Cloning and expression of Mtb20SOG have been described.27 (link) Briefly, a PACYCDuet-1 vector (Novagen, Madison, WI, USA) consisting of the Mtb prcAOG (encoding truncated-PrcA) and prcB genes was expressed in the BL21(DE3) strain of E. coli. Cells were grown at 37 °C to OD600 = 0.5–0.6 before being induced with 0.2 mM IPTG. The Mtb proteasome was expressed at 37 °C for 18 h to ensure full processing of β-subunits. Cells were harvested by centrifugation, resuspended in 10 mM potassium phosphate (pH 8.0), 10 mM imidazole, and 0.25 M NaCl, and lysed by passing through a Microfluidizer cell disruptor. The homogenate was clarified by spinning at 27,000 × g, and the supernatant was applied to a HiTrap-Ni column (GE Healthcare) pre-equilibrated with the lysis buffer. His-tagged proteins were eluted with a 10–300 mM imidazole gradient in 10 mM potassium phosphate (pH 8.0) and 0.25 M NaCl. Eluted fractions containing Mtb proteasomes were applied to a Hi-Trap Q column pre-equilibrated with 10 mM Tris (pH 8.0) and 50 mM NaCl and were eluted with a 50–500 mM NaCl gradient. Mtb20SOG was further purified with a Superdex 200 column (16 × 600 mm, GE Healthcare) in buffer containing 10 mM HEPES (pH 7.5), 1 mM dithiothreitol, and 0.15 M NaCl. For crystallization, the purified Mtb20SOG complex was concentrated to 10 mg/mL.