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LINKER FOR UNIMOLECULAR FRET BIOSENSOR BASED ON PRINCIPLE OF FLUORESCENCE RESONANCE ENERGY TRANSFER

外国特許コード F120006680
掲載日 2012年5月24日
出願国 世界知的所有権機関(WIPO)
国際出願番号 2011JP071891
国際公開番号 WO 2012043477
国際出願日 平成23年9月26日(2011.9.26)
国際公開日 平成24年4月5日(2012.4.5)
優先権データ
  • 特願2010-215738 (2010.9.27) JP
発明の名称 (英語) LINKER FOR UNIMOLECULAR FRET BIOSENSOR BASED ON PRINCIPLE OF FLUORESCENCE RESONANCE ENERGY TRANSFER
発明の概要(英語) [Problem] To provide a linker for a unimolecular FRET biosensor, as well as a unimolecular FRET biosensor and such generally containing a linker, which enable the measurement of non-invasive serine-threonine protein kinase activity, tyrosine kinase activity, and small GTP-binding protein activity, and are based on the principle of fluorescence resonance energy transfer.
[Solution] Provided is: a linker that is a polypeptide containing between 52 and 400 amino acid residues, with at least 95% of the total number of amino acid residues comprising glycine, serine, threonine, and alanine, and which contains 35-65% of glycine, 10-40% of serine and/or threonine, and 10-40% of alanine; unimolecular FRET biosensors mostly containing linkers; a transformed cell which holds an expression vector containing a gene which codes the biosensors; and a transgenic nonhuman animal.
従来技術、競合技術の概要(英語) BACKGROUND ART
Fluorescence resonance energy transfer (Fluorescence resonance energy transfer, hereinafter sometimes referred to as FRET) fluorescent protein utilizing the principle of the biosensor in the field of life science has spread rapidly (for example see non-patent document 1-4).
Is FRET, a fluorescent molecule in an excited state (donor: energy donor) from the immediate vicinity of the fluorescent molecule (acceptor: energy acceptor) phenomenon to be a transferring excitation energy. Using FRET for the dissemination of the biosensor, color mutants of green fluorescent protein (GFP) appearance and greatly contributes to the improvement thereof. Currently, in many cases, GFP CFP (cyan fluorescent protein) is a mutant of YFP (yellow fluorescent protein) and the donor and the acceptor and used as a fluorescent protein.
Fluorescent protein biosensor system using FRET, two types of molecules (Fig. 1) and biosensor FRET FRET (Fig. 2) single-molecule-roughly divided into the biosensor. A second molecule type FRET biosensor and detecting interactions between molecules, single-molecule-FRET biosensor detects a change in the structure of the molecule (for example see non-patent document 1-4).
Among them, two-molecule-1 (single-molecule-FRET biosensor) biosensor is, ion, sugar, and quantitative analysis of low molecules such as lipids, low molecular weight GTP binding protein, phosphorylation is used to measure such as an enzyme activity has been developed (for example see non-patent document 4). However, in order to produce this biosensor is, at least one 3, 4 in many cases needs to be bonded to one or more protein domains and, simply combining them is usually only, have sufficient sensitivity to single-molecule-FRET biosensor cannot be created. That is, such a single-molecule-FRET biosensor creation, i) and the light emission spectrum of the donor fluorescent protein and the overlap of the absorption spectrum of the acceptor fluorescent protein, ii) the distance between the donor fluorescent protein and the acceptor fluorescent protein, iii) the donor fluorescent protein acceptor emission and the moment of the orientation of the fluorescent protein 3 absorbing moment must be factors are taken into consideration. In addition, a fluorescent protein when fuzed with other proteins, other proteins fuzed with a fluorescent protein misfolding of stress occurs and, as a result, can reduce the efficiency of chromophore formation, the possibility that the non-fluorescence must be also taken into account. In this way, the donor fluorescent protein and the acceptor fluorescent protein using FRET between them to create the successful expression of the stringent condition is present, the arrangement of the between the two does not have also been found in a certain rule, create a single-molecule-FRET for each biosensor, the length of the query protein domains respectively, various changes in the inter-domain linker sequences and the like, to create optimal are performed, this includes, such as a lot of trial and requires complicated and advanced experiments, single-molecule-having sensitivity sufficient for the development of biosensor FRET Breeden and was accompanied by.
Connecting each of the domains linkers include, 9 of the amino acids glycine, serine, threonine or particles (for example see non-patent document 5), 72 amino acid glycine linker (for example see non-patent document 6) are reported, and is sufficiently optimized cannot. In addition, these linkers in, many types of single-molecule-FRET biosensor can be optimized in common were not.
  • 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
  • Kyoto University
  • 発明者(英語)
  • MATSUDA, Michiyuki
  • KOMATSU, Naoki
  • AOKI, Kazuhiro
  • KAMIOKA, Yuuji
  • YUKINAGA, Hiroko
  • INAOKA, Yoshie
  • SAKURAI, Aturou
  • KIYOKAWA, Etuko
  • SUMIYAMA, Kenta
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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