In-Cell Fluorescence Activation and Labeling of Proteins Mediated by FRET-Quenched Split Inteins

Borra, Radhika; Dong, Dezheng; Elnagar, Ashraf; Woldemariam, Getachew A.; Camarero, Julio A.

doi:10.1021/ja300209u

Cited by 69 publications

(70 citation statements)

References 58 publications

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“…Hence, the use of orthogonal split inteins (i.e. split inteins that do not cross-react with each other) might allow simultaneous multicolor labeling of proteins within living cells (56).…”

Section: Site-specific Labeling Of Proteinsmentioning

confidence: 99%

“…The PTS reaction ligates the fluorophore to the protein of interest while simultaneously dissociating the dye from the quencher and activating its fluorescence. This approach was used for site-specific in-cell labeling of the DNA-binding domain (DBD) of the transcription factor YY1 in several human cell lines, showing that this method can be used for modifying proteins to control their cellular localization and alter their biological activity (56).…”

Section: Site-specific Labeling Of Proteinsmentioning

confidence: 99%

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Intein Applications: From Protein Purification and Labeling to Metabolic Control Methods

Wood

Camarero

2014

Journal of Biological Chemistry

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124

102

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The discovery of inteins in the early 1990s opened the door to a wide variety of new technologies. Early engineered inteins from various sources allowed the development of self-cleaving affinity tags and new methods for joining protein segments through expressed protein ligation. Some applications were developed around native and engineered split inteins, which allow protein segments expressed separately to be spliced together in vitro. More recently, these early applications have been expanded and optimized through the discovery of highly efficient trans-splicing and trans-cleaving inteins. These new inteins have enabled a wide variety of applications in metabolic engineering, protein labeling, biomaterials construction, protein cyclization, and protein purification.The ability of inteins to form and cleave specific peptide bonds in a variety of contexts has enabled the development of powerful new tools in molecular biology. Initial applications focused on self-cleaving affinity tags and protein modification and labeling, and utilized mutations that altered the native splicing reactions described by Perler and co-workers (89) in this series. An important advantage of intein-based methods is that they are generally enzymatic in nature, and therefore exhibit highly specific activities under physiological conditions. Although thiol compounds are used in some applications, the chemistries involved in most intein methods are innocuous to their various target proteins. Intein-based methods have greatly expanded in subsequent years through the discovery of hundreds of additional inteins, and new applications in protein activity regulation and modification have followed. An important underlying theme in this work has been the discovery and development of highly efficient split inteins for advanced applications in vitro and in vivo. These trans-splicing and -cleaving inteins have been employed for applications ranging from the purification of recombinant products to the in vivo control of protein function and labeling. Protein PurificationOne of the first major applications of inteins was the development of self-cleaving affinity tags for the recovery of untagged target proteins in recombinant expression systems (1-3). In these applications, a modified intein is expressed in fusion to an affinity tag and target protein, and once the fusion is affinity-purified, the intein is induced to cleave the target protein from the intein and tag (Fig. 1). The first commercial intein system was released by New England Biolabs in 1997 and employed a modified Sce VMA1 3 intein that is triggered to cleave at its N terminus (IMPACT system), or both N and C termini (IMPACT-CN system), by the addition of thiol compounds (e.g. Refs. 4 -7) (Fig. 1A, left). Shortly thereafter, inteins with rapid C-terminal cleaving activity were published. These inteins exhibit suppressed cleavage activity at pH 8.5, allowing purification of the tagged target, and are then induced to cleave by a shift to pH 6.5. The cleavage activity of these inteins is als...

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“…Hence, the use of orthogonal split inteins (i.e. split inteins that do not cross-react with each other) might allow simultaneous multicolor labeling of proteins within living cells (56).…”

Section: Site-specific Labeling Of Proteinsmentioning

confidence: 99%

Section: Site-specific Labeling Of Proteinsmentioning

confidence: 99%

Intein Applications: From Protein Purification and Labeling to Metabolic Control Methods

Wood

Camarero

2014

Journal of Biological Chemistry

Self Cite

124

102

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“…Protein semisynthesis has been demonstrated inside living mammalian cells [110,111] and Xenopus embryos [102], as well as on the surface of eukaryotic cells [99,101]. While these studies mostly have a proof-of-principle character, it is to be expected that with improved inteins and improved methods for cellular delivery of exogenously added proteins or peptides exciting progress will be feasible.…”

Section: Intein-based Protein Engineering and Covalent Manipulationmentioning

confidence: 99%

Recent progress in intein research: from mechanism to directed evolution and applications

Volkmann

Mootz

2012

Cell. Mol. Life Sci.

102

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Inteins catalyze a post-translational modification known as protein splicing, where the intein removes itself from a precursor protein and concomitantly ligates the flanking protein sequences with a peptide bond. Over the past two decades, inteins have risen from a peculiarity to a rich source of applications in biotechnology, biomedicine, and protein chemistry. In this review, we focus on developments of intein-related research spanning the last 5 years, including the three different splicing mechanisms and their molecular underpinnings, the directed evolution of inteins towards improved splicing in exogenous protein contexts, as well as novel applications of inteins for cell biology and protein engineering, which were made possible by a clearer understanding of the protein splicing mechanism.

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“…3 In addition, the interdependence of spectral and structural properties arising from the covalent linkage between the fluorophore and protein matrix poses challenges to fine-tuning FPs’ color, brightness and photostability. 4 Furthermore, the current lack of photostable near-infrared-fluorescing proteins precludes their use for deep tissue imaging. 5 These limitations have motivated the development of alternative technologies that rely on the binding of exogenous fluorescent dyes to genetically encoded peptide or small-protein tags.…”

Section: Introductionmentioning

confidence: 99%

Bichromophoric dyes for wavelength shifting of dye-protein fluoromodules

et al. 2015

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Dye-protein fluoromodules consist of fluorogenic dyes and single chain antibody fragments that form brightly fluorescent noncovalent complexes. This report describes two new bichromophoric dyes that extend the range of wavelengths of excitation or emission of existing fluoromodules. In one case, a fluorogenic thiazole orange (TO) was attached to an energy acceptor dye, Cy5. Upon binding to a protein that recognizes TO, red emission due to efficient energy transfer from TO to Cy5 replaces the green emission observed for monochromophoric TO bound to the same protein. Separately, TO was attached to a coumarin that serves as an energy donor. The same green emission is observed for coumarin-TO and TO bound to a protein, but efficient energy transfer allows violet excitation of coumarin-TO, versus longer wavelength, blue excitation of monochromophoric TO. Both bichromophores exhibit low nanomolar KD values for their respective proteins, >95% energy transfer efficiency and high fluorescence quantum yields.

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In-Cell Fluorescence Activation and Labeling of Proteins Mediated by FRET-Quenched Split Inteins

Cited by 69 publications

References 58 publications

Intein Applications: From Protein Purification and Labeling to Metabolic Control Methods

Intein Applications: From Protein Purification and Labeling to Metabolic Control Methods

Recent progress in intein research: from mechanism to directed evolution and applications

Bichromophoric dyes for wavelength shifting of dye-protein fluoromodules

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