High-Level Expression, Purification, and In Vitro Refolding of Soluble Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL)
| العنوان: | High-Level Expression, Purification, and In Vitro Refolding of Soluble Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) |
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| المؤلفون: | Dongming Wang, Linmei Shi |
| المصدر: | Applied Biochemistry and Biotechnology. 157:1-9 |
| بيانات النشر: | Springer Science and Business Media LLC, 2008. |
| سنة النشر: | 2008 |
| مصطلحات موضوعية: | Enteropeptidase, Protein Folding, Recombinant Fusion Proteins, Apoptosis, Bioengineering, Biology, Applied Microbiology and Biotechnology, Biochemistry, Inclusion bodies, law.invention, TNF-Related Apoptosis-Inducing Ligand, law, Cell Line, Tumor, Protein purification, Escherichia coli, Humans, Molecular Biology, Cell Proliferation, Inclusion Bodies, Tumor Necrosis Factor-alpha, General Medicine, Ligand (biochemistry), Fusion protein, Molecular biology, Gene Expression Regulation, Cell culture, Recombinant DNA, RNA, Thioredoxin, HeLa Cells, Plasmids, Biotechnology |
| الوصف: | 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. |
| تدمد: | 1559-0291 0273-2289 |
| الوصول الحر: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::b314e5362f2d7eb7b87dab9bcfe46ff9Test https://doi.org/10.1007/s12010-007-8079-xTest |
| حقوق: | CLOSED |
| رقم الانضمام: | edsair.doi.dedup.....b314e5362f2d7eb7b87dab9bcfe46ff9 |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 15590291 02732289 |
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