Site-Specific N- and C-Terminal Labeling of a Single Polypeptide Using Sortases of Different Specificity
John M. Antos,Guo-Liang Chew,Carla P. Guimaraes,Nicholas C. Yoder,Gijsbert M. Grotenbreg,Maximilian Wei-Lin Popp,Hidde L. Ploegh +6 more
TLDR
The generality of N-terminal labeling with SrtAstaph is demonstrated by near-quantitative labeling of multiple protein substrates with excellent site specificity.Abstract:
The unique reactivity of two sortase enzymes, SrtAstaph from Staphylococcus aureus and SrtAstrep from Streptococcus pyogenes, is exploited for site-specific labeling of a single polypeptide with different labels at its N and C termini. SrtAstrep is used to label the protein’s C terminus at an LPXTG site with a fluorescently labeled dialanine nucleophile. Selective N-terminal labeling of proteins containing N-terminal glycine residues is achieved using SrtAstaph and LPXT derivatives. The generality of N-terminal labeling with SrtAstaph is demonstrated by near-quantitative labeling of multiple protein substrates with excellent site specificity.read more
Citations
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Cellular Incorporation of Unnatural Amino Acids and Bioorthogonal Labeling of Proteins
Kathrin Lang,Jason W. Chin +1 more
Journal ArticleDOI
Structural Basis for dsRNA Recognition, Filament Formation, and Antiviral Signal Activation by MDA5.
Bin Wu,Alys Peisley,Alys Peisley,Claire Richards,Hui Yao,Xiaohui Zeng,Cecilie Lin,Feixia Chu,Thomas Walz,Thomas Walz,Sun Hur,Sun Hur +11 more
TL;DR: The crystal structure of MDA5 bound to dsRNA is reported, which shows how, using the same domain architecture, MDA5 recognizes the internal duplex structure, whereas RIG-I recognizes the terminus of ds RNA.
Journal ArticleDOI
Genetically encoded norbornene directs site-specific cellular protein labelling via a rapid bioorthogonal reaction
Kathrin Lang,Lloyd S. Davis,Jessica Torres-Kolbus,Chungjung Chou,Alexander Deiters,Jason W. Chin +5 more
TL;DR: The genetic encoding of a norbornene amino acid using the pyrrolysyl tRNA synthetase/tRNA(CUA) pair in Escherichia coli and mammalian cells and the rapid bioorthogonal site-specific labelling of a protein on the mammalian cell surface is demonstrated.
Journal ArticleDOI
Developments and recent advancements in the field of endogenous amino acid selective bond forming reactions for bioconjugation
Oleksandr Koniev,Alain Wagner +1 more
TL;DR: The utility of each endogenous amino acid-selective conjugation methodology in the fields of biology and protein science has been surveyed with emphasis on the most relevant among reported transformations.
Journal ArticleDOI
Designer proteins: applications of genetic code expansion in cell biology.
Lloyd S. Davis,Jason W. Chin +1 more
TL;DR: Designer amino acids, beyond the canonical 20 that are normally used by cells, can now be site-specifically encoded into proteins in cells and organisms using 'orthogonal' aminoacyl-tRNA synthetase–tRNA pairs that direct amino acid incorporation in response to an amber stop codon placed in a gene of interest.
References
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Journal ArticleDOI
Sortases and the Art of Anchoring Proteins to the Envelopes of Gram-Positive Bacteria
TL;DR: This review focuses on the mechanisms of surface protein anchoring to the cell wall envelope by sortases and the role that these enzymes play in bacterial physiology and pathogenesis.
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Sortagging: a versatile method for protein labeling
TL;DR: sortagging (sortase-mediated transpeptidation) is a versatile chemoenzymatic system for site-specific labeling of proteins with small (<2 kDa) probes and is applied to proteins in vitro and on the surface of living cells.
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An iron-regulated sortase anchors a class of surface protein during Staphylococcus aureus pathogenesis
TL;DR: Cell wall-anchored surface proteins and the isd locus seem involved in a novel mechanism of iron acquisition that is important for bacterial pathogenesis.
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An embarrassment of sortases - a richness of substrates?
TL;DR: In this article, the authors search for sortase homologues among complete and incomplete genome sequences, and find them to be present in almost all Gram-positives, a single Gram-negative bacterium and an archaean.
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Sortase‐Mediated Ligation: A Gift from Gram‐Positive Bacteria to Protein Engineering
Shinya Tsukiji,Teruyuki Nagamune +1 more
TL;DR: 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.