Immobilization of proteins to biacore sensor chips using Staphylococcus aureus sortase A

Clow, Fiona; Fraser, John D.; Proft, Thomas

doi:10.1007/s10529-008-9718-1

Cited by 65 publications

(53 citation statements)

References 13 publications

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“…Very recently, Proft and co-workers also used SML to fabricate a protein sensor chip on which fibronectin-binding protein (Fba) was covalently attached. [52] The chip allowed the researchers to analyze the interaction A C H T U N G T R E N N U N G between the Fba and its ligand, factor H, by surface plasmon resonance.…”

Section: Covalent Immobilization Of Proteins Onto Solid Supportsmentioning

confidence: 99%

Sortase‐Mediated Ligation: A Gift from Gram‐Positive Bacteria to Protein Engineering

2009

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A new enzymatic protein ligation tool, sortase, has recently emerged from Gram‐positive bacteria. This article outlines the technique, sortase‐mediated ligation, and its applications in protein engineering, which include the introduction of unnatural molecules into proteins, protein immobilization, protein–protein conjugation, protein cyclization, as a self‐cleavable tag for protein expression, protein–PNA hybrids, neoglycoconjugates, and cell‐surface protein labeling, etc.magnified image

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Section: Covalent Immobilization Of Proteins Onto Solid Supportsmentioning

confidence: 99%

Sortase‐Mediated Ligation: A Gift from Gram‐Positive Bacteria to Protein Engineering

2009

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“…c Samantaray et al (2008) generated neoglycoconjugates be ligating a LPXTG-tagged polypeptide to amino-methylene groups present in 6-aminohexoses. d LPXTG-tagged proteins can be immobilised onto oligoglycine-coated solid surfaces, such as polystyrene beads (Parthasarathy et al 2007) or biosensor chips (Clow et al 2008) Biotechnol Lett (2010) 32:1-10 5 into the active site crossover loop of UCHL3, a member of the ubiquitin C-terminal hydrolase family was reported ). This allowed the authors to selectively modify the loop structure in order to study its role in substrate filtering.…”

Section: Specific Incorporation Of Novel Functionality Into Proteinsmentioning

confidence: 99%

Sortase-mediated protein ligation: an emerging biotechnology tool for protein modification and immobilisation

Proft

2009

Biotechnol Lett

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115

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Sortases are transpeptidases produced by Gram-positive bacteria to anchor cell surface proteins covalently to the cell wall. The Staphylococcus aureus sortase A (SrtA) cleaves a short C-terminal recognition motif (LPXTG) on the target protein followed by the formation of an amide bond with the pentaglycine cross-bridge in the cell wall. Over recent years, several researchers have exploited this specific reaction for a range of biotechnology applications, including the incorporation of non-native peptides and non-peptidic molecules into proteins, the generation of nucleic acid-peptide conjugates and neoglycoconjugates, protein circularisation, and labelling of cell surface proteins on living cells.

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“…As this mechanism is unique to Gram-positive pathogens, inhibiting the reaction is an attractive target for the development of novel antibacterials (3,4). The sortase-mediated transpeptidation reaction is also being increasingly used in a variety of biotechnology applications (5)(6)(7)(8).…”

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confidence: 99%

Crystal Structure of Streptococcus pyogenes Sortase A

Race

Bentley

Melvin

et al. 2009

Journal of Biological Chemistry

121

162

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Sortases are a family of Gram-positive bacterial transpeptidases that anchor secreted proteins to bacterial cell surfaces. These include many proteins that play critical roles in the virulence of Gram-positive bacterial pathogens such that sortases are attractive targets for development of novel antimicrobial agents. All Gram-positive pathogens express a "housekeeping" sortase that recognizes the majority of secreted proteins containing an LPXTG wall-sorting motif and covalently attaches these to bacterial cell wall peptidoglycan. Many Gram-positive pathogens also express additional sortases that link a small number of proteins, often with variant wall-sorting motifs, to either other surface proteins or peptidoglycan. To better understand the mechanisms of catalysis and substrate recognition by the housekeeping sortase produced by the important human pathogen Streptococcus pyogenes, the crystal structure of this protein has been solved and its transpeptidase activity established in vitro. The structure reveals a novel arrangement of key catalytic residues in the active site of a sortase, the first that is consistent with kinetic analysis. The structure also provides a complete description of residue positions surrounding the active site, overcoming the limitation of localized disorder in previous structures of sortase A-type proteins. Modification of the active site Cys through oxidation to its sulfenic acid form or by an alkylating reagent supports a role for a reactive thiol/ thiolate in the catalytic mechanism. These new insights into sortase structure and function could have important consequences for inhibitor design.Cell wall-anchored proteins play critical roles in the virulence of most Gram-positive bacterial pathogens by acting as adhesins or invasins and/or interfering with various arms of the host innate or specific immune defenses. The vast majority of these virulence proteins are retained at the bacterial surface after secretion by a mechanism that involves the covalent linkage of target proteins to the peptidoglycan layer of the cell wall. This linkage is catalyzed by membrane-associated transpeptidases called sortases (1, 2). Proteins destined for cell-surface attachment contain a sorting signal recognized by these enzymes. As this mechanism is unique to Gram-positive pathogens, inhibiting the reaction is an attractive target for the development of novel antibacterials (3, 4). The sortase-mediated transpeptidation reaction is also being increasingly used in a variety of biotechnology applications (5-8).The sorting signal that targets proteins for cell surface attachment is located at the C terminus of substrates and comprises a pentapeptide motif, typically LPXTG (where X is any amino acid), followed by a hydrophobic region and a tail of positively charged residues that locates the substrate to the cell surfacefollowingsecretion(2,9).Inonecurrentmodelofsortasedependent transpeptidation, the LPXTG motif is specifically recognized by the enzyme (10), and the thiolate group of an essential active sit...

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Immobilization of proteins to biacore sensor chips using Staphylococcus aureus sortase A

Cited by 65 publications

References 13 publications

Sortase‐Mediated Ligation: A Gift from Gram‐Positive Bacteria to Protein Engineering

Sortase‐Mediated Ligation: A Gift from Gram‐Positive Bacteria to Protein Engineering

Sortase-mediated protein ligation: an emerging biotechnology tool for protein modification and immobilisation

Crystal Structure of Streptococcus pyogenes Sortase A

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