Click-Tag and Amine-Tag: Chemical Tag Approaches for Efficient Protein Labeling In Vitro and on Live Cells using the Naturally Split Npu DnaE Intein
Protein labeling with synthetic moieties remains in many cases a technically challenging or unresolved task. Two new and simple concepts are presented. In both approaches, a very short tag of only a few amino acids is prepared with the desired chemical modification and, in a second step, it is trans...
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Veröffentlicht in: | Angewandte Chemie International Edition 2014-04, Vol.53 (16), p.4113-4117 |
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description | Protein labeling with synthetic moieties remains in many cases a technically challenging or unresolved task. Two new and simple concepts are presented. In both approaches, a very short tag of only a few amino acids is prepared with the desired chemical modification and, in a second step, it is transferred to the protein of interest by protein trans‐splicing. For the amine‐tag, a recombinant intein fragment free of lysine residues was generated such that the amine group of the N terminus could be selectively modified with regular amine‐reactive reagents. Thus, standard bioconjugation procedures without any chemical synthesis could be applied without modification of lysines in the protein of interest. For the click‐tag, protein trans‐splicing was combined with unnatural amino acid mutagenesis and subsequent bioorthogonal side chain modification, as demonstrated for click chemistry using p‐azidophenylalanine. By the two‐step strategy, exposure of the protein of interest to the copper catalyst was avoided.
Click and splice: Short tags of only a few amino acids fused to a recombinantly produced split intein fragment were modified with synthetic moieties and then transferred to a protein of interest by protein trans‐splicing. These two‐step procedures circumvent exposure of the protein of interest to copper ions for click chemistry and undesired modification of lysines, even though standard bioconjugation reagents are used. |
doi_str_mv | 10.1002/anie.201309396 |
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Click and splice: Short tags of only a few amino acids fused to a recombinantly produced split intein fragment were modified with synthetic moieties and then transferred to a protein of interest by protein trans‐splicing. These two‐step procedures circumvent exposure of the protein of interest to copper ions for click chemistry and undesired modification of lysines, even though standard bioconjugation reagents are used.</description><edition>International ed. in English</edition><identifier>ISSN: 1433-7851</identifier><identifier>EISSN: 1521-3773</identifier><identifier>DOI: 10.1002/anie.201309396</identifier><identifier>PMID: 24615830</identifier><identifier>CODEN: ACIEAY</identifier><language>eng</language><publisher>Weinheim: WILEY-VCH Verlag</publisher><subject>Amines - chemistry ; Amino acids ; bioconjugation ; bioorthogonality ; Copper ; Exposure ; Fragmentation ; fusion tags ; Humans ; In Vitro Techniques - methods ; Inteins - genetics ; Lysine ; lysine modification ; Marking ; Protein Splicing - genetics ; Proteins ; Proteins - chemistry ; Recombinant ; unnatural amino acids</subject><ispartof>Angewandte Chemie International Edition, 2014-04, Vol.53 (16), p.4113-4117</ispartof><rights>2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim</rights><rights>2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.</rights><rights>2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c4446-1f9802295d9874664f59de29a2d2151ebded1bd7ccac4b5a951ec9caa6a8a2ba3</citedby><cites>FETCH-LOGICAL-c4446-1f9802295d9874664f59de29a2d2151ebded1bd7ccac4b5a951ec9caa6a8a2ba3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://onlinelibrary.wiley.com/doi/pdf/10.1002%2Fanie.201309396$$EPDF$$P50$$Gwiley$$H</linktopdf><linktohtml>$$Uhttps://onlinelibrary.wiley.com/doi/full/10.1002%2Fanie.201309396$$EHTML$$P50$$Gwiley$$H</linktohtml><link.rule.ids>314,780,784,1417,27924,27925,45574,45575</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/24615830$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Schütz, Vivien</creatorcontrib><creatorcontrib>Mootz, Henning D.</creatorcontrib><title>Click-Tag and Amine-Tag: Chemical Tag Approaches for Efficient Protein Labeling In Vitro and on Live Cells using the Naturally Split Npu DnaE Intein</title><title>Angewandte Chemie International Edition</title><addtitle>Angew. Chem. Int. Ed</addtitle><description>Protein labeling with synthetic moieties remains in many cases a technically challenging or unresolved task. Two new and simple concepts are presented. In both approaches, a very short tag of only a few amino acids is prepared with the desired chemical modification and, in a second step, it is transferred to the protein of interest by protein trans‐splicing. For the amine‐tag, a recombinant intein fragment free of lysine residues was generated such that the amine group of the N terminus could be selectively modified with regular amine‐reactive reagents. Thus, standard bioconjugation procedures without any chemical synthesis could be applied without modification of lysines in the protein of interest. For the click‐tag, protein trans‐splicing was combined with unnatural amino acid mutagenesis and subsequent bioorthogonal side chain modification, as demonstrated for click chemistry using p‐azidophenylalanine. By the two‐step strategy, exposure of the protein of interest to the copper catalyst was avoided.
