Sortagging: a versatile method for protein labeling

Popp, Maximilian W.; Antos, John M.; Grotenbreg, Gijsbert M.; Spooner, Eric; Ploegh, Hidde L.

doi:10.1038/nchembio.2007.31

Cited by 490 publications

(499 citation statements)

References 15 publications

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“…Sortase reactions with SrtA Staph were performed as described previously (11). For either cyclization or PEGylation, reactions containing 50 μM substrate, 50 μM SrtA Staph and 1 mM probe (for PEGylation) were incubated overnight at 25°C without agitation.…”

Section: Discussionmentioning

confidence: 99%

“…This acyl-enzyme is resolved by nucleophilic attack by the N terminus of an oligoglycine peptide, resulting in formation of an amide bond between the substrate protein and the incoming nucleophile (10). Sortase A tolerates C-terminal extensions of the oligoglycine nucleophile, allowing diverse functionalized nucleophiles to be installed site specifically onto proteins equipped with an LPXTG motif (11,12). The related Streptococcus pyogenes sortase accepts di-alanine based nucleophiles, which the S. aureus enzyme does not.…”

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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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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%

mentioning

confidence: 99%

Sortase-catalyzed transformations that improve the properties of cytokines

Popp

Dougan

Chuang

et al. 2011

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

Self Cite

177

150

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“…Future studies will show if probe selectivity can be further expanded to more attractive fluorescent probes, allowing for widespread use of PRIME as tagging approach for fluorescence imaging. The shortest known enzyme-mediated peptide tag is the sortase tag [54][55][56]. The bacterial enzyme sortase A from Staphylococcus aureus recognizes the 5 amino acid peptide tag sequence LPXTG, cleaves between the T and G residues and subsequently forms a new peptide bond to a polyG-probe conjugate.…”

Section: Enzyme Mediated Peptide Tagsmentioning

confidence: 99%

Chemical tags: Applications in live cell fluorescence imaging

Wombacher

Cornish

2011

Journal of Biophotonics

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Technologies to visualize cellular structures and dynamics enable cell biologists to gain insight into complex biological processes. Currently, fluorescent proteins are used routinely to investigate the behavior of proteins in live cells. Chemical biology techniques for selective labeling of proteins with fluorescent labels have become an attractive alternative to fluorescent protein labeling. In the last ten years the progress in the development of chemical tagging methods have been substantial offering a broad palette of applications for live cell fluorescent microscopy. Several methods for protein labeling have been established, using protein tags, peptide tags and enzyme mediated tagging. This review focuses on the different strategies to achieve the attachment of fluorophores to proteins in live cells and cast light on the advantages and disadvantages of each individual method. Selected experiments in which chemical tags have been successfully applied to live cell imaging will be discussed and evaluated. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

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“…D espite the many attractive features of protein enzymes as catalysts for organic synthesis (1), as research tools (2)(3)(4), and as an important class of human therapeutics (5,6), the extent and diversity of their applications remain limited by the difficulty of finding in nature or creating in the laboratory highly active proteins that catalyze chemical reactions of interest. A significant fraction of protein catalysts currently used for research and industrial applications was obtained through the directed evolution of natural enzymes (7).…”

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confidence: 99%

A general strategy for the evolution of bond-forming enzymes using yeast display

Chen

Dorr

Liu

2011

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

511

584

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The ability to routinely generate efficient protein catalysts of bondforming reactions chosen by researchers, rather than nature, is a long-standing goal of the molecular life sciences. Here, we describe a directed evolution strategy for enzymes that catalyze, in principle, any bond-forming reaction. The system integrates yeast display, enzyme-mediated bioconjugation, and fluorescence-activated cell sorting to isolate cells expressing proteins that catalyze the coupling of two substrates chosen by the researcher. We validated the system using model screens for Staphylococcus aureus sortase A-catalyzed transpeptidation activity, resulting in enrichment factors of 6,000-fold after a single round of screening. We applied the system to evolve sortase A for improved catalytic activity. After eight rounds of screening, we isolated variants of sortase A with up to a 140-fold increase in LPETG-coupling activity compared with the starting wild-type enzyme. An evolved sortase variant enabled much more efficient labeling of LPETG-tagged human CD154 expressed on the surface of HeLa cells compared with wild-type sortase. Because the method developed here does not rely on any particular screenable or selectable property of the substrates or product, it represents a powerful alternative to existing enzyme evolution methods.

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Sortagging: a versatile method for protein labeling

Cited by 490 publications

References 15 publications

Sortase-catalyzed transformations that improve the properties of cytokines

Sortase-catalyzed transformations that improve the properties of cytokines

Chemical tags: Applications in live cell fluorescence imaging

A general strategy for the evolution of bond-forming enzymes using yeast display

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