Document Detail


Genetically engineered clostridial C2 toxin as a novel delivery system for living mammalian cells.
MedLine Citation:
PMID:  20030334     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
The C2 toxin of Clostridium botulinum is a binary bacterial protein toxin, comprising the enzyme component C2I and the separate binding/translocation component C2IIa. C2IIa mediates the transport of C2I into the host cell cytosol. The N-terminal domain of C2I (C2IN) is enzymatically inactive but essential for C2IIa-mediated internalization of C2I. Here, we exploit the C2IIa/C2IN system to generate a recombinant C2IN-streptavidin fusion protein allowing for the delivery of biotinylated molecules into the cytosol of mammalian cells. C2IN-streptavidin overproduced in E. coli was affinity-purified and capable of binding biotinylated proteins in a concentration-dependent manner. Real-time surface plasmon resonance confirmed the biotin-mediated interaction yielding a K(D)-value of approximately 0.75 muM. Internalization of C2IN-streptavidin into the cytosol of epithelial cells and macrophages was demonstrated by immunoblot analysis and confirmed by confocal microscopy. Cell viability studies showed no cytotoxic effects of the novel transporter. Furthermore, Vero cells treated with biotin-fluorescein or biocytin-Alexa488 as model cargo displayed a specific C2IN-streptavidin/C2IIa-dependent uptake, providing proof-of-principle for the functionality of this novel delivery system.
Authors:
J?rg Fahrer; Rainer Plunien; Ulrike Binder; Torben Langer; Hartmut Seliger; Holger Barth
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Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  Bioconjugate chemistry     Volume:  21     ISSN:  1520-4812     ISO Abbreviation:  Bioconjug. Chem.     Publication Date:  2010 Jan 
Date Detail:
Created Date:  2010-01-20     Completed Date:  2010-05-20     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  9010319     Medline TA:  Bioconjug Chem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  130-9     Citation Subset:  IM    
Affiliation:
Institute of Pharmacology and Toxicology, University of Ulm, Albert-Einstein-Allee 47, D-89081 Ulm, Germany.
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MeSH Terms
Descriptor/Qualifier:
Animals
Binding Sites
Biotin / chemistry,  metabolism
Biotinylation
Botulinum Toxins / chemistry,  genetics*,  metabolism*,  toxicity
Cell Line
Cell Survival
Cercopithecus aethiops
Cricetinae
Cytosol / metabolism
Drug Delivery Systems / methods*
Epithelial Cells / metabolism
Escherichia coli / chemistry,  metabolism
Fluoresceins / chemistry,  metabolism
Genetic Engineering*
Humans
Immunoblotting
Macrophages / metabolism
Microscopy, Confocal
Protein Transport
Recombinant Fusion Proteins / chemistry,  genetics*,  metabolism*,  toxicity
Streptavidin / chemistry,  genetics,  metabolism
Vero Cells
Chemical
Reg. No./Substance:
0/Botulinum Toxins; 0/Fluoresceins; 0/Recombinant Fusion Proteins; 58-85-5/Biotin; 9013-20-1/Streptavidin

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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