2.5. Purification of the rMELEISH Protein

The frozen pellet was resuspended in 1 mL lysis buffer (8 M urea, 50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole, pH 8.0) following incubation at 4 °C for 16 h. Cell suspension was then sonicated (5 pulses of 10 sec with 1 min intervals) using a Vibra Cell sonicator (Sonics & Materials, Inc, Newtown, CT, USA) and incubated on ice for 2 h following centrifugation at 6000× g for 15 min at 4 °C. The supernatant was added to 0.5 mL Ni-Sepharose 6 Fast Flow resin (Sigma,St. Louis, MO, USA) (resuspended in lysis buffer), which was then incubated at 4 °C for 90 min on a vertical disc rotator. Next, the resin was sedimented and washed 4 times with 1 mL washing buffer (8 M urea, 50 mM NaH₂PO₄, 300 mM NaCl, 5 mM imidazole, pH 8.0). The protein was eluted in 3 fractions using 0.5 mL elution buffer (8 M urea, 50 mM NaH₂PO₄, 300 mM NaCl, 50, 100, and 200 mM imidazole, pH 8.0).