In this study, four deubiquitinases from legionella (Lpg1621 (LotB), Lpg2529 (LotC), Lpg2411, and Lpg2907) were cloned into either pParallelGST2 or pParallelHis2 vector (Sheffield et al., 1999) and expressed in the E.coli.

We used the T7 express Escherichia coli competent cells (NEB) for expressing deubiquitinases. Cells were transformed with plasmids. A single colony was picked and grown in 5 ml of LB medium overnight at 37°C. The 5 ml of cultured cells are transferred into 1L LB medium and further grown until the OD₆₀₀ reaches to 0.6–0.8 at 37°C. Protein expression was induced by the addition of 0.5 mM IPTG (isopropyl D-thiogalactopyranoside), and the cells were further grown overnight at 18°C and harvested by centrifugation (4,000 rpm, 10 mins). The cell pellet was resuspended in 30 ml of lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, and 2 mM DTT) and lysed by sonication and centrifuged at 13,000 rpm, 1 hr to clarify the supernatant. The supernatant of GST-tagged protein was incubated for 1 hr with glutathione-S-sepharose, which is pre-equilibrated with washing buffer (50 mM Tris-HCl pH 7.5, 500 mM NaCl, and 2 mM DTT), and nonspecific proteins were cleared by washing the beads 3 times with washing buffer. GST-proteins were eluted with elution buffer (50 mM Tris-HCl pH 8.0, 50 mM NaCl, 2 mM DTT, and 15 mM reduced glutathione). Finally, the buffer of the sample is exchanged to storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, and 1 mM DTT).

For His-tagged proteins, the supernatant was incubated with Ni-NTA pre-equilibrated with washing buffer (50 mM Tris-HCl pH 7.5, 500 mM NaCl, and 20 mM Imidazole) for 2 hr and eluted with elution buffer (50 mM Tris-HCl pH 7.5, 500 mM NaCl, and 300 mM imidazole) and the buffer was exchanged to the storage buffer. For LotC_14-310, instead of using the elution buffer, glutathione beads were incubated with sfGFP-TEV protease (Wu et al., 2009) overnight at 4°C. Cleaved protein was buffer exchanged to IEX buffer A (20 mM Tris-HCl pH 8.0, 20 mM NaCl, and 1 mM DTT) and purified by anion-exchange chromatography on HitrapQ (GE Healthcare) with gradient elution with IEX buffer B (20 mM Tris-HCl pH 8.0, 1 M NaCl, and 1 mM DTT) and fractions contacting samples were loaded onto size-exclusion column (Superdex 75 16/60, GE Healthcare) pre-equilibrated with 50 mM Tris-HCl pH 7.5, 50 mM NaCl, and 1 mM TCEP. Proteins were concentrated to 20 mg/ml and stored for crystallization.