Irreversible Sortase A-Mediated Ligation Driven by Diketopiperazine Formation

Liu, Fa; Luo, Ethan Y.; Flora, David B.; Mezo, Adam R.

doi:10.1021/jo4024914

Cited by 41 publications

(39 citation statements)

References 21 publications

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“…Wiliamson et al reported the synthesis of a depsipeptide that releases a relatively unreactive hydroxyacetyl moiety instead of a nucleophilic aminoglycine [40, 41• 42]. A second depsipeptide was synthesized by Liu and coworkers that involves release of a fragment that spontaneously deactivates via diketopiperazine formation [43]. These systems, which are best suited for N-terminal labeling, give excellent ligation yields with a variety of protein and peptide targets using only 1.0–3.0 equivalents of depsipeptide acyl donor.…”

Section: Driving Ligation Product Formationmentioning

confidence: 99%

Recent advances in sortase-catalyzed ligation methodology

Antos

Truttmann

Ploegh

2016

Current Opinion in Structural Biology

139

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The transpeptidation reaction catalyzed by bacterial sortases continues to see increasing use in the construction of novel protein derivatives. In addition to growth in the number of applications that rely on sortase, this field has also seen methodology improvements that enhance reaction performance and scope. In this opinion, we present an overview of key developments in the practice and implementation of sortase-based strategies, including applications relevant to structural biology. Topics include the use of engineered sortases to increase reaction rates, the use of redesigned acyl donors and acceptors to mitigate reaction reversibility, and strategies for expanding the range of substrates that are compatible with a sortase-based approach.

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Section: Driving Ligation Product Formationmentioning

confidence: 99%

Recent advances in sortase-catalyzed ligation methodology

Antos

Truttmann

Ploegh

2016

Current Opinion in Structural Biology

139

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“…Therefore, we explored next whether MPI-SrtA could be used to modify proteins. In this case, bovine insulin was used as the model protein, as there is a glycine residue located at its N-terminus that has been established as a nucleophile in SML 56 . Bz-LPETGGS was used as the peptide donor as shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

One-step purification and immobilization of extracellularly expressed sortase A by magnetic particles to develop a robust and recyclable biocatalyst

Zhao

Hong

Cheng

et al. 2017

Sci Rep

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Sortase A (SrtA) is a transpeptidase widely used to site-specifically modify peptides and proteins and shows promise for industrial applications. In this study, a novel strategy was developed for constructing immobilized-SrtA as a robust and recyclable enzyme via direct immobilization of extracellularly expressed SrtA in the fermentation supernatant using magnetic particles. Efficient extracellular SrtA expression was achieved in Escherichia coli through molecular engineering, including manipulation of the protein transport pathway, codon optimization, and co-expression of molecular chaperones to promote expressed SrtA secretion into the medium at high levels. Subsequently, a simple one-step protocol was established for the purification and immobilization of SrtA containing a His-tag from the fermentation supernatant onto a nickel-modified magnetic particle. The immobilized SrtA was proved to retain full enzymatic activity for peptide-to-peptide ligation and protein modification, and was successfully reused for five cycles without obvious activity loss.

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“…In terms of reaction reversibility, a handful of strategies have been introduced that drive reaction equilibrium through selective removal or deactivation of transpeptidation products. These strategies may be as simple as running the reaction under dialysis conditions to separate out low-molecular-weight byproducts (Kobashigawa, Kumeta, Ogura, & Inagaki, 2009; Refaei et al, 2011; Pritz et al, 2007), but also include the use of β-hairpins (Yamamura, Hirakawa, Yamaguchi, & Nagamune, 2011), depsipeptides (Liu, Luo, Flora, & Mezo, 2014; Williamson, Fascione, Webb, & Turnbull, 2012), or masked metal-binding peptides (Row et al, 2015) that prevent certain transpeptidation products from re-entering the catalytic cycle. Finally, advances have been made in expanding the substrate scope of sortase-catalyzed transpeptidations beyond the LPXTG motif.…”

Section: Commentarymentioning

confidence: 99%

Site‐Specific Protein Labeling via Sortase‐Mediated Transpeptidation

et al. 2017

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Strategies for site-specific protein modification are highly desirable for the construction of conjugates containing non-genetically-encoded functional groups. Ideally, these strategies should proceed under mild conditions, and be compatible with a wide range of protein targets and non-natural moieties. The transpeptidation reaction catalyzed by bacterial sortases is a prominent strategy for protein derivatization that possesses these features. Naturally occurring or engineered variants of sortase A from Staphylococcus aureus catalyze a ligation reaction between a five-amino-acid substrate motif (LPXTG) and oligoglycine nucleophiles. By pairing proteins and synthetic peptides that possess these ligation handles, it is possible to install modifications onto the protein N- or C-terminus in site-specific fashion. As described in this unit, the successful implementation of sortase-mediated labeling involves straightforward solid-phase synthesis and molecular biology techniques, and this method is compatible with proteins in solution or on the surface of live cells.

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Irreversible Sortase A-Mediated Ligation Driven by Diketopiperazine Formation

Cited by 41 publications

References 21 publications

Recent advances in sortase-catalyzed ligation methodology

Recent advances in sortase-catalyzed ligation methodology

One-step purification and immobilization of extracellularly expressed sortase A by magnetic particles to develop a robust and recyclable biocatalyst

Site‐Specific Protein Labeling via Sortase‐Mediated Transpeptidation

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