Site-Specific Fluorogenic Protein Labelling Agent for Bioconjugation.
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.
Palavras-chave
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