Genetically Encoded 1,2-Aminothiols Facilitate Rapid and Site-Specific Protein Labeling via a Bio-orthogonal Cyanobenzothiazole Condensation

Nguyen, Duy; Elliott, T. S. J.; Holt, Matthew T.; Muir, Tom W.; Chin, Jason W.

doi:10.1021/ja203111c

Cited by 155 publications

(153 citation statements)

References 32 publications

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“…1,2-Aminothiols also react with cyanobenzothiazole derivatives, which has been reported as a very fast bioorthogonal labeling reaction (Fig. 5b) [86].…”

Section: Bioorthogonal Labeling Of Proteinsmentioning

confidence: 76%

Biochemical analysis with the expanded genetic lexicon

2012

Anal Bioanal Chem

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The information used to build proteins is stored in the genetic material of every organism. In nature, ribosomes use 20 native amino acids to synthesize proteins in most circumstances. However, laboratory efforts to expand the genetic repertoire of living cells and organisms have successfully encoded more than 80 nonnative amino acids in E. coli, yeast, and other eukaryotic systems. The selectivity, fidelity, and site-specificity provided by the technology have enabled unprecedented flexibility in manipulating protein sequences and functions in cells. Various biophysical probes can be chemically conjugated or directly incorporated at specific residues in proteins, and corresponding analytical techniques can then be used to answer diverse biological questions. This review summarizes the methodology of genetic code expansion and its recent progress, and discusses the applications of commonly used analytical methods.

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“…1,2-Aminothiols also react with cyanobenzothiazole derivatives, which has been reported as a very fast bioorthogonal labeling reaction (Fig. 5b) [86].…”

Section: Bioorthogonal Labeling Of Proteinsmentioning

confidence: 76%

Biochemical analysis with the expanded genetic lexicon

2012

Anal Bioanal Chem

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“…In particular, fluorescently labeled positions must be precisely known in a single-molecule FRET which observes energy transfer efficiency on a single protein, as exemplified in enzyme dynamics studies using T4 lysozyme and DHFR (Chen et al, 2013;Lemke, 2011). Site-specific incorporation of a reactive NNAA, in combination with an engineered cysteine, allowed mutually orthogonal dual labeling of model proteins with a donor and an acceptor dye through CuAAC and thiol-maleimide coupling, respectively Nguyen et al, 2011). However, this approach is limited when a protein has multiple cysteines, motivating the incorporation of two distinct NNAAs.…”

Section: Biochemical Analysismentioning

confidence: 99%

Bioconjugation of therapeutic proteins and enzymes using the expanded set of genetically encoded amino acids

Lim

Kwon

2015

Critical Reviews in Biotechnology

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The last decade has witnessed striking progress in the development of bioorthogonal reactions that are strictly directed towards intended sites in biomolecules while avoiding interference by a number of physical and chemical factors in biological environment. Efforts to exploit bioorthogonal reactions in protein conjugation have led to the evolution of protein translational machineries and the expansion of genetic codes that systematically incorporate a range of non-natural amino acids containing bioorthogonal groups into recombinant proteins in a site-specific manner. Chemoselective conjugation of proteins has begun to find valuable applications to previously inaccessible problems. In this review, we describe bioorthogonal reactions useful for protein conjugation, and biosynthetic methods that produce proteins amenable to those reactions through an expanded genetic code. We then provide key examples in which novel protein conjugates, generated by the genetic incorporation of a non-natural amino acid and the chemoselective reactions, address unmet needs in protein therapeutics and enzyme engineering.

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“…1) (White et al, 1963). Recently, others have reported novel applications of this reaction for the selective labeling of proteins on N-terminal cysteines (Nguyen et al, 2011; Ren et al, 2009), as well as the controlled assembly of polymers in physiological solutions and living cells (Ye et al, 2011; Liang et al, 2010). Remarkably, the rate of this reaction was found to be three orders of magnitude faster than Staudinger ligation (Ren et al, 2009; Yuana and Liang, 2014).…”

Section: Commentarymentioning

confidence: 99%

A Biocompatible “Split Luciferin” Reaction and Its Application for Non‐Invasive Bioluminescent Imaging of Protease Activity in Living Animals

Godinat

Budin

Morales

et al. 2014

CP Chemical Biology

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The great complexity of many human pathologies, such as cancer, diabetes, and neurodegenerative diseases, requires new tools for studies of biological processes on the whole organism level. The discovery of novel biocompatible reactions has tremendously advanced our understanding of basic biology; however, no efficient tools exist for real‐time non‐invasive imaging of many human proteases that play very important roles in multiple human disorders. We recently reported that the “split luciferin” biocompatible reaction represents a valuable tool for evaluation of protease activity directly in living animals using bioluminescence imaging (BLI). Since BLI is the most sensitive in vivo imaging modality known to date, this method can be widely applied for the evaluation of the activity of multiple proteases, as well as identification of their new peptide‐specific substrates. In this unit, we describe several applications of this “split luciferin” reaction for quantification of protease activities in test tube assays and living animals. Curr. Protoc. Chem. Biol. 6:169‐189 © 2014 by John Wiley & Sons, Inc.

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Genetically Encoded 1,2-Aminothiols Facilitate Rapid and Site-Specific Protein Labeling via a Bio-orthogonal Cyanobenzothiazole Condensation

Cited by 155 publications

References 32 publications

Biochemical analysis with the expanded genetic lexicon

Biochemical analysis with the expanded genetic lexicon

Bioconjugation of therapeutic proteins and enzymes using the expanded set of genetically encoded amino acids

A Biocompatible “Split Luciferin” Reaction and Its Application for Non‐Invasive Bioluminescent Imaging of Protease Activity in Living Animals

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