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PROBE REAGENT FOR MEASUREMENT OF PROTEIN DECOMPOSITION ACTIVITY

外国特許コード F110004996
整理番号 E08804WO
掲載日 2011年8月3日
出願国 世界知的所有権機関(WIPO)
国際出願番号 2011JP051089
国際公開番号 WO 2011090159
国際出願日 平成23年1月21日(2011.1.21)
国際公開日 平成23年7月28日(2011.7.28)
優先権データ
  • 特願2010-012084 (2010.1.22) JP
発明の名称 (英語) PROBE REAGENT FOR MEASUREMENT OF PROTEIN DECOMPOSITION ACTIVITY
発明の概要(英語) A probe reagent comprising, from the N-terminal to the C-terminal, an amino acid sequence for a fluorescent protein I, an amino acid sequence for a peptide capable of terminating the decomposition of a protein (i.e., a decomposition termination peptide), an amino acid sequence for a spacer peptide, an amino acid sequence for a fluorescent protein II, and an amino acid sequence for a protein to be decomposed, wherein the probe reagent is characterized in