Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a new member of the TNF superfamily. Here, a recombinant form of the extracellular domain of the TRAIL (sTRAIL) was expressed in Escherichia coli BL21(DE3) under the control of a T7 promoter. The resulting insoluble bodies were separated from cellular debris by centrifugation and solubilized with 8 M urea. A rapid and simple on-column refolding procedure was developed. It was applied and then the refolded sTRAIL was purified by anion-exchange chromatography. The purified final product was > 98% pure by SDS-PAGE stained with Coomassie brilliant blue R-250. Mass spectroscopic analysis indicated the protein to be 19.2 kDa, which equalled the theoretically expected mass. N-terminal sequencing of refolding sTRAIL showed the sequence which corresponded to the designed protein. The renatured protein displayed its immunoreactivity with the a ritibodies, to TRAIL protein by Western blotting. The purified sTRAIL had a strong cytotoxic activity against human cervical cancer HeLa cells with ED50 about 1.5 mg/L. Circular dichroism and fluorescence spectrum analysis showed that the refolded sTRAIL had a structure similar to that of native protein with P-sheet secondary structure. This efficient procedure of sTRAIL renaturation may be useful for the mass production of this therapeutically important protein. (c) 2006 Elsevier Inc. All rights reserved.