Recombinant HSP60 Protein Purification

rHSP60 was expressed and purified as described by Fernandes et al [15 (link)]. Briefly, E. coli transformed with pET28a– HSP60 vector were grown in LB medium supplemented with kanamycin sulfate (50 μg/mL). After inducing with 0.5 mM isopropyl-β-d-thiogalactopyranoside for 6 hours, bacterial cells were lysed by sonication in lysing buffer (50 mM NaH₂PO₄, 300 mM NaCl, 5 mM 2-mercaptoethanol, and 0.5% Triton X-100, pH 8.0). The sample was centrifuged at 7,000 × g and the pellet washed 5 times with the lysing buffer. Pellet inclusion bodies was solubilized in a denaturing buffer (50 mM NaH₂PO₄, 300 mM NaCl, 30 mM imidazole, 7 M urea, 5 mM 2-mercaptoethanol, and 0.5% Tween 20, pH 8.0) for 1 hour. After centrifugation at 7,500 × g, the supernatant was clarified through 0.22 μm filter, and submitted to a Ni²⁺–Sepharose affinity column (His-Trap; GE Healthcare). rHSP60 purified was concentrated and refolded by dialysis against phosphate-buffered saline (PBS) in a Amicon Ultra 15 device with a molecular weight cut-off of 10-kDa (Merck, Cork, Ireland). Protein concentration was determined using Quick Start Bradford Protein Assay (Bio-Rad, Hercules, USA). Purified rHSP60 sample was analyzed by SDS-PAGE. The sample contained less than 0.05 ng/mL of bacterial endotoxin, as determined by the Limulus amoebocyte lysate assay (Sigma-Aldrich, St. Louis, USA).