Efficient N‐Terminal Labeling of Proteins by Use of Sortase

Williamson, Daniel J.; Fascione, M. A.; Webb, Michael E.; Turnbull, W. Bruce

doi:10.1002/anie.201204538

Cited by 117 publications

(109 citation statements)

References 38 publications

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“…With respect to by-product deactivation, LPXTG variants have been designed that release unreactive fragments during formation of the desired sortagging product (Figure 2c). 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].…”

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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“…Proteins can also serve as nucleophiles, provided they display a suitably exposed (stretch of) glycine(s) at their N-terminus (Gly 1-5 ). Such modification allows the proteins to be N-terminally labeled with functionalized peptides 24,25 or to form protein-protein adducts 1,26,27 .…”

Section: Introductionmentioning

confidence: 99%

“…Relying on a common mechanistic principle, sortagging affords ready access to a wealth of site-specific modifications: C-terminal 10,19,20 , internal loop regions 1,28 , N-terminal 24,25 , and formation of cyclized (poly)peptides 29,30,31 . We have mainly used sortases A derived from Staphylococcus aureus and Streptococcus pyogenes .…”

Section: Introductionmentioning

confidence: 99%

Site-specific C-terminal and internal loop labeling of proteins using sortase-mediated reactions

et al. 2013

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Methods for site-specific modification of proteins are in high demand. Reactions that yield bioconjugates should be quantitative, site-specific, and versatile with respect to nature and size of the biological/chemical targets involved, require minimal modification of the target, display acceptable kinetics under physiological conditions, and be orthogonal to other labeling methods. Sortase-mediated transpeptidation reactions meet these criteria. Here we describe the expression and purification conditions for two orthogonal sortase A enzymes and provide the protocol that allows functionalization of any given protein at its C-terminus or for select proteins at an internal site. Sortase-mediated reactions take only a few minutes, but reaction times can be extended to increase yields.

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“…This enzyme cleaves the amide bond between Thr and Gly and catalyzes the ligation of the cleaved sequence to a Gly-Gly-Gly motif, resulting in a contiguous LeuPro-X-Thr-Gly-Gly-Gly polypeptide chain [20][21][22]. The Sortase A reaction which occurs primarily as an intermolecular reaction in S. aureus, has been recently exploited for generating circular proteins or creating fusions of different proteins, as well as labeling proteins at the N-or C-termini [23][24][25]. To generate cyclic MCoT-I-II, we engineered a MCoTI-II construct (referred to as rMCoTI-II) that comprises a Gly-Gly-Gly motif at the N-terminus and LeuPro-Glu-Thr-Gly-Gly at the C-terminus (Fig.…”

Section: Biosynthesis Of Recombinant Mcoti-iimentioning

confidence: 99%

Backbone cyclization of a recombinant cystine‐knot peptide by engineered Sortase A

Stanger¹,

Maurer²,

Kaluarachchi³

et al. 2014

FEBS Letters

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Edited by Miguel De la RosaKeywords: Cyclotide Recombinant expression Sortase Protein engineering MCoTI-II Cystine-knot peptide a b s t r a c t Cyclotides belong to the family of cyclic cystine-knot peptides and have shown promise as scaffolds for protein engineering and pharmacological modulation of cellular protein activity. Cyclotides are characterized by a cystine-knotted topology and a head-to-tail cyclic polypeptide backbone. While they are primarily produced in plants, cyclotides have also been obtained by chemical synthesis. However, there is still a need for methods to generate cyclotides in high yields to near homogeneity. Here, we report a biomimetic approach which utilizes an engineered version of the enzyme Sortase A to catalyze amide backbone cyclization of the recombinant cyclotide MCoTI-II, thereby allowing the efficient production of active homogenous species in high yields. Our results provide proof of concept for using engineered Sortase A to produce cyclic MCoTI-II and should be generally applicable to generating other cyclic cystine-knot peptides.

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Efficient N‐Terminal Labeling of Proteins by Use of Sortase

Cited by 117 publications

References 38 publications

Recent advances in sortase-catalyzed ligation methodology

Recent advances in sortase-catalyzed ligation methodology

Site-specific C-terminal and internal loop labeling of proteins using sortase-mediated reactions

Backbone cyclization of a recombinant cystine‐knot peptide by engineered Sortase A

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