Development of a Small Peptide Tag for Covalent Labeling of Proteins

Tanaka, Fujie; Fuller, Roberta P.; Asawapornmongkol, Lily; Warsinke, Axel; Gobuty, and Sarah; Barbas, Carlos F.

doi:10.1021/bc070080x

Cited by 24 publications

(31 citation statements)

References 53 publications

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“…34,36,37 To ascertain whether the reaction conditions for Cu-free Sonogashira cross-coupling is compatible with phage display, M13KE phage was incubated with the palladium catalyst at 37 °C for 30 min and the phage titer was then determined. No significant decrease in phage titer was detected (Table S1 in the Supporting Information), suggesting that phage was tolerant to the palladium treatment.…”

Section: Resultsmentioning

confidence: 99%

Fast and Sequence-Specific Palladium-Mediated Cross-Coupling Reaction Identified from Phage Display

Lim

Ramil

et al. 2014

ACS Chem. Biol.

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Fast and specific bioorthogonal reactions are highly desirable because they provide efficient tracking of biomolecules that are present in low abundance and/or involved in fast dynamic process in living systems. Toward this end, classic strategy involves the optimization of substrate structures and reaction conditions in test tubes, testing their compatibility with biological systems, devising synthetic biology schemes to introduce the modified substrates into living cells or organisms, and finally validating the superior kinetics for enhanced capacity in tracking biomolecules in vivo—a lengthy process often mired by unexpected results. Here, we report a streamlined approach in which the “microenvironment” of a bioorthogonal chemical reporter is exploited directly in biological systems via phage-assisted interrogation of reactivity (PAIR) to optimize not only reaction kinetics but also specificity. Using the PAIR strategy, we identified a short alkyne-containing peptide sequence showing fast kinetics (k2 = 13 000 ± 2000 M–1 s–1) in a palladium-mediated cross-coupling reaction. Site-directed mutagenesis studies suggested that the residues surrounding the alkyne moiety facilitate the assembly of a key palladium–alkyne intermediate along the reaction pathway. When this peptide sequence was inserted into the extracellular domain of epidermal growth factor receptor (EGFR), this reactive sequence directed the specific labeling of EGFR in live mammalian cells.

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Section: Resultsmentioning

confidence: 99%

Fast and Sequence-Specific Palladium-Mediated Cross-Coupling Reaction Identified from Phage Display

Lim

Ramil

et al. 2014

ACS Chem. Biol.

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“…Previous studies have included the selection of enzymes or antibodies by using mechanismbased inhibitors or reactive ligands for evolving catalytic activity, [14][15][16][17] and the selection of peptides based on enaminone formation by using a reactive ligand for developing a small peptide tag. [18] However, phage display has several limitations for directed evolution experiments because of the cellular transformation step-it takes 3-4 days for each round of selection, and the library size is limited to 10 9 -10 10 . Moreover, protein expression in vivo can be problematic and varies between different proteins.…”

Section: Discussionmentioning

confidence: 99%

In Vitro Selection of Proteins that Undergo Covalent Labeling with Small Molecules by Thiol‐Disulfide Exchange by Using Ribosome Display

et al. 2011

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There is a great deal of interest in proteins that can bind covalently to target molecules, as they allow unambiguous experiments by tight binding to molecules of interest. Here, we report the generation of proteins that undergo covalent labeling with small molecules through in vitro selection by using ribosome display. Selection was performed from a mutant library of the WW domain with a biotinylated peptide as its binding target, in which the biotin and the peptide are connected by a disulfide bond. After five rounds of selection, we identified mutants carrying a particular cysteine mutation. The binding target reacted specifically with the selected mutant, even in the presence of other proteins, and resulted in the generation of biotin- or peptide-labeled WW domains by thiol-disulfide exchange. When the mutant was fused to a protein of interest, the fusion protein was also labeled with biotin. Thus, the characteristics of the selected mutant should be suitable as a tag sequence that can be covalently labeled with small synthetic molecules. These results indicate that the rapid and efficient generation of such proteins is possible by ribosome display.

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“…Phage display has been used to identify short peptides that react with functional groups (7). For example, we have recently selected from a phage-displayed peptide library a set of peptides that react with the hydrazide functional group under biological conditions (8).…”

Section: Introductionmentioning

confidence: 99%

Identifying Reactive Peptides from Phage-Displayed Libraries

Eldridge

Weiss

2014

Peptide Libraries

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Summary Phage display enables the synthesis, selection and screening of large, polypeptide libraries (>1 × 1010). Selections from such libraries can identify binding partners to essentially any desired target (1, 2). Peptide with affinity or reactivity to small molecule probes are attractive for numerous uses including the targeted, site-specific labeling of proteins. Here, we describe selection and screening protocols for the identification of short peptides that can selectively bind to and/or react with small molecules.

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Development of a Small Peptide Tag for Covalent Labeling of Proteins

Cited by 24 publications

References 53 publications

Fast and Sequence-Specific Palladium-Mediated Cross-Coupling Reaction Identified from Phage Display

Fast and Sequence-Specific Palladium-Mediated Cross-Coupling Reaction Identified from Phage Display

In Vitro Selection of Proteins that Undergo Covalent Labeling with Small Molecules by Thiol‐Disulfide Exchange by Using Ribosome Display

Identifying Reactive Peptides from Phage-Displayed Libraries

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