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  Screening of fusion partners for high yield expression and purification of bioactive viscotoxins

Bogomolovas, J., Simon, B., Sattler, M., & Stier, G. (2009). Screening of fusion partners for high yield expression and purification of bioactive viscotoxins. Protein Expression and Purification, 64(1), 16-23. doi:10.1016/j.pep.2008.10.003.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-002E-8AA0-6 Version Permalink: http://hdl.handle.net/21.11116/0000-0000-E39E-E
Genre: Journal Article

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ProtExprPurif_64_2009_16.pdf (Any fulltext), 567KB
 
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Bogomolovas, Julius, Author
Simon, Bernd, Author
Sattler, Michael, Author
Stier, Gunter1, Author              
Affiliations:
1Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Max Planck Society, ou_1497700              

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Free keywords: Viscotoxins; Thionins; Fusion tag; Expression screen; Escherichia coli; Isotopic labeling; Cytotoxicity
 Abstract: Viscotoxins are small cationic proteins found in European mistletoe Viscum album. They are highly toxic towards phytopathogenic fungi and cancer cells. Heterologous expression of viscotoxins would broaden the spectrum of methods to be applied for better understanding of their structure and function and satisfy possible biopharmaceutical needs. Here, we evaluated 13 different proteins as a fusion partners for expression in Escherichia coli cells: His6 tag and His6-tagged versions of GB1, ZZ tag, Z tag, maltose binding protein, NusA, glutathione S-transferase, thioredoxin, green fluorescent protein, as well as periplasmic and cytosolic versions of DsbC and DsbA. The fusion to thioredoxin gave the highest yield of soluble viscotoxin. The His6-tagged fusion protein was captured with Ni(2+) affinity chromatography, subsequently cleaved with tobacco etch virus protease. Selective precipitation by acidification of the cleavage mixture was followed by cation exchange chromatography. This protocol yielded 5.2mg of visctoxin A3 from 1l of culture medium corresponding to a recovery rate of 68%. Mass spectrometry showed a high purity of the sample and the presence of three disulfide bridges in the recombinant viscotoxin. Proper folding of the protein was confirmed by heteronuclear NMR spectra recorded on a uniformly 15N-labeled sample. Recombinant viscotoxins prepared using this protocol are toxic to HeLa cells and preserve the activity differences between isoforms B and A3 found in native proteins.

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Language(s): eng - English
 Dates: 2008-06-202008-10-022008-10-172009-03
 Publication Status: Published in print
 Pages: 8
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Degree: -

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Title: Protein Expression and Purification
Source Genre: Journal
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Publ. Info: San Diego : Academic Press
Pages: - Volume / Issue: 64 (1) Sequence Number: - Start / End Page: 16 - 23 Identifier: ISSN: 1046-5928
CoNE: https://pure.mpg.de/cone/journals/resource/954922650158