Preparation of 20S proteasome

Preparation of 20S proteasome

20S proteasomes from T. acidophilum were expressed from pRSETA containing the bicistronic gene including psmA and psmB. Transformed BL21 cells were induced with 0.1 mM IPTG and incubated for 18 hours at 37°C. Harvested cells were resuspended in 3 ml of lysis buffer (50 mM Na₂HPO₄ pH 8.0, 300 mM NaCl) per 1 g of cells and lysed with the French press. The lysate was incubated at 65°C for 15 min. Heat-denatured proteins were removed by centrifugation at 30,000gat 4°C. Polyethylenimine (0.1%, w/v) was added to the supernatant to precipitate contaminating nucleic acids. Precipitated nucleic acids were removed by centrifugation at 100,000g for 1 hour. The supernatant was subjected to differential precipitation with polyethylene glycol 400 (PEG; number signifies the mean molecular weight of the PEG polymer). PEG400 was added to a concentration of 20% (v/v) to the supernatant under stirring at 18°C and incubated for 30 min. Precipitated proteins were removed by centrifugation at 30,000g for 30 min at 4°C. The supernatant was then precipitated by raising the concentration of PEG400 to 40% (v/v). The precipitate of this step contained the 20S proteasomes and was recovered by centrifugation at 30,000g for 30 min at 4°C and resuspended in purification buffer (0.05 M BisTris pH 6.5, 0.05 M K(OAc), 0.01 M Mg(OAc)2, 0.01 M β-Glycerophosphate) containing 5% (w/v) sucrose, 10 mM DTT, and 0.01% (w/v) lauryl maltose neopentyl glycol (LMNG) on an orbital shaker at 18°C. The resuspended material was loaded on 10 to 30% (w/v) sucrose gradients in purification buffer containing 5 mM DTT, which are centrifuged at 284,000g for 16 hours at 4°C. Gradients were harvested in 400 μl of fractions. SDS-PAGE was used to identify fractions containing 20Sproteasomes. Selected fractions were pooled and precipitated by the addition of 40% (v/v) PEG400. After centrifugation (30,000g, 20 min), the supernatant was removed and the precipitate was resuspended in purification buffer containing 5% (w/v) sucrose, 10 mM DTT, and 0.01% (w/v) LMNG. The resuspended material was loaded on linear 10 to 40% (w/v) sucrose gradients in purification buffer containing 5 mM DTT, which are centrifuged at 284,000g for 18 hours at 4°C. Fractions containing 20S proteasomes are yet again identified by SDS-PAGE, precipitated and concentrated by the addition of 40% PEG400, and resuspended in purification buffer containing 5% (w/v) sucrose and 5 mM DTT, yielding the final purified protein preparation at 26 mg/ml. Protein concentrations were determined by the Bradford assay (Bio-Rad, Munich, Germany) using bovine serum albumin (BSA) as a standard.