The expression and purification of rSj-Cys has been described previously (27 (link)). Briefly, DNA encoding Sj-Cys was cloned into the pET-28a vector (Promega Corporation). The sequence was confirmed to be inserted successfully before transformation of the restructured plasmid into Escherichia coli (BL 21). Next, the transformants were induced for large-scale protein expression using 1 mM isopropyl-β-D-1-thiogalactopyranoside (Sigma-Aldrich; Merck KGaA) at 37°C for 5 h. After purification with the HisPur™ Ni-NTA Spin Column (Thermo Fisher Scientific, Inc.), endotoxin contamination was mostly eliminated using the ToxOut™ High-Capacity Endotoxin Removal kit (BioVision, Inc.), and residual endotoxin detection was performed using the ToxinSensor™ Chromogenic Limulus Amebocyte Lysate Endotoxin Assay kit (GenScript), in accordance with the manufacturer's protocols. Finally, a bicinchoninic acid (BCA) protein assay kit (Beyotime Institute of Biotechnology) was used to determine the concentration of rSj-Cys.