Ni sepharose fast flow resin

Supernatant after centrifugation was applied to a disposable column filled with Ni-Sepharose Fast Flow resin (GE Healthcare, Chicago, IL, USA) which was equilibrated with buffer A containing 10 mM imidazole. The column was extensively washed with buffer A containing 20 mM imidazole. The target proteins were eluted with buffer B (20 mM Tris-HCl, 200 mM NaCl, 100 mM imidazole, pH 8.0). Combined fractions containing the target protein with a purity of more than 90% were dialyzed against buffer C (20 mM Tris-HCl, 100 mM NaCl) and sterilized. Protein concentration was measured using a Protein Assay Kit (Bio-Rad) with bovine serum albumin as a standard.

Full length human PAD4 with N-terminal His-tag was expressed in E. coli BL21 (DE3) cells using the pET-16b vector. HisTag-PAD4 expression was induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at OD_600nm = 0.6 and the culture was incubated at 25 °C for 16 h. After this time cells were harvested, lysed and soluble protein fraction was applied on Ni Sepharose Fast Flow resin (GE Healthcare, Life Science, USA). Fractions containing HisTag-PAD4 were further purified using combination of ion exchange and gel filtration chromatography on MonoQ 4.6/100PE and HiLoad Superdex S75 16/600 columns respectively (GE Healthcare, Life Science). Finally, HisTag-PAD4 was concentrated and frozen in storage buffer (50 mM Hepes pH 7.5 with 0.25 M NaCl, 3% glycerol). PAD4 activity was determined on N-α-benzoyl-L-arginine ethyl ester hydrochloride (BAEE) substrate with colorimetric assay [33 (link)].

The bacterial cell pellet was resuspended in 100 ml resuspension buffer (50 mM Na₂HPO₄, 0,5 M NaCl pH 7.5). 25 μg/mL DNase, 0,1 mg/mL lysozyme, 1% Triton X-100, 1 mM PMSF, 10 mM MgCl₂ were added and the solution was sonicated and then centrifuged at 13000 x g for 20 min at 4°C. The precipitated insoluble intracellular fraction was separated and the pellet was dissolved by sonication followed by 2 hours incubation at room temperature in 50 mL of solubilization buffer (8 M urea, 50 mM Na₂HPO₄, 0.5 M NaCl pH 7.5).
Solubilized proteins were purified through a 2 mL Ni-Sepharose Fast-Flow resin (GE Healthcare) followed by elution of bound protein with the same solubilization buffer containing 500 mM imidazole.
Refolding of urea-denatured proteins from inclusion bodies was attained by multi-step dialysis in refolding buffer (50 mM TrisHCl, 0.5 M NaCl, 0.4 M L-Arg, pH 7.5) that gradually decreased the concentration of denaturant. The final dialysis step was carried out in PBS (pH 7.2) for 12 hours.