TNFα protein expression and purification

E. coli TOP10 cells (Thermo Fisher Scientific) transformed with each of the expression constructs were cultured in terrific broth (TB) (ampicillin, +100 μg/ml) to an OD₆₀₀ (optical density at 600 nm) = 0.6, expression was induced by adding arabinose (0.1%), and cultures were incubated for a further 16 hours at 20°C. Cells were harvested by centrifugation and stored at −80°C. Cells were resuspended (1 g in 4 ml) in 25 mM tris-HCL (pH 8.0), 200 mM NaCl, 0.02% CHAPS, 50 mM l-arginine, 125 U of Benzonaze (Novagen), 100 mg of lysozyme, and one cOmplete EDTA-free protease inhibitor tablet (Roche) and lysed by sonication. Insoluble material was removed by centrifugation, and His-tagged protein was captured from the soluble fraction by immobilized metal affinity chromatography (IMAC) (HiTrap Chelating HP, GE Healthcare) eluted with a 500 mM imidazole step or gradient. The 6His-Smt tag was removed with ubiquitin-like–specific protease 1 while dialyzing against 2 liters of 25 mM tris (pH 8.0) and 200 mM NaCl overnight at 4°C in 10-kDa molecular weight cut-off (MWCO) snakeskin dialysis tubing. Cleaved protein was further purified by a second IMAC step, followed by size exclusion chromatography (HiPrep 16/60 Sephacryl S-100 HR, GE Healthcare) in 10 mM Hepes (pH 7.5) and 150 mM NaCl.