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Protein Expr Purif. 2010 Aug;72(2):238-43. doi: 10.1016/j.pep.2010.03.022. Epub 2010 Apr 2.

Recombinant production of bioactive human TNF-alpha by SUMO-fusion system--high yields from shake-flask culture.

Author information

1
NWG Künstliche Bindeproteine, Institut für Biochemie und Biotechnologie, Technische Biochemie, Martin-Luther-Universität Halle-Wittenberg, Heinrich-Damerow-Str. 4, 06120 Halle, Germany.

Abstract

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.

PMID:
20363332
DOI:
10.1016/j.pep.2010.03.022
[Indexed for MEDLINE]

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