Highly fluorescent protein labeling using dendritic peptide derivatives.
Non-specific fluorescent dyes and photosensitizers are routinely used in clinical practice for the photodetection and photoablation of superficial lesions. Future applications in photomedicine are likely to rely on the selective delivery of photoactive compounds to diseased areas, using specific targeting agents such as antibodies. This fact underlines the need for methods that allow the chemically defined conjugation of several photoactive molecules to a single protein 'vehicle', with full retention of binding affinity. Here, we present methods for the site-specific fluorescent labeling of proteins using dendritic peptides, which had been chemically modified with multiple molecules of fluorescein. Branched peptide derivatives can be stably conjugated to proteins either by reaction with suitable free reactive groups or by using the high-affinity non-covalent interaction between calmodulin and a specific binding peptide. Chemical modification of proteins with one, two or four molecules of fluorescein resulted in a proportional increase in protein fluorescence.
Giovannoni
L
L
Philogen S.r.l. La Lizza, Siena, Italy.
Lozzi
L
L
Neri
D
D
Neri
P
P
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D019793:Fluorescein / Q000737:chemistry*; D005456:Fluorescent Dyes; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010455:Peptides / Q000138:chemical synthesis*; D011485:Protein Binding; D013050:Spectrometry, Fluorescence
D000911:Antibodies, Monoclonal; D002147:Calmodulin; D005456:Fluorescent Dyes; D010455:Peptides; D019793:Fluorescein