Synthesis and application of a N-1' fluorescent biotinyl derivative inducing the specific carboxy-terminal dual labeling of a novel RhoB-selective scFv.
Bioconjug Chem
; 20(5): 847-55, 2009 May 20.
Article
em En
| MEDLINE
| ID: mdl-19348471
ABSTRACT
The fluorescent site-specific labeling of protein would provide a new, easy-to-use alternative to biochemical and immunochemical methods. We used an intein-mediated strategy for covalent labeling of the carboxy-terminal amino acid of a RhoB-selective scFv previously isolated from a phage display library (a human synthetic V(H) + V(L) scFv phage library). The scFv fused to the Mxe intein was produced in E. coli and purified and was then labeled with a newly synthesized fluorescent biotinyl cysteine derivative capable of inducing scFv-Mxe intein splicing. In this study, we investigated the splicing and labeling properties of various amino acids in the hinge domain between scFv and Mxe under thiol activation. In this dual labeling system, the fluorescein is used for antibody detection and biotin is used for purification, resulting in a high specific activity for fluorescence. We then checked that the purified biotinylated fluorescent scFv retained its selectivity for RhoB without modification of its affinity.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Coloração e Rotulagem
/
Biotina
/
Proteína rhoB de Ligação ao GTP
/
Anticorpos de Cadeia Única
/
Especificidade de Anticorpos
/
Nitrogênio
Limite:
Humans
Idioma:
En
Revista:
Bioconjug Chem
Assunto da revista:
BIOQUIMICA
Ano de publicação:
2009
Tipo de documento:
Article
País de afiliação:
França