High-Level Expression, Purification, and In VitroRefolding of Soluble Tumor Necrosis Factor-RelatedApoptosis-Inducing Ligand (TRAIL)

摘要

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a newmember of the TNF superfamily. In this paper, we report the expression, purification, andpreparation of a recombinant form of the extracelluar domain of the TRAIL (sTRAIL)without posttranslational modifications, which may selectively induce apoptosis of tumorcells in vitro. To obtain recombinant nonfusion sTRAIL protein, the encoding region forsTRAIL was cloned between KpnI and BamHI in pET32a. The Trx (thioredoxin)/sTRAILfusion proteins were expressed in the form of inclusion bodies in Escherichia coli hoststrain BL21 (DE3). The expression level was more than 35% of total cell lysate. Inclusionbodies were disrupted, washed, and isolated at pH 9.0, and were completely dissolved in abuffer containing 2 M urea at pH 9.0. After nickel ion metal affinity chromatography, gelfiltration chromatography, and renaturation, the refolded fusion proteins with a purity of>98% were obtained. Trx/sTRAIL L proteins were digested by enterokinase to both Trxand sTRAIL fragments, which then were separated by cation exchange chromatography.Cell proliferation experiments proved that the rsTRAIL (98% purity) retains its cancer-selective apoptosis-inducing properties. This result suggested that the recombinant sTRAILmay have cancer therapeutic applications.