A genetically encoded photosensitizer

Bulina, Maria E; Chudakov, Dmitriy M.; Britanova, Olga V.; Yanushevich, Yurii G.; Staroverov, Dmitry B; Chepurnykh, T. V.; Merzlyak, Ekaterina M.; Shkrob, Maria; Lukyanov, Sergey; Lukyanov, Konstantin A.

doi:10.1038/nbt1175

Cited by 516 publications

(587 citation statements)

References 34 publications

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“…However, the high-energy requirements of EGFP-CALI hold a hidden benefit: routine imaging and protein inactivation can be performed separately because very distinct excitation regimes are required for normal imaging and target inactivation. Newly developed fluorescent proteins such as Killer Red produce reactive photoproducts much more efficiently, and therefore illumination must be carefully controlled to avoid unintended photodamage during imaging (28). Moreover, because many functional EGFP fusions already exist, this technique can readily be applied to many proteins with minimal time spent generating new reagents.…”

Section: Discussionmentioning

confidence: 99%

Enhanced EGFP-chromophore-assisted laser inactivation using deficient cells rescued with functional EGFP-fusion proteins

Vitriol

Uetrecht

Shen

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Chromophore-assisted laser inactivation (CALI) is a light-mediatedtechnique that offers precise spatiotemporal control of protein inactivation, enabling better understanding of the protein's role in cell function. EGFP has been used effectively as a CALI chromophore, and its cotranslational attachment to the target protein avoids having to use exogenously added labeling reagents. A potential drawback to EGFP-CALI is that the CALI phenotype can be obscured by the endogenous, unlabeled protein that is not susceptible to light inactivation. Performing EGFP-CALI experiments in deficient cells rescued with functional EGFP-fusion proteins permits more complete loss of function to be achieved. Here, we present a modified lentiviral system for rapid and efficient generation of knockdown cell lines complemented with physiological levels of EGFP-fusion proteins. We demonstrate that CALI of EGFP-CapZ␤ increases uncapped actin filaments, resulting in enhanced filament growth and the formation of numerous protrusive structures. We show that these effects are completely dependent upon knocking down the endogenous protein. We also demonstrate that CALI of EGFP-Mena in Mena/VASP-deficient cells stabilizes lamellipodial protrusions.capping protein ͉ Mena/VASP ͉ spatiotemporal ͉ lentivirus

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Section: Discussionmentioning

confidence: 99%

Enhanced EGFP-chromophore-assisted laser inactivation using deficient cells rescued with functional EGFP-fusion proteins

Vitriol

Uetrecht

Shen

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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“…Fluorescent proteins do not serve as good sources for ROS, presumably because the barrel structure screens the chromophore. Nevertheless, the FP called "killer red" was found to be a good photosensitizer but has the drawback of dimerization [71]. Chemical tags offer the ideal technology to localize photosensitizers in close proximity to the target protein.…”

Section: Fluorescent Chemical Tags For Chromophore Assisted Light Inamentioning

confidence: 99%

Chemical tags: Applications in live cell fluorescence imaging

Wombacher

Cornish

2011

Journal of Biophotonics

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Technologies to visualize cellular structures and dynamics enable cell biologists to gain insight into complex biological processes. Currently, fluorescent proteins are used routinely to investigate the behavior of proteins in live cells. Chemical biology techniques for selective labeling of proteins with fluorescent labels have become an attractive alternative to fluorescent protein labeling. In the last ten years the progress in the development of chemical tagging methods have been substantial offering a broad palette of applications for live cell fluorescent microscopy. Several methods for protein labeling have been established, using protein tags, peptide tags and enzyme mediated tagging. This review focuses on the different strategies to achieve the attachment of fluorophores to proteins in live cells and cast light on the advantages and disadvantages of each individual method. Selected experiments in which chemical tags have been successfully applied to live cell imaging will be discussed and evaluated. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

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“…Moreover, the main disadvantage of chemical antibody-photosensitizer chemical conjugates may be the impossibility of intracellular production of the molecule with subsequent secretion into the bloodstream [as presented by Wang et al (12)], thus generating an intraorganism photosensitizer-producing ''factory.' ' Recently, a phototoxic red f luorescent protein KillerRed has been reported (13). KillerRed can be used as a fully genetically encoded photosensitizer to kill bacterial and eukaryotic cells.…”

Section: P Hotodynamic Therapy (Pdt) Is a Promising Approach Tomentioning

confidence: 99%

Targeting cancer cells by using an antireceptor antibody-photosensitizer fusion protein

Serebrovskaya

Edelweiss²,

Stremovskiy³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

133

128

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Antibody-photosensitizer chemical conjugates are used successfully to kill cancer cells in photodynamic therapy. However, chemical conjugation of photosensitizers presents several limitations, such as poor reproducibility, aggregation, and free photosensitizer impurities. Here, we report a fully genetically encoded immunophotosensitizer, consisting of a specific anti-p185 HER-2-ECD antibody fragment 4D5scFv fused with the phototoxic fluorescent protein KillerRed. Both parts of the recombinant protein preserved their functional properties: high affinity to antigen and light activation of sensitizer. 4D5scFv-KillerRed showed fine targeting properties and efficiently killed p185 HER-2-ECD -expressing cancer cells upon light irradiation. It also showed a remarkable additive effect with the commonly used antitumor agent cisplatin, further demonstrating the potential of the approach.reactive oxygen species ͉ phototoxicity ͉ KillerRed

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A genetically encoded photosensitizer

Cited by 516 publications

References 34 publications

Enhanced EGFP-chromophore-assisted laser inactivation using deficient cells rescued with functional EGFP-fusion proteins

Enhanced EGFP-chromophore-assisted laser inactivation using deficient cells rescued with functional EGFP-fusion proteins

Chemical tags: Applications in live cell fluorescence imaging

Targeting cancer cells by using an antireceptor antibody-photosensitizer fusion protein

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