In vivo, site-specific, covalent conjugation of quantum dots to proteins via split-intein splicing
Date
2012Author
Charalambous, AnnaAndreou, Maria I.
Antoniades, Ioanna
Christodoulou, Neophytos
Skourides, Paris A.
ISSN
1064-3745Source
Methods in Molecular BiologyVolume
906Pages
157-169Google Scholar check
Keyword(s):
Metadata
Show full item recordAbstract
The ability to target proteins with nanostructures and/or nanodevices in vivo is important for understanding and controlling their biological function. Quantum dots (QDs) serve as an ideal model nanostructure due to their superior optical properties that permit visual confirmation of in vivo targeting and localization and due to their potential as a bio-imaging tool. Here, we describe the site-specific covalent conjugation of quantum dots to target proteins in vivo using an intein-based method. Experimental procedure includes the following steps: (1) fusion of Pleckstrin-homology (PH) domains with the N-terminus half of a split intein (I N) (2) conjugation of the C-terminal (I C) intein-derived peptide to streptavidin-coated QDs in vitro and (3) in vivo expression of PH-I N following microinjection of PH-I N RNA and I C-QDs into Xenopus embryos. Intein-splicing results in covalent conjugation of QDs with the C-terminus of the PH domain without interfering with protein localization or function. Such produced QD-PH conjugates could be monitored in real time within live embryos. The use of near infrared-emitting QDs allows monitoring of QD conjugates within the embryo at depths where EGFP is undetectable demonstrating the advantages of QDs for this type of experiment. The reported approach therefore allows the covalent conjugation of QDs or other similar nanostructures to proteins in vivo and the targeting of such nanomaterial to any intracellular compartment or signaling -complex within the cells of the developing embryo. © 2012 Springer Science+Business Media, LLC.
Collections
Cite as
Related items
Showing items related by title, author, creator and subject.
-
Article
Addressing the Functional Determinants of FAK during Ciliogenesis in Multiciliated Cells
Antoniades, Ioanna; Stylianou, Panayiota; Christodoulou, Neophytos; Skourides, Paris A. (2017)We previously identified focal adhesion kinase (FAK) as an important regulator of ciliogenesis in multiciliated cells. FAK and other focal adhesion (FA) proteins associate with the basal bodies and their striated rootlets ...
-
Article
Split-Inteins for Simultaneous, site-specific conjugation of Quantum Dots to multiple protein targets In vivo
Charalambous, Anna; Antoniades, Ioanna; Christodoulou, Neophytos; Skourides, Paris A. (2011)Background: Proteins labelled with Quantum Dots (QDs) can be imaged over long periods of time with ultrahigh spatial and temporal resolution, yielding important information on the spatiotemporal dynamics of proteins within ...
-
Article
The nucleotide-binding proteins Nubp1 and Nubp2 are negative regulators of ciliogenesis
Kypri, Elena; Christodoulou, A.; Maimaris, G.; Lethan, M.; Markaki, M.; Lysandrou, C.; Lederer, C. W.; Tavernarakis, N.; Geimer, S.; Pedersen, L. B.; Santama, Niovi (2014)Nucleotide-binding proteins Nubp1 and Nubp2 are MRP/MinD-type P-loop NTPases with sequence similarity to bacterial division site-determining proteins and are conserved, essential proteins throughout the Eukaryotes. They ...