Cell disruptor at 25 kpsi

GST-SENP1 was expressed in C41(DE3) cells grown in 2xTY medium, supplemented with 100 μg/ml ampicillin, at 37°C. Cells were induced with 0.5 mM IPTG at OD₆₀₀ = 0.6-0.8, grown over night at 18°C and harvested by centrifugation. Cells were lysed in buffer SA (50 mM Tris/HCl, 150 mM NaCl, 2 mM TCEP, 1 mM EDTA, 5 % glycerol, pH 8.5), supplemented with DNase, RNase and cOmplete EDTA-free Protease Inhibitor Cocktail (Roche), using a cell disruptor at 25 kpsi (Constant Systems). The lysate was centrifuged at 100,000x g for 30 min at 4°C. The supernatant was added to Glutathione Sepharose 4B beads (Cytiva) and incubated for 2h at 4°C. The beads were thoroughly washed in buffer SA, followed by buffer SA with 500 mM NaCl and, again, buffer SA. The protein was eluted in buffer SA with 10 mM reduced glutathione. Peak fractions were concentrated with a Vivaspin 20 concentrator (30 kDa MWCO, Sartorius). The protein was further purified by size exclusion chromatography on a HiLoad 26/600 Superdex 200 pg column (Cytiva), equilibrated in buffer SEC-S (50 mM Tris/HCl, 50 mM NaCl, 5 mM TCEP, 1 mM EDTA, 1 mM NaN₃, 5 % glycerol, pH 8.0). Peak fractions were concentrated to ~15-20 mg/ml, frozen in aliquots and stored at −80°C.

6H-TEV protease was expressed in C41(DE3) cells grown in 2xTY medium, supplemented with 30 μg/ml kanamycin, at 37°C. Cells were induced with 1 mM IPTG at OD₆₀₀ = 0.6-0.8, grown over night at 19°C and harvested by centrifugation. Cells were lysed in TEV lysis buffer (20 mM Tris/HCl, 500 mM NaCl, 1 mM TCEP, 1 mM NaN₃, pH 8.0), supplemented with DNase and RNase, using a cell disruptor at 25 kpsi (Constant Systems). The lysate was centrifuged at 100,000x g for 30 min at 4°C. The supernatant was supplemented with 20 mM imidazole and loaded onto a HisTrap FF column (Cytiva). The column was washed with TEV lysis buffer supplemented with 20 mM imidazole. The protein was eluted in TEV lysis buffer with increasing concentrations of imidazole, concentrated with Vivaspin 20 concentrators (10 kDa MWCO, Sartorius) and further purified by size exclusion chromatography on a HiLoad 16/600 Superdex 75 pg column (Cytiva), equilibrated in TEV lysis buffer. Peak fractions were concentrated to 30 mg/ml, frozen in aliquots and stored at −80°C.