High-level expression, purification, and in vitro refolding of soluble tumor necrosis factor-related apoptosis-inducing ligand (TRAIL).
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a new member of the TNF superfamily. In this paper, we report the expression, purification, and preparation of a recombinant form of the extracelluar domain of the TRAIL (sTRAIL) without posttranslational modifications, which may selectively induce apoptosis of tumor cells in vitro. To obtain recombinant nonfusion sTRAIL protein, the encoding region for sTRAIL was cloned between KpnI and BamHI in pET32a. The Trx (thioredoxin)/sTRAIL fusion proteins were expressed in the form of inclusion bodies in Escherichia coli host strain BL21 (DE3). The expression level was more than 35% of total cell lysate. Inclusion bodies were disrupted, washed, and isolated at pH 9.0, and were completely dissolved in a buffer containing 2 M urea at pH 9.0. After nickel ion metal affinity chromatography, gel filtration chromatography, and renaturation, the refolded fusion proteins with a purity of >98% were obtained. Trx/sTRAIL L proteins were digested by enterokinase to both Trx and 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 sTRAIL may have cancer therapeutic applications.
Wang
Dongming
D
School of Environmental Engineering, Lishui Vocational and Technical College, Lishui, Zhejiang, People's Republic of China. dongmingwang2002@126.com
Shi
Linmei
L
D017209:Apoptosis / Q000502:physiology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics*; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D002479:Inclusion Bodies; D010957:Plasmids / Q000235:genetics; D017510:Protein Folding; D012313:RNA / Q000097:blood; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry*
D011993:Recombinant Fusion Proteins; D053221:TNF-Related Apoptosis-Inducing Ligand; D014409:Tumor Necrosis Factor-alpha; D012313:RNA