Protein-protein interaction analysis by C-terminally specific fluorescence labeling and fluorescence cross-correlation spectroscopy.
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.
Oyama
Rieko
R
Department of Biosciences and Informatics, Keio University, Yokohama 223-8522, Japan.
Takashima
Hideaki
H
Yonezawa
Masato
M
Doi
Nobuhide
N
Miyamoto-Sato
Etsuko
E
Kinjo
Masataka
M
Yanagawa
Hiroshi
H
D002147:Calmodulin / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D002846:Chromatography, Affinity; D005456:Fluorescent Dyes / Q000737:chemistry*; D052139:Intracellular Calcium-Sensing Proteins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D063146:Polycomb-Group Proteins; D014176:Protein Biosynthesis; D025941:Protein Interaction Mapping / Q000379:methods*; D016760:Proto-Oncogene Proteins c-fos / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D016755:Proto-Oncogene Proteins c-jun / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D011691:Puromycin / Q000031:analogs & derivatives; D012097:Repressor Proteins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence / Q000379:methods*
D002147:Calmodulin; D005456:Fluorescent Dyes; D052139:Intracellular Calcium-Sensing Proteins; D063146:Polycomb-Group Proteins; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D012097:Repressor Proteins; D011691:Puromycin