Synthesis and application of a N-1' fluorescent biotinyl derivative inducing the specific carboxy-terminal dual labeling of a novel RhoB-selective scFv.
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.
Chaisemartin
L
L
INSERM U563, Departement Oncogenese, Signalisation et Innovation Therapeutique, Institut Claudius Regaud, 31052 Toulouse, France.
Chinestra
P
P
Favre
G
G
Blonski
C
C
Faye
J C
JC
D000918:Antibody Specificity; D001665:Binding Sites; D001710:Biotin / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D003545:Cysteine / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D004952:Esters; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D047668:Inteins; D009584:Nitrogen / Q000737:chemistry*; D019154:Protein Splicing; D057127:Single-Chain Antibodies / Q000032:analysis / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods*; D013379:Substrate Specificity; D020743:rhoB GTP-Binding Protein / Q000276:immunology*
D004952:Esters; D005456:Fluorescent Dyes; D057127:Single-Chain Antibodies; D001710:Biotin; D020743:rhoB GTP-Binding Protein; D003545:Cysteine; D009584:Nitrogen