Protein-protein interaction analysis by C-terminally specific fluorescence labeling and fluorescence cross-correlation spectroscopy.
Protein-protein interaction analysis by C-terminally specific fluorescence labeling and fluorescence cross-correlation spectroscopy.
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DOI：
10.1093/nar/gkl477
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发表时间：
2006
影响因子：
14.9
通讯作者：
Yanagawa H
中科院分区：
文献类型：
--
作者：
Oyama R;Takashima H;Yonezawa M;Doi N;Miyamoto-Sato E;Kinjo M;Yanagawa H
在这里，我们描述了新的嘌呤霉素衍生物共轭亚氨基生物素和荧光染料，可以共价连接到C-末端的全长蛋白质在无细胞翻译。亚氨基生物素标记的蛋白质可以通过用链霉亲和素珠的亲和纯化来高度纯化。我们证实，纯化的荧光标记的蛋白质是有用的定量蛋白质-蛋白质相互作用分析的基础上的荧光互相关光谱（FCCS）。模型蛋白对如原癌基因c-Fos/c-Jun和Ca 2+调节的钙调素/相关结合蛋白家族的原型的表观解离常数与报道的值一致。此外，通过对所有组合对的相互作用测定，成功地对polycomb组复合物的组分Bmi 1、M33、Ring 1A和RYBP的相互作用进行了详细的分析。结果表明，基于嘌呤霉素的蛋白质标记和纯化的FCCS分析是一种有效和方便的体外蛋白质-蛋白质相互作用分析方法，该方法将有助于利用现有的核苷酸序列资源更好地了解蛋白质的功能。
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.
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影响因子：
64.5
作者：
Du, CY;Fang, M;Wang, XD
通讯作者：
Wang, XD
影响因子：
2.9
作者：
Heuer, KH;Mackay, JP;EasterbrookSmith, SB
通讯作者：
EasterbrookSmith, SB
DOI：
10.1016/s0021-9673(00)93969-4
发表时间：
1987-12-18
期刊：
JOURNAL OF CHROMATOGRAPHY
影响因子：
--
作者：
HOCHULI, E;DOBELI, H;SCHACHER, A
通讯作者：
SCHACHER, A
影响因子：
14.9
作者：
Miyamoto-Sato, E;Takashima, H;Yanagawa, H
通讯作者：
Yanagawa, H
影响因子：
7
作者：
Doi, N;Takashima, H;Yanagawa, H
通讯作者：
Yanagawa, H