Click and splice: Short tags of only a few amino acids fused to a recombinantly produced split intein fragment were modified with synthetic moieties and then transferred to a protein of interest by protein trans‐splicing. These two‐step procedures circumvent exposure of the protein of interest to copper ions for click chemistry and undesired modification of lysines, even though standard bioconjugation reagents are used.</description><subject>Amines - chemistry</subject><subject>Amino acids</subject><subject>bioconjugation</subject><subject>bioorthogonality</subject><subject>Copper</subject><subject>Exposure</subject><subject>Fragmentation</subject><subject>fusion tags</subject><subject>Humans</subject><subject>In Vitro Techniques - methods</subject><subject>Inteins - genetics</subject><subject>Lysine</subject><subject>lysine modification</subject><subject>Marking</subject><subject>Protein Splicing - genetics</subject><subject>Proteins</subject><subject>Proteins - chemistry</subject><subject>Recombinant</subject><subject>unnatural amino acids</subject><issn>1433-7851</issn><issn>1521-3773</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2014</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkkuP0zAUhSMEYh6wZYkssWGT4kcSx-xKKEOhKkgMsLRuHGfqGccJdjLQ_8EPxqFDhdiw8uN-59xrHSfJE4IXBGP6ApzRC4oJw4KJ4l5ySnJKUsY5ux_3GWMpL3NykpyFcB35ssTFw-SEZgXJS4ZPk5-VNeomvYQrBK5By844PZ9eomqnO6PAorm2HAbfg9rpgNreo1XbGmW0G9FH34_aOLSBWlvjrtDaoS9m9P1vuz4WzK1GlbY2oCnMwLjTaAvj5MHaPfo0WDOi7TCh1w5WUT27PUoetGCDfny3nief36wuq7fp5sPFulpuUpVlWZGSVpSYUpE3ouRZUWRtLhpNBdCGkpzoutENqRuuFKiszkHEOyUUQAEl0BrYefL84Bsf923SYZSdCSrOCk73U5CEY4IFLgmP6LN_0Ot-8i5OJ2OrvGCc4SxSiwOlfB-C160cvOnA7yXBcs5LznnJY15R8PTOdqo73RzxPwFFQByA78bq_X_s5HK7Xv1tnh60Joz6x1EL_kYWnPFcft1eyPhT3lOyeSXfsV-4L7A4</recordid><startdate>20140414</startdate><enddate>20140414</enddate><creator>Schütz, Vivien</creator><creator>Mootz, Henning D.</creator><general>WILEY-VCH Verlag</general><general>WILEY‐VCH Verlag</general><general>Wiley Subscription Services, Inc</general><scope>BSCLL</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7TM</scope><scope>K9.</scope><scope>7SR</scope><scope>8BQ</scope><scope>8FD</scope><scope>JG9</scope></search><sort><creationdate>20140414</creationdate><title>Click-Tag and Amine-Tag: Chemical Tag Approaches for Efficient Protein Labeling In Vitro and on Live Cells using the Naturally Split Npu DnaE Intein</title><author>Schütz, Vivien ; Mootz, Henning D.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c4446-1f9802295d9874664f59de29a2d2151ebded1bd7ccac4b5a951ec9caa6a8a2ba3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2014</creationdate><topic>Amines - chemistry</topic><topic>Amino acids</topic><topic>bioconjugation</topic><topic>bioorthogonality</topic><topic>Copper</topic><topic>Exposure</topic><topic>Fragmentation</topic><topic>fusion tags</topic><topic>Humans</topic><topic>In Vitro Techniques - methods</topic><topic>Inteins - genetics</topic><topic>Lysine</topic><topic>lysine modification</topic><topic>Marking</topic><topic>Protein Splicing - genetics</topic><topic>Proteins</topic><topic>Proteins - chemistry</topic><topic>Recombinant</topic><topic>unnatural amino acids</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Schütz, Vivien</creatorcontrib><creatorcontrib>Mootz, Henning D.</creatorcontrib><collection>Istex</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Nucleic Acids Abstracts</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Engineered Materials Abstracts</collection><collection>METADEX</collection><collection>Technology Research Database</collection><collection>Materials Research Database</collection><jtitle>Angewandte Chemie International Edition</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Schütz, Vivien</au><au>Mootz, Henning D.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Click-Tag and Amine-Tag: Chemical Tag Approaches for Efficient Protein Labeling In Vitro and on Live Cells using the Naturally Split Npu DnaE Intein</atitle><jtitle>Angewandte Chemie International Edition</jtitle><addtitle>Angew. Chem. Int. Ed</addtitle><date>2014-04-14</date><risdate>2014</risdate><volume>53</volume><issue>16</issue><spage>4113</spage><epage>4117</epage><pages>4113-4117</pages><issn>1433-7851</issn><eissn>1521-3773</eissn><coden>ACIEAY</coden><abstract>Protein labeling with synthetic moieties remains in many cases a technically challenging or unresolved task. Two new and simple concepts are presented. In both approaches, a very short tag of only a few amino acids is prepared with the desired chemical modification and, in a second step, it is transferred to the protein of interest by protein trans‐splicing. For the amine‐tag, a recombinant intein fragment free of lysine residues was generated such that the amine group of the N terminus could be selectively modified with regular amine‐reactive reagents. Thus, standard bioconjugation procedures without any chemical synthesis could be applied without modification of lysines in the protein of interest. For the click‐tag, protein trans‐splicing was combined with unnatural amino acid mutagenesis and subsequent bioorthogonal side chain modification, as demonstrated for click chemistry using p‐azidophenylalanine. By the two‐step strategy, exposure of the protein of interest to the copper catalyst was avoided.
Click and splice: Short tags of only a few amino acids fused to a recombinantly produced split intein fragment were modified with synthetic moieties and then transferred to a protein of interest by protein trans‐splicing. These two‐step procedures circumvent exposure of the protein of interest to copper ions for click chemistry and undesired modification of lysines, even though standard bioconjugation reagents are used.</abstract><cop>Weinheim</cop><pub>WILEY-VCH Verlag</pub><pmid>24615830</pmid><doi>10.1002/anie.201309396</doi><tpages>5</tpages><edition>International ed. in English</edition></addata></record> |
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subjects | Amines - chemistry Amino acids bioconjugation bioorthogonality Copper Exposure Fragmentation fusion tags Humans In Vitro Techniques - methods Inteins - genetics Lysine lysine modification Marking Protein Splicing - genetics Proteins Proteins - chemistry Recombinant unnatural amino acids |
title | Click-Tag and Amine-Tag: Chemical Tag Approaches for Efficient Protein Labeling In Vitro and on Live Cells using the Naturally Split Npu DnaE Intein |
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