Abstract
As a new member of tumor necrosis factor (TNF) superfamily, TNF-related apoptosis-inducing ligand (Apo2L/TRAIL) was produced mainly as inclusion bodies by recombinant Escherichia coli with a temperature-inducible expression system. High concentrations of both biomass (65 g dry cells l(-1)) and inactive TRAIL (4.8 g l(-1)) were obtained by applying a high-cell-density cultivation procedure. After the inclusion bodies were washed and solubilized. TRAIL refolded when at 1 mg ml(-1) by a simple pulse dilution method with a 35% yield. Renatured TRAIL was purified to electrophoretic homogeneity by one-step immobilized metal affinity chromatography. The purified TRAIL showed strong cytotoxicity activity against human pancreatic 1990 tumor cells, with ED50 about 1.6 microg ml(-1).
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Apoptosis Regulatory Proteins
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Cell Culture Techniques / methods*
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Cell Line, Tumor / drug effects
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Cell Survival / drug effects
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Chromatography, Affinity / methods
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Dose-Response Relationship, Drug
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Escherichia coli / genetics
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Escherichia coli / metabolism*
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Humans
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Inclusion Bodies / genetics
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Inclusion Bodies / metabolism*
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Membrane Glycoproteins / biosynthesis*
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Membrane Glycoproteins / genetics
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Membrane Glycoproteins / isolation & purification*
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Membrane Glycoproteins / pharmacology
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Pancreatic Neoplasms / physiopathology
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Protein Engineering / methods
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Protein Folding
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Recombinant Proteins / genetics
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Recombinant Proteins / isolation & purification
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Recombinant Proteins / pharmacology
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TNF-Related Apoptosis-Inducing Ligand
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Tumor Necrosis Factor-alpha / biosynthesis*
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Tumor Necrosis Factor-alpha / genetics
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Tumor Necrosis Factor-alpha / isolation & purification*
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Tumor Necrosis Factor-alpha / pharmacology
Substances
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Apoptosis Regulatory Proteins
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Membrane Glycoproteins
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Recombinant Proteins
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TNF-Related Apoptosis-Inducing Ligand
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TNFSF10 protein, human
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Tumor Necrosis Factor-alpha