Recombinant-protein expression and purification

The positive clone verified by DNA sequencing was inoculated in 5 mL LB medium with kanamycin (50 μg/mL) with shaking at 220 rpm at 37 °C. After 4 to 6 h, the culture was diluted to 1 L LB medium and grown to an OD₆₀₀ of 0.4 to 0.6, after which 0.1 mM isopropyl-beta D-thiogalactopyranoside (IPTG) was added, followed by culturing for 4 h at 37 °C. The expressed protein presented as inclusion bodies was solubilized by addition of 10 mL 8 M urea in 50 mM Tris buffer (pH 7.4) and incubated in 1 mM dithiothreitol. We then added 200 μL of 100 mM cysteine in 0.5 M NaOH and 15 mL of 5 mM cysteine in 100 mM Tris buffer (pH 8.0). Before purification, the protein solution was dialyzed eight times every 2 h into 30 mM Tris buffer (pH 7.4). The recombinant protein was purified by an affinity Ni-chromatography column (GE Healthcare, Uppsala, Sweden). Recombinant bovine enterokinase was added to the eluted proteins and incubated at 26 °C for 10 h to remove the His-tags from the recombinant proteins. Protein expression and purification was assessed by 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The purified protein was dialyzed in Tris buffer (pH 7.4) and (pH 5.0), and the concentration was determined prior to performing fluorescence-binding assays. The purified proteins were stored at −80 °C until use.