samples 5-fold with NMR buffer and exchanging the buffer to NMR buffer using centrifugal filters. The purified proteins and the immobilized His₆-Scc1 were analyzed using SDS-PAGE, and the purified Scc4 and Scc1-FLAG proteins were analyzed using the Commassie protein assay.

Scc4 and His₆-Scc1 were expressed individually in E. coli BL21-Gold (DE3) cells using the same method as the expression of the Scc4:His₆-Scc1 and His₆-Scc4:Scc1-FLAG complexes. Scc4 in clarified lysate and His₆-Scc1 inclusion bodies were prepared by suspending the harvested cells in 20 mL of Tris buffer with 2 Pierce EDTA-free protease inhibitor mini tablets, 2.3 mL of 10X BugBuster protein extraction reagent, and 1 μL of Benzonase nuclease and lysing with a French press (SLM Instruments Inc./American Instrument Co., Urbana, IL, USA) 3 times. The Scc4 cell lysate was clarified by centrifugation (25,000× g for 20 min at 4 °C). His₆-Scc1 inclusion bodies were purified by washing the lysed pellet with 20 mL each of 1× BugBuster in Tris buffer, 1X BugBuster in Tris buffer with 200 μg/mL of hen egg white lysozyme, and 1X BugBuster in Tris buffer using centrifugation at 15,000× g for 20 min at 4 °C to remove the supernatant between each step. The purified His₆-Scc1 inclusion bodies were centrifuged at 25,000× g for 20 min at 4 °C to remove residual supernatant and analyzed using SDS-PAGE.