Affinity Purification of Recombinant Proteins

The cell culture pellets were re-suspended with 50 ml lysis buffer (100 mM Tris-HCl [pH 8], 20 mM, Imidazole, 500 mM NaCl, 1 mM TCEP-HCl (Tris(2-carboxyethyl)phosphine hydrochloride), 2% (V/V) Glycerol), supplemented with 1 ml lysozyme (50 mg/ml), 50 μl DNase I (5 mg/ml) and one tablet of protease inhibitor. Bacterial cells were lysed with a microfluidizer or French Press at ~ 20,000 psi. Lysis was considered complete when the cloudy cell suspension becomes translucent. The lysate was centrifuged for 30 min at 16,000 rpm at 4 °C. Soluble protein (supernatant) was removed into a fresh 50 ml centrifuge tube. The supernatant was then filtered through a 0.22 μm filter and kept on ice. Affinity chromatography purification was performed using a HisTrap™ FF column (5 ml) in the ÄKTA protein purification system. The column was washed with Wash buffer 1 (100 mM Tris-HCl [pH 8], 20 mM Imidazole, 2 M NaCl, 2% Glycerol, 1 mM TCEP-HCl, 0.1. mM AEBSF (4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride)) to remove nonspecifically bound DNA. Then the column was washed using Wash buffer 2 (100 mM Tris-HCl [pH 8], 20 mM Imidazole, 50 mM NaCl, 2% Glycerol, 1 mM TCEP-HCl, 0.1 mM AEBSF). Elution was carried out with Elution buffer 1 (100 mM Tris-HCl [pH 8], 500 mM Imidazole, 500 mM NaCl, 2% Glycerol, 1 mM TCEP-HCl, 0.1 mM AEBSF) using a linear gradient with a set target concentration of Elution buffer 1 of 50%. Protein-containing fractions were run on a 12% polyacrylamide gel. Visualization of protein bands was achieved by incubating the gel with InstantBlue stain for 5–10 min and the protein-containing fractions pooled. The protein sample was stored at 4 °C.