Source:http://linkedlifedata.com/resource/pubmed/id/20363332
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| Predicate | Object |
|---|---|
| rdf:type | |
| lifeskim:mentions | |
| pubmed:issue |
2
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| pubmed:dateCreated |
2010-5-20
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| pubmed:abstractText |
Tumor necrosis factor (TNF-alpha) inhibitors, used for the treatment of common inflammatory diseases, currently belong among the most important biotechnologically produced pharmaceuticals. So far four TNF-alpha antagonists have been approved by regulatory authorities for defined subsets of applications. Furthermore, numerous approaches are being taken to develop new protein-based pharmaceuticals and to broaden their application areas in the treatment of TNF-alpha -related diseases. Both the fundamental understanding of disease-related TNF-alpha activity and the subsequent development of corresponding drug candidates demand the availability of large amounts of TNF-alpha as a bioactive protein. We have therefore established a protocol for the rapid high-level synthesis of recombinant human TNF-alpha in Escherichia coli shake-flask cultures and the subsequent purification of the mature protein. Using the advantages of SUMO-fusion technology we were able to produce protein with an authentic N-terminus in high yield. Two immobilized metal ion-affinity chromatography steps with a protease cleavage step in between and subsequent size-exclusion chromatography were utilized to purify the protein. The protein was obtained from the last chromatography step as a trimer, while purity was at least 96% as estimated by SDS-PAGE. The identity of the protein was confirmed by MALDI-TOF mass spectrometry. Recombinant mature TNF-alpha was correctly folded as assessed by CD spectroscopy and its biological activity was confirmed by an L929 cell assay.
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| pubmed:language |
eng
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| pubmed:journal | |
| pubmed:citationSubset |
IM
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| pubmed:chemical | |
| pubmed:status |
MEDLINE
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| pubmed:month |
Aug
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| pubmed:issn |
1096-0279
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| pubmed:author | |
| pubmed:copyrightInfo |
Copyright 2010 Elsevier Inc. All rights reserved.
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| pubmed:issnType |
Electronic
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| pubmed:volume |
72
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| pubmed:owner |
NLM
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| pubmed:authorsComplete |
Y
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| pubmed:pagination |
238-43
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| pubmed:meshHeading |
pubmed-meshheading:20363332-Animals,
pubmed-meshheading:20363332-Cell Culture Techniques,
pubmed-meshheading:20363332-Cell Death,
pubmed-meshheading:20363332-Cell Line, Tumor,
pubmed-meshheading:20363332-Chromatography, Affinity,
pubmed-meshheading:20363332-Chromatography, Gel,
pubmed-meshheading:20363332-Chromatography, High Pressure Liquid,
pubmed-meshheading:20363332-Circular Dichroism,
pubmed-meshheading:20363332-Electrophoresis, Polyacrylamide Gel,
pubmed-meshheading:20363332-Escherichia coli,
pubmed-meshheading:20363332-Humans,
pubmed-meshheading:20363332-Mice,
pubmed-meshheading:20363332-Recombinant Fusion Proteins,
pubmed-meshheading:20363332-SUMO-1 Protein,
pubmed-meshheading:20363332-Solubility,
pubmed-meshheading:20363332-Spectrometry, Mass, Matrix-Assisted Laser...,
pubmed-meshheading:20363332-Tumor Necrosis Factor-alpha
|
| pubmed:year |
2010
|
| pubmed:articleTitle |
Recombinant production of bioactive human TNF-alpha by SUMO-fusion system--high yields from shake-flask culture.
|
| pubmed:affiliation |
NWG Künstliche Bindeproteine, Institut für Biochemie und Biotechnologie, Technische Biochemie, Martin-Luther-Universität Halle-Wittenberg, Heinrich-Damerow-Str. 4, 06120 Halle, Germany.
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| pubmed:publicationType |
Journal Article,
Research Support, Non-U.S. Gov't
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