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METHOD FOR FLUORESCENT LABELING OF PROTEIN

外国特許コード F130007439
掲載日 2013年7月4日
出願国 世界知的所有権機関(WIPO)
国際出願番号 2012JP052227
国際公開番号 WO 2012105596
国際出願日 平成24年2月1日(2012.2.1)
国際公開日 平成24年8月9日(2012.8.9)
優先権データ
  • 特願2011-020804 (2011.2.2) JP
発明の名称 (英語) METHOD FOR FLUORESCENT LABELING OF PROTEIN
発明の概要(英語) [Problem] To provide a method for the fluorescent labeling of a protein that can even be used for the fluorescent labeling of a biological molecule (protein) in vivo .
[Solution] A method for the fluorescent labeling of a target protein, comprising a step for the intracellular formation of a fused protein of the protein to be labeled and a mutant β-lactamase, a step for using intracellular esterase for the conversion of a compound represented by the following formula (I) or a salt thereof to a compound represented by the following formula (II) or a salt thereof, and a step for the reaction of the fused protein and the compound represented by formula (II) or a salt thereof.
従来技術、競合技術の概要(英語) BACKGROUND ART
In recent years, in living organism, the organism to observe the functioning of biomolecules such as proteins is bio-imaging has attracted attention. The observation of the biomolecule function, fluorescently labeled biomolecules and, in the localization of the biological molecule in an organism or to visualize the movement is performed by a general-purpose. As a method of fluorescently labeled biomolecules, using genetic engineering techniques, an object of the fluorescent protein at the end of the biomolecule (protein) to be co-expressed in the commonly-used method. This fluorescent protein, large molecular size, in addition, tissue penetration of near-infrared light fluorescence observation that there is an inconvenience such as difficult. Therefore, small size molecules from the fluorescent protein, and, an organic small molecule fluorescent property has attracted attention. Small organic molecules as a technique for the label to a protein, HaloTag (registered trademark) (manufactured by Promega KK) (for example see non-patent document 1) and SNAP-tag (registered trademark) (for example see non-patent document 2) using a labeling kit has been commercialized. These labeling method, the enzyme reaction using fluorescently labeled biomolecules to the purpose. Therefore, an object of the small organic molecules and the specificity of labeling of the biomolecules is very high when, not binding to a biomolecule fluorescence from small organic molecules, that is observed as the background signal. In order to reduce this background signal, the target biomolecules labeled with a fluorescent small organic molecules, washed, unreacted small organic molecules and needs to be removed. However, in a cell or an organism present in a biological molecule to clean, and is generally difficult, often cannot be washed. Therefore, in vivo cells to observe the biological function of molecules, a general purpose labeling method described above is not efficient. In addition, small organic molecules and biological molecules having the specific sequence for the purpose of linking the peptides may be used to, fluorescently labeled biomolecules has been developed is a method, the method, the specificity of the peptide and small organic molecules and deteriorate.
In order to solve this problem, the inventors of the present invention, already, a compound having the following formulA is used, in vivo cells biomolecule (protein) can be used to label fluorescence, protein provides a method of fluorescently labeling (patent document 1). In this method, mutant β-lactamase protein to be labeled to obtain a fusion protein, the fusion protein and the compound represented by the following formulA or a salt thereof by the reaction, the protein of interest comprises a fluorescent label, and the variant β lactamase, the compound represented by the formulA or a salt thereof to ring-opening so that β - lactam ring, and, wherein the compound or a salt thereof of the β - lactam ring is ring-opening portion is shared with a protein capable of binding, provides the fluorescent labeling of the protein.
In the formulA, X 'is, fluorescent groups and, L1 'is, X' for linker, R1 'is, H, lower alkylcarbonyloxy substituted by a lower alkylene group or a lower alkyl group.
However, a method of using the compound (A) a cell-membrane permeable is low and, as a result, the fluorescence of the biomolecule (protein) in the cell label the poor efficiency in some cases.
  • 出願人(英語)
  • ※2012年7月以前掲載分については米国以外のすべての指定国
  • OSAKA UNIVERSITY
  • 発明者(英語)
  • KIKUCHI Kazuya
  • MIZUKAMI Shin
  • WATANABE Shuji
  • AKIMOTO Yuri
国際特許分類(IPC)
指定国 National States: AE AG AL AM AO AT AU AZ BA BB BG BH BR BW BY BZ CA CH CL CN CO CR CU CZ DE DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IS JP KE KG KM KN KP KR KZ LA LC LK LR LS LT LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PE PG PH PL PT QA RO RS RU RW SC SD SE SG SK SL SM ST SV SY TH TJ TM TN TR TT TZ UA UG US UZ VC VN ZA ZM ZW
ARIPO: BW GH GM KE LR LS MW MZ NA RW SD SL SZ TZ UG ZM ZW
EAPO: AM AZ BY KG KZ MD RU TJ TM
EPO: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
OAPI: BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG
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