Recombinant Leptospiral Ligand Purification

The rLigAc was produced as described previously [11 (link)]. Briefly, inclusion bodies were isolated by centrifugation, washed with Tris buffer, pH 8.0 (50 mM Tris and 200 mM NaCl) containing 0.5% Triton X-100 and 1 M urea at 4 °C for 3 h, and solubilized in Tris buffer containing 6 M urea and 5 mM DTT overnight at 4 °C. The extracted proteins were purified by Ni²⁺ Chelating Sepharose column (GE Healthcare, Buckinghamshire, UK) under denaturing conditions. Purified rLigAc was refolded by dialysis with Tris buffer containing stepwise decreasing concentrations of urea (5 to 0 M). The secondary structure of purified rLigAc was evaluated by Jasco J-815 Circular Dichroism (CD) Spectropolarimeter (Jasco Incorporated, MD, USA) and analyzed with CDPro software. The factor H binding activity of the purified rLigAc was evaluated as described previously [11 (link)].