{"doi":"10.1038/nprot.2014.003","title":"Depsipeptide substrates for sortase-mediated N-terminal protein ligation","abstract":null,"journal":"Nature Protocols","year":2014,"id":682773,"datarank":0.5806801516361837,"base_score":3.8712010109078907,"endowment":3.8712010109078907,"self_citation_contribution":0.5806801516361837,"citation_network_contribution":0.0,"self_endowment_contribution":0.5806801516361837,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":47,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":null,"is_data_producer":false,"deposit_databanks":null,"is_oa":false,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":null,"fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1783668,"name":"Michael E Webb","orcid":null,"position":1,"is_corresponding":false},{"id":1783669,"name":"W Bruce Turnbull","orcid":null,"position":2,"is_corresponding":false},{"id":1783665,"name":"Daniel J Williamson","orcid":null,"position":0,"is_corresponding":false}],"reference_count":0,"raw_metadata":{"has_enrichment":true,"resolved":true,"title":"Depsipeptide substrates for sortase-mediated N-terminal protein ligation","abstract":"Technologies that allow the efficient chemical modification of proteins under mild conditions are widely sought after. Sortase-mediated peptide ligation provides a strategy for modifying the N or C terminus of proteins. This protocol describes the use of depsipeptide substrates (containing an ester linkage) with sortase A (SrtA) to completely modify proteins carrying a single N-terminal glycine residue under mild conditions in 4-6 h. The SrtA-mediated ligation reaction is reversible, so most labeling protocols that use this enzyme require a large excess of both substrate and sortase to produce high yields of ligation product. In contrast, switching to depsipeptide substrates effectively renders the reaction irreversible, allowing complete labeling of proteins with a small excess of substrate and catalytic quantities of sortase. Herein we describe the synthesis of depsipeptide substrates that contain an ester linkage between a threonine and glycolic acid residue and an N-terminal FITC fluorophore appended via a thiourea linkage. The synthesis of the depsipeptide substrate typically takes 2-3 d.","is_dataset_classified":null,"base_score":3.8712010109078907,"endowment":3.8712010109078907,"datacite_reuse_total":0,"file_count":0,"downloads":0,"views":0,"has_version_chain":false,"is_dataset":false,"is_oa":false,"pmid":"24407354","pmcid":null,"openalex_id":"https://openalex.org/W2084863947","authors":[],"funders":[{"funder_name":"Engineering and Physical Sciences Research Council","grant_id":"EP/K03135X/1","title":null},{"funder_name":"Biotechnology and Biological Sciences Research Council","grant_id":"BB/G004145/1","title":"Molecular characterisation of an ADP-dependent regulatory protein"},{"funder_name":"Engineering and Physical Sciences Research Council","grant_id":"EP/G043302/1","title":"Self-assembling virus-like particles"},{"funder_name":"Engineering and Physical Sciences Research Council","grant_id":"EP/I013083/1","title":"Synthetic probes of histidine phosphorylation: new reagents for systems biology and proteomics"}],"total_grants":4,"fwci":2.599,"citation_percentile":0.9037417,"influential_citations":0,"citation_trend":[{"year":2014,"count":3},{"year":2015,"count":2},{"year":2016,"count":7},{"year":2017,"count":8},{"year":2018,"count":2},{"year":2019,"count":5},{"year":2020,"count":1},{"year":2021,"count":3},{"year":2022,"count":3},{"year":2023,"count":8},{"year":2024,"count":2},{"year":2025,"count":2},{"year":2026,"count":1}],"oa_status":"closed","license":"Springer TDM","oa_locations":[{"url":"http://www.nature.com/articles/nprot.2014.003.pdf","host_type":"publisher"},{"url":"http://www.nature.com/articles/nprot.2014.003","host_type":"publisher"},{"url":"https://doi.org/10.1038/nprot.2014.003","host_type":"journal"},{"url":"https://pubmed.ncbi.nlm.nih.gov/24407354","host_type":"repository"},{"url":"http://eprints.whiterose.ac.uk/121578/1/author%20accepted%20manuscriptNP-PFV130247%20Webb%20Turnbull.pdf","host_type":"repository"},{"url":"https://dx.doi.org/10.1038/nprot.2014.003","host_type":""},{"url":"https://eprints.whiterose.ac.uk/id/eprint/121578/","host_type":""}],"fields_of_study":["Biochemical and Structural Characterization","Peptidase Inhibition and Analysis","Glycosylation and Glycoproteins Research","0301 basic medicine","01 natural sciences","0104 chemical sciences","03 medical and health sciences"],"mesh_terms":["Bacterial Proteins","Cysteine Endopeptidases","Glycine","Models, Chemical","Thiourea","Protein Engineering","Molecular Structure","Aminoacyltransferases","Depsipeptides"],"keywords":["Sortase","Sortase A","Depsipeptide","Chemical ligation","Chemistry","Residue (chemistry)","Biochemistry","Native chemical ligation","Stereochemistry","Combinatorial chemistry","Peptide","Cysteine","Enzyme","Molecular Structure","Glycine","Thiourea","Chemical Modification","Biosynthesis","Aminoacyltransferases","Protein Engineering","Cysteine Endopeptidases","Bacterial Proteins","Models, Chemical","Depsipeptides","Peptides","Sensors and probes"],"sdg_mappings":[{"sdg_number":3,"sdg_label":"3. 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