Charalambous A et al. (2009), Intein-mediated site-specific conjugation of Qu...

XB-ART-40831

J Nanobiotechnology 2009 Dec 10;7:9. doi: 10.1186/1477-3155-7-9.

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Intein-mediated site-specific conjugation of Quantum Dots to proteins in vivo.

Charalambous A , Andreou M , Skourides PA .

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We describe an intein based method to site-specifically conjugate Quantum Dots (QDs) to target proteins in vivo. This approach allows the covalent conjugation of any nanostructure and/or nanodevice to any protein and thus the targeting of such material to any intracellular compartment or signalling complex within the cells of the developing embryo. We genetically fused a pleckstrin-homology (PH) domain with the N-terminus half of a split intein (IN). The C-terminus half (IC) of the intein was conjugated to QDs in vitro. IC-QD's and RNA encoding PH-IN were microinjected into Xenopus embryos. In vivo intein-splicing resulted in fully functional QD-PH conjugates that could be monitored in real time within live embryos. Use of Near Infra Red (NIR)-emitting QDs allowed monitoring of QD-conjugates within the embryo at depths where EGFP is undetectable demonstrating the advantages of QD's for this type of experiment. In conclusion, we have developed a novel in vivo methodology for the site-specific conjugation of QD's and other artificial structures to target proteins in different intracellular compartments and signaling complexes.

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Species referenced: Xenopus
Genes referenced: akt1 btk pik3ca

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	Figure 1. In vivo conjugation of QD's to Akt-PH-EGFP via intein mediated protein splicing. (a) Schematic representation of site-specific intein-mediated conjugation of QD's to target protein. (b) Co-localization of QDot585 with Akt-PH-EGFP on the cell membrane. Fluorescence images of stage 10 Xenopus embryos microinjected with the probe (IC-QDot585) shown in red, in one blastomere at the two-cell stage, and then injected with RNA encoding the target protein (Akt PH-EGFP-IN) shown in green, in three of four blastomeres. Yellow shows the overlap between red QDot585 and green EGFP indicating successful QD-protein conjugation in a live embryo. (c) Biochemical characterization of protein-QD conjugates. Xenopus embryos were injected with either probe (Ic-QD's) only or Btk-PH-EGFP-IN RNA only or both, lysed at stage 10 and loaded onto a 0.5% agarose gel. QDot655 were visualized with a band pass 650/30 emission filter under UV excitation and GFP was imaged with a band pass 500/50 filter set on UVP iBox Imaging System. The ligation product appears as a smeary band under both the GFP and QD filters, only in lysates of Xenopus embryos injected with both the RNA and the probe, and is denoted as Btk-PH-EGFP-QD conjugate. A single band corresponding to the Btk-PH-EGFP protein fusion that is not conjugated to QD's is also detectable under the GFP filter, in lysates of Xenopus embryos injected with RNA only or RNA and probe, but not QD's only.
	Figure 2. UV-inducible and wortmannin-sensitive translocation of QD-Akt-PH-EGFP conjugates to the membrane. (a) Akt-PH-EGFP protein fusions and Akt-PH-QDot585 conjugates translocate to the cell membrane upon exposure of injected Xenopus embryos to UV radiation. Live Xenopus embryos injected as described were imaged on a Zeiss Axioimager to visualize the localization of Akt-PH-EGFP and Akt-PH-QD conjugate before and after exposure to UV radiation for 5 min. Both Akt-PH-EGFP and Akt-PH-QD conjugates translocate to the cell membrane following brief exposure to UV radiation. (b) Translocation of Akt-PH-QD conjugates (QDot655) to the cell membrane is Wortmanin sensitive. Live Xenopus embryos were imaged on a Zeiss Axioimager to visualize the localization of Akt-PH-QD conjugate before and after treatment with Wortmannin (400 nM), a PI3-K inhibitor, for 1 hour. The Akt-PH QD conjugates become diffusely localized in the cytosol after treatemnt.
	Figure 3. QD-Akt-PH conjugates are resistant to photobleaching, unlike Akt-PH-EGFP fusions. Fluorescence images of stage 10 Xenopus embryos microinjected with the probe (IC-QD525) and with RNA encoding the target protein (Akt PH-EGFP-IN), both shown in green since their emission spectra are closely matched. Embryos were exposed to continuous excitation (~ 480 nm) for > 20 min. This led to gradual loss of the EGFP signal but did not affect the QDot525 signal.
	Figure 4. Increased NIR-QDot size imposes constraints on Akt-PH-QD conjugate translocation efficiency but NIR-QD's allow visualization in deeper cell layers in a live Xenopus embryo, unlike Akt-PH-EGFP (a) Co-localization of QDot705 with Akt-PH-EGFP on the cell membrane. Note that unlike the QDot585, the QDot705 are not recruited as effectively to the cell membrane. (b) QDot800 allow visualization of the Akt-PH ~ two to three layers below the superficial cell layer, where the GFP signal was either undetectable or too diffuse. The images are of the same region of the embryo imaged with a GFP (left) and a QDot800 (right) filter set.

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Evans, Mechanistic and kinetic considerations of protein splicing. 2002, Pubmed