Site-Specific N- and C-Terminal Labeling of a Single Polypeptide Using Sortases of Different Specificity

Antos, John M.; Chew, Guo-Liang; Guimarães, Carla P.; Yoder, Nicholas C.; Grotenbreg, Gijsbert M.; Popp, Maximilian W.; Ploegh, Hidde L.

doi:10.1021/ja902681k

Cited by 236 publications

(264 citation statements)

References 9 publications

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“…Immunogenicity of this site is very likely reduced by the PEG moiety shielding this area (3). We and others have labeled proteins with an exposed N-terminal glycine using sortase (14,24). This approach should therefore be readily extended to site-specific PEGylation at the N terminus as well.…”

Section: Discussionmentioning

confidence: 99%

“…This sortase (SrtA strep ) cleaves the LPXTA motif between theronine and alanine and allows installation of modified alanine-based nucleophiles. SrtA strep also recognizes and cleaves LPXTG motifs, albeit with reduced efficiency, however the LPXTA motif is refractory to cleavage by SrtA Staph (13,14).Here we present a general strategy for site-specific modification of therapeutic recombinant proteins at their termini, an approach that is particularly well suited to the four-helix bundle cytokines, where the termini are distant from the receptor interaction site. We show improvements in thermal stability by cova- Fig.…”

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Sortase-catalyzed transformations that improve the properties of cytokines

Popp

Dougan

Chuang

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Recombinant protein therapeutics often suffer from short circulating half-life and poor stability, necessitating multiple injections and resulting in limited shelf-life. Conjugation to polyethylene glycol chains (PEG) extends the circulatory half-life of many proteins, but the methods for attachment often lack specificity, resulting in loss of biological activity. Using four-helix bundle cytokines as an example, we present a general platform that uses sortasemediated transpeptidation to facilitate site-specific attachment of PEG to extend cytokine half-life with full retention of biological activity. Covalently joining the N and C termini of proteins to obtain circular polypeptides, again executed using sortase, increases thermal stability. We combined both PEGylation and circularization by exploiting two distinct sortase enzymes and the use of a molecular suture that allows both site-specific PEGylation and covalent closure. The method developed is general, uses a set of easily accessible reagents, and should be applicable to a wide variety of proteins, provided that their termini are not involved in receptor binding or function.M any clinically relevant cytokines share a four-helix bundle structure, typified by IFNα2, Granulocyte colony-stimulating factor 3 (GCSF-3), Erythropoietin (EPO), IL-2, IL-4, IL-7, IL-9, and IL-15. Cocrystal structures of cytokines with receptor fragments and biochemical studies that map residues critical for interaction of a cytokine with its receptor show that the receptor contacts the sides of the helical bundles (1). This mode of interaction positions the N and C termini of the cytokine away from the receptor.Polyethylene glycol chains attached to therapeutically important proteins increase circulatory half-life, reduce clearance by kidney filtration, reduce proteolysis, and reduce the generation of neutralizing antibodies (2-4). The attachment of PEG commonly employs standard chemistries that target reactive amino acid side chains (e.g., cysteine and lysine). This strategy often generates a heterogeneous mixture in which multiple amino acids in the target are modified with a PEG chain, necessitating cumbersome separations and characterization (5). Such PEGylated molecules often show decreased biological activity, likely due to attachment of a PEG chain to a residue important for interaction with the receptor (6)-this problem may be overcome by sitespecific PEGylation. Although engineering of a carefully placed unpaired cysteine residue allows site-specific PEGylation (7, 8), this method must be tailored to the specific protein target.Sortase A from Staphylcoccus aureus (SrtA Staph ) is a thiolcontaining transpeptidase that recognizes an LPXTG motif in multiple structurally unrelated substrates (9). SrtA Staph cleaves the peptide bond between the threonine and glycine residues with concomitant formation of a thioacyl enzyme intermediate that involves the catalytic cysteine and the substrate threonine. This acyl-enzyme is resolved by nucleophilic attack by the N terminus of an oligo...

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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Sortase-catalyzed transformations that improve the properties of cytokines

Popp

Dougan

Chuang

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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177

150

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“…PMT-DTa and PMT were N-terminally conjugated to fluorescein by using the sortase method (35,36) and the fluorophore 5(6)-fluorescein-NH-Ahx-LPRT-COOMe (Thermo Scientific, Ulm, Germany).…”

Section: Methodsmentioning

confidence: 99%

“…Sortase-mediated labeling of PMT was performed according to the method described by Antos et al (36), with slight alterations. PMT or PMT-DTa was incubated with 5(6)-fluorescein-NH-Ahx-LPRT-COOMe (500 M) and SrtA Staph (20 M) for 1 h at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Pasteurella multocida Toxin as a Transporter of Non-Cell-Permeating Proteins

et al. 2013

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bThe protein toxin Pasteurella multocida toxin (PMT) is the causative agent of atrophic rhinitis in pigs, leading to atrophy of the nasal turbinate bones by affecting osteoblasts and osteoclasts. The mechanism of PMT-induced intoxication is a deamidation of ␣-subunits of heterotrimeric G proteins, including G␣ q , G␣ 13 , and G␣ i , thereby causing persistent activation of the G proteins. Here we utilized PMT as a transporter of the non-cell-permeating A domain of diphtheria toxin (DTa). Fusion proteins of PMT and DTa ADP-ribosylated elongation factor 2, the natural target of diphtheria toxin, leading to cell toxicity. PMT-DTa effects were competed by PMT, indicating binding to the same cell surface receptor. Fluorescently labeled PMT-DTa and PMT colocalized with specific markers of early and late endosomes. Bafilomycin A, which inhibits vacuolar H ؉ -ATPase, blocked PMT-DTainduced intoxication of HEK-293 cells. By constructing various PMT-DTa chimeras, we identified a minimal region of PMT necessary for uptake of DTa. The data suggest that PMT is able to transport cargo proteins into eukaryotic cells by utilizing the PMT-specific uptake route.

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Site‐Specific Protein Labeling via Sortase‐Mediated Transpeptidation

2009

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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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Site-Specific N- and C-Terminal Labeling of a Single Polypeptide Using Sortases of Different Specificity

Cited by 236 publications

References 9 publications

Sortase-catalyzed transformations that improve the properties of cytokines

Sortase-catalyzed transformations that improve the properties of cytokines

Pasteurella multocida Toxin as a Transporter of Non-Cell-Permeating Proteins

Site‐Specific Protein Labeling via Sortase‐Mediated Transpeptidation

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