Recombinant proteins were purified using Ni-NTA affinity columns (Novagen) and dialyzed against phosphate buffered
saline (PBS) at pH 7.2–7.4 through dialysis membrane (SnakeSkin™ Pleated Dialysis Tubing, Pierce) for 4 h at 4 °C and stored at −20 °C until used. Protein concentration was determined by the method of Bradford [15].