Protein - Protein interaction analysis by C-terminally specific fluorescence labeling and fluorescence cross-correlation spectroscopy

Rieko Oyama, Hideaki Takashima, Masato Yonezawa, Nobuhide Doi, Etsuko Miyamoto-Sato, Masataka Kinjo, Hiroshi Yanagawa

Research output: Contribution to journalArticle

37 Citations (Scopus)

Abstract

Here, we describe novel puromycin derivatives conjugated with iminobiotin and a fluorescent dye that can be linked covalently to the C-terminus of full-length proteins during cell-free translation. The iminobiotin-labeled proteins can be highly purified by affinity purification with streptavidin beads. We confirmed that the purified fluorescence-labeled proteins are useful for quantitative protein-protein interaction analysis based on fluorescence cross-correlation spectroscopy (FCCS). The apparent dissociation constants of model protein pairs such as proto-oncogenes c-Fos/c-Jun and archetypes of the family of Ca2+-modulated calmodulin/related binding proteins were in accordance with the reported values. Further, detailed analysis of the interactions of the components of polycomb group complex, Bmi1, M33, Ring1A and RYBP, was successfully conducted by means of interaction assay for all combinatorial pairs. The results indicate that FCCS analysis with puromycin-based labeling and purification of proteins is effective and convenient for in vitro protein-protein interaction assay, and the method should contribute to a better understanding of protein functions by using the resource of available nucleotide sequences.

Original languageEnglish
Article numbere102
JournalNucleic acids research
Volume34
Issue number14
DOIs
Publication statusPublished - 2006

ASJC Scopus subject areas

  • Genetics

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