title: Antibody-based fluorescent and fluorescent ratiometric indicators for detection of phosphotyrosine.
abstract: Fluorescent indicators for protein phosphorylation are very important in not only fundamental biology but also biomedical applications. In this study, we developed novel fluorescent and fluorescent ratiometric indicators for detection of phosphotyrosine (pTyr) derivatives. A single-chain antibody variable fragment (scFv) against phosphotyrosine was fluorescent-labeled by incorporation of tetramethylrhodamine (TAMRA)-linked nonnatural amino acid at the N- or C-terminus. The TAMRA-labeled scFv showed fluorescence enhancement upon addition of pTyr-containing peptides based on antigen-dependent fluorescence quenching effect on TAMRA. The TAMRA-labeled scFv was further fused with enhanced green fluorescent protein (EGFP) to generate a double-labeled scFv for pTyr. In the absence of antigen, fluorescence resonance energy transfer (FRET) occurred from EGFP to TAMRA but TAMRA was quenched. The antigen-binding removed the quenching of TAMRA while FRET occurred without altering its efficiency. As a result of the FRET and antigen-dependent fluorescence quenching effect, the double-labeled scFv exhibited fluorescence ratio enhancement upon the antigen-binding. The fluorescent and fluorescent ratiometric indicators obtained in this study will become a novel tool for analysis of protein phosphorylation. Moreover, this strategy utilizes antibody derivatives, and therefore, can be easily applied to other antigen-antibody pairs to generate fluorescent ratiometric indicators for various target molecules.
authors.affiliation: School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi, Ishikawa 923-1292, Japan.
authors.forename: Kim Phuong
authors.initials: KP
authors.lastname: Huynh Nhat
authors.affiliation: School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi, Ishikawa 923-1292, Japan.
authors.forename: Takayoshi
authors.initials: T
authors.lastname: Watanabe
authors.affiliation: School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi, Ishikawa 923-1292, Japan.
authors.forename: Kensuke
authors.initials: K
authors.lastname: Yoshikoshi
authors.affiliation: School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi, Ishikawa 923-1292, Japan. Electronic address: hohsaka@jaist.ac.jp.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Hohsaka
mesh_terms: D000941:Antigens; D005453:Fluorescence; D005455:Fluorescent Antibody Technique / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis* / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D019000:Phosphotyrosine / Q000032:analysis*; D012235:Rhodamines / Q000032:analysis* / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence
chemical_list: D000941:Antigens; D005456:Fluorescent Dyes; D012235:Rhodamines; D057127:Single-Chain Antibodies; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D019000:Phosphotyrosine; C005358:tetramethylrhodamine
keywords: Cell-free translation; Fluorescent ratiometric indicator; Nonnatural amino acid; Protein phosphorylation; Single-chain antibody