Detecting Cytosolic Peptide Delivery with the GFP Complementation Assay in the Low Micromolar Range

Transfection of cells with a plasmid encoding for the first ten strands of the GFP protein (GFP1‐10) provides the means to detect cytosolic peptide import at low micromolar concentrations. Cytosolic import of the eleventh strand of the GFP protein either by electroporation or by cell‐penetrating pep...

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Bibliographic Details
Published in:Angewandte Chemie 2015-12, Vol.127 (50), p.15320-15323
Main Authors: Schmidt, Samuel, Adjobo-Hermans, Merel J. W., Wallbrecher, Rike, Verdurmen, Wouter P. R., Bovée-Geurts, Petra H. M., van Oostrum, Jenny, Milletti, Francesca, Enderle, Thilo, Brock, Roland
Format: Article
Language:eng ; ger
Subjects:
Chemistry
Electroporation
Encoding
Fluorescence
Formations
Fragmentvervollständigung
Imports
Lipidmembranen
Peptides
Proteins
Strands
Wirkstofftransport
Zellaufnahme
Zellgängige Peptide
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Summary:Transfection of cells with a plasmid encoding for the first ten strands of the GFP protein (GFP1‐10) provides the means to detect cytosolic peptide import at low micromolar concentrations. Cytosolic import of the eleventh strand of the GFP protein either by electroporation or by cell‐penetrating peptide‐mediated import leads to formation of the full‐length GFP protein and fluorescence. An increase in sensitivity is achieved through structural modifications of the peptide and the expression of GFP1‐10 as a fusion protein with mCherry. Peptidbasierte Vervollständigung von GFP: In Zellen, die das GFP1‐10‐Fragment von GFP (grauer Kreis) exprimieren, wurde GFP‐11 (graues Quadrat) eingeschleust, das mit einem Linker (blau) an CPP (orange) konjugiert war. Daraus resultierte die Vervollständigung von GFP (grüner Kreis) im Zytosol, die sich durch Fluoreszenz zu erkennen gab.
ISSN:0044-8249
1521-3757
DOI:10.1002/ange.201505913