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Site-Specific Fluorogenic Protein Labelling Agent for Bioconjugation.
Tsao, Kelvin K; Lee, Ann C; Racine, Karl É; Keillor, Jeffrey W.
Afiliação
  • Tsao KK; Department of Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa, ON K1N 6N5, Canada.
  • Lee AC; Department of Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa, ON K1N 6N5, Canada.
  • Racine KÉ; Department of Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa, ON K1N 6N5, Canada.
  • Keillor JW; Department of Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa, ON K1N 6N5, Canada.
Biomolecules ; 10(3)2020 02 28.
Article em En | MEDLINE | ID: mdl-32121143
ABSTRACT
Many clinically relevant therapeutic agents are formed from the conjugation of small molecules to biomolecules through conjugating linkers. In this study, two novel conjugating linkers were prepared, comprising a central coumarin core, functionalized with a dimaleimide moiety at one end and a terminal alkyne at the other. In our first design, we developed a protein labelling method that site-specifically introduces an alkyne functional group to a dicysteine target peptide tag that was genetically fused to a protein of interest. This method allows for the subsequent attachment of azide-functionalized cargo in the facile synthesis of novel protein-cargo conjugates. However, the fluorogenic aspect of the reaction between the linker and the target peptide was less than we desired. To address this shortcoming, a second linker reagent was prepared. This new design also allowed for the site-specific introduction of an alkyne functional group onto the target peptide, but in a highly fluorogenic and rapid manner. The site-specific addition of an alkyne group to a protein of interest was thus monitored in situ by fluorescence increase, prior to the attachment of azide-functionalized cargo. Finally, we also demonstrated that the cargo can also be attached first, in an azide/alkyne cycloaddition reaction, prior to fluorogenic conjugation with the target peptide-fused protein.
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Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Rodaminas / Azidas / Proteínas / Alcinos / Corantes Fluorescentes Idioma: En Revista: Biomolecules Ano de publicação: 2020 Tipo de documento: Article País de afiliação: Canadá

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Rodaminas / Azidas / Proteínas / Alcinos / Corantes Fluorescentes Idioma: En Revista: Biomolecules Ano de publicação: 2020 Tipo de documento: Article País de afiliação: Canadá