Semienzymatic Cyclization of Disulfide-rich Peptides Using Sortase A

Jia, Xinying; Kwon, Soohyun; Wang, Ching-I Anderson; Huang, Yen‐Hua; Chan, Lai Yue; Tan, Chia Chia; Rosengren, K. Johan; Mulvenna, Jason; Schroeder, Christina I.; Craik, David J.

doi:10.1074/jbc.m113.539262

Cited by 86 publications

(91 citation statements)

References 82 publications

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“…Notwithstanding these limitations, the method described here provides a robust and efficient approach for in vitro production of cyclic cystine-knot peptides and can potentially be extended to produce backbone-cyclized cystine-knot peptides in vivo by coexpression with Sortase A. Finally, while this manuscript was in preparation, a study demonstrated the use of wild-type Sortase A to cyclize another cystine-knot peptide, the cyclotide kalata B1 [32], which was obtained via chemical synthesis and oxidative H NOESY spectrum of cyclic rMCoTI-II (black) overlaid with the back-calculated data (red). The back-calculation of the 300 ms NOESY using a distance cut-off of 0.5 nm was done in Auremol (Bruker Reports 2004, 154/155, 11-14) utilizing the complete relaxation matrix approach.…”

Section: Resultsmentioning

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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Section: Resultsmentioning

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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“…After an acyl shift from the thioester to the thiol side chain of cysteine, an S→N rearrangement takes place, driving the reaction to completion and resulting in an N→C backbone-cyclized peptide with a cysteine insert at the ligation site ( Figure 1D). Cyclization of θ-defensins, containing two anti-parallel motifs of three cysteine residues separated by a single residue (CXCXC), is accelerated due to formation of an intermediate so-called cysteine zipper Tam and Wong, 2012;Borra and Camarero, 2013;Jia et al, 2014). The drawback of the NPL strategy is that it has been incompatible with standard Fmoc-protocols for many years, as C-terminal thioesters are not resistant to the repetitive alkaline conditions during Fmoc removal by piperidine .…”

Section: Chemical Cyclization Of Peptidesmentioning

confidence: 99%

“…This has several benefits. In the first place, it offers the possibility to position the LPXTGGlinker at a safe distance from the known active domain or recognition site of the peptide (Jia et al, 2014). If present, existing motifs within the sequence can be used, so that the size of the inserts is diminished, which minimizes even more the risk on deleterious biological side effects.…”

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confidence: 99%

Sortase-mediated backbone cyclization of proteins and peptides

Hof

Maňásková²,

Veerman

et al. 2015

Biological Chemistry

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Backbone cyclization has a profound impact on the biological activity and thermal and proteolytic stability of proteins and peptides. Chemical methods for cyclization are not always feasible, especially for large peptides or proteins. Recombinant Staphylococcus aureus sortase A shows potential as a new tool for the cyclization of both proteins and peptides. In this review, the scope and background of the sortase-mediated cyclization are discussed. High efficiency, versatility, and easy access make sortase A a promising cyclization tool, both for recombinant and chemo-enzymatic production methods.

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“…[7] Thus,d ue to their favourable properties such as excellent chemo-selectivity,t he use of enzymesf or the head-totail cyclization of peptides has been extensively examined and providesa ne legant link between chemistry and biology. [8] Thec urrently existing set of enzymes used for peptide cyclization is comprisedo fe nzymes such as sortases, [9,10] trypsin, [11] asparaginyl endoproteases (AEP)l ike butelase-1 [12,13] or OaAEP1b [14] and subtilisin variants like peptiligase. [15,16] Most of the enzymatic approachesi nvestigated sufferf rom incomplete ligation reactions and low catalytic efficiency, and in addition leave al igase "footprint", an unavoidable enzyme recognition sequence at the coupling site.I nc ontrast, peptiligase based enzyme variants have recently emerged as av ery powerful tool for traceless (footprint free) enzyme-mediated peptide bond formation.…”

mentioning

confidence: 99%

Omniligase‐1: A Powerful Tool for Peptide Head‐to‐Tail Cyclization

Schmidt

Toplak²,

Quaedflieg³

et al. 2017

Adv Synth Catal

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Strategies for the efficients ynthesis of peptide macrocyclesh ave been al ong-standing goal. In this paper, we demonstrate the use of the peptide ligase termedo mniligase-1 as av ersatile and broadly applicable enzymatic toolf or peptide cyclization. Severalh ead-to-tail (multi)cyclic peptides have been synthesized, including the cyclotide MCoTI-II. Cyclization ando xidative foldingo ft he cyclotide MCoTI-II were efficiently performed in ao ne-pot reactiono na1 -gram scale.T he native cyclotide was isolated andt he correct disulfide bonding pattern was confirmed by NMRs tructure determination. Furthermore,c ompatibility of chemo-enzymatic peptide synthesis( CEPS) using omniligase1w ith methods such as chemical ligation of peptides onto scaffolds( CLIPS)w as successfully demonstrated by synthesizing ak inase-inhibitor derived tricyclic peptide.O ur studies indicate that the minimal ring size for omniligase-1 mediated cyclization is 11 amino acids,w hereas the cyclization of peptides longer than 12 amino acids proceeds with remarkablee fficiency.I na ddition,s everal macrocycles containing non-peptidic backbones( e.g.,p olyethylene glycol), isopeptide bonds (aminoa cid sidechain attachment) as well as d-amino acids couldbe efficiently cyclized.Keywords: chemo-enzymatic peptide synthesis (CEPS);c yclic peptides; cyclization;c yclotide synthesis;c yclotides;e nzyme catalysis;h ead-to-tail cyclization; ligases;m acrocycles;o mniligase-1;p eptides Peptide macrocyclesr epresent an extremely diverse class of molecules that attracti ncreased attentiona s drug leads and prospective pharmaceuticals,w ith currently over 30 cyclic peptides registered or in clinical trials.[1-3] Togetherw ith linearp eptides, they fill the gap between small-molecule drugs (less than 500 Da) and biologics (over 5000 Da) andh avet he potential to address previously "undruggable" targets. Many cyclicp eptides are characterized by their unique structural and enhancedb iopharmaceutical properties,s uch as an improved metabolic stability due to ar educed sensitivity to proteolytic cleavage.T he increasing number of cyclic peptidesu sed as therapeutics is accompanied by the need for efficient andc osteffectiver outes that enable their synthesis on al arge scale.M oreover, efficients ynthesiso fl arge libraries of cyclic peptides (e.g.,f or screeningp urposes) or of cyclicp eptides containing non-canonical amino acids (e.g., d-o ru nnatural amino acids to increase stability and diversity) is of importance.H owever, efficient cyclization of peptidesu sing traditional synthetic methodsi ss till ac hallenge.[4] Classical coupling reagents are often used to cyclize side-chain protected peptides in anhydrous organic solvents.H owever, heavy dilution to prevent polymerization,r isk of epimerization and ap oors olubility of side-chain protected peptideslimitthis approach, especially for peptides longer than 20 amino acids.[5] Native chemical ligation (NCL) [6] is often used to cyclize unprotected peptides in aqueous solution. However, not all pep...

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Semienzymatic Cyclization of Disulfide-rich Peptides Using Sortase A

Cited by 86 publications

References 82 publications

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

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

Sortase-mediated backbone cyclization of proteins and peptides

Omniligase‐1: A Powerful Tool for Peptide Head‐to‐Tail Cyclization

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