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Nucleic Acids Research Advance Access published online on August 16, 2006
Nucleic Acids Research, doi:10.1093/nar/gkl477
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© 2006 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

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 Kinjo1 and Hiroshi Yanagawa*

Department of Biosciences and Informatics, Keio University Yokohama 223-8522, Japan 1 Research Institute for Electronic Science, Hokkaido University Sapporo 060-0812, Japan

*To whom correspondence should be addressed. Tel: +81 45 566 1775; Fax: +81 45 566 1440; Email: hyana{at}bio.keio.ac.jp

Received May 22, 2006. Accepted June 20, 2006.

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.

Present addresses:

Rieko Oyama, RIKEN, Genome Science Laboratory, 2-1 Hirosawa, Wako 351-0198, Japan

Masato Yonezawa, Research Institute of Molecular Pathology (IMP), Vienna, Austria


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