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Issue 5, 2013
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Chemical biology-based approaches on fluorescent labeling of proteins in live cells

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Abstract

Recently, significant advances have been made in live cell imaging owing to the rapid development of selective labeling of proteins in vivo. Green fluorescent protein (GFP) was the first example of fluorescent reporters genetically introduced to protein of interest (POI). While GFP and various types of engineered fluorescent proteins (FPs) have been actively used for live cell imaging for many years, the size and the limited windows of fluorescent spectra of GFP and its variants set limits on possible applications. In order to complement FP-based labeling methods, alternative approaches that allow incorporation of synthetic fluorescent probes to target POIs were developed. Synthetic fluorescent probes are smaller than fluorescent proteins, often have improved photochemical properties, and offer a larger variety of colors. These synthetic probes can be introduced to POIs selectively by numerous approaches that can be largely categorized into chemical recognition-based labeling, which utilizes metal-chelating peptide tags and fluorophore-carrying metal complexes, and biological recognition-based labeling, such as (1) specific non-covalent binding between an enzyme tag and its fluorophore-carrying substrate, (2) self-modification of protein tags using substrate variants conjugated to fluorophores, (3) enzymatic reaction to generate a covalent binding between a small molecule substrate and a peptide tag, and (4) split-intein-based C-terminal labeling of target proteins. The chemical recognition-based labeling reaction often suffers from compromised selectivity of metal–ligand interaction in the cytosolic environment, consequently producing high background signals. Use of protein–substrate interactions or enzyme-mediated reactions generally shows improved specificity but each method has its limitations. Some examples are the presence of large linker protein, restriction on the choice of introducible probes due to the substrate specificity of enzymes, and competitive reaction mediated by an endogenous analogue of the introduced protein tag. These limitations have been addressed, in part, by the split-intein-based labeling approach, which introduces fluorescent probes with a minimal size (∼4 amino acids) peptide tag. In this review, the advantages and the limitations of each labeling method are discussed.

Graphical abstract: Chemical biology-based approaches on fluorescent labeling of proteins in live cells

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Publication details

The article was received on 09 Oct 2012, accepted on 10 Dec 2012 and first published on 12 Dec 2012


Article type: Review Article
DOI: 10.1039/C2MB25422K
Citation: Mol. BioSyst., 2013,9, 862-872
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    Chemical biology-based approaches on fluorescent labeling of proteins in live cells

    D. Jung, K. Min, J. Jung, W. Jang and Y. Kwon, Mol. BioSyst., 2013, 9, 862
    DOI: 10.1039/C2MB25422K

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