Screening of fusion partners for high yield expression and purification of bioactive viscotoxins.
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.
Bogomolovas
Julius
J
Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
Simon
Bernd
B
Sattler
Michael
M
Stier
Gunter
G
D002352:Carrier Proteins / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D058619:Maltose-Binding Proteins; D010445:Peptide Elongation Factors / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D019704:Protein Disulfide-Isomerases / Q000378:metabolism; D017510:Protein Folding; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D013879:Thioredoxins / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D035602:Transcriptional Elongation Factors
D002352:Carrier Proteins; D004220:Disulfides; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D010445:Peptide Elongation Factors; D010940:Plant Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; C068727:nusA protein, E coli; D013879:Thioredoxins; C488038:viscotoxin; D005982:Glutathione Transferase; D019704:Protein Disulfide-Isomerases; C526648:dsbA protein, E coli; C526649:dsbC protein, E coli