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J Biosci Bioeng. 2016 Aug;122(2):146-54. doi: 10.1016/j.jbiosc.2016.01.010. Epub 2016 Feb 16.

Antibody-based fluorescent and fluorescent ratiometric indicators for detection of phosphotyrosine.

Author information

1
School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi, Ishikawa 923-1292, Japan.
2
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.

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.

KEYWORDS:

Cell-free translation; Fluorescent ratiometric indicator; Nonnatural amino acid; Protein phosphorylation; Single-chain antibody

PMID:
26896314
DOI:
10.1016/j.jbiosc.2016.01.010
[Indexed for MEDLINE]

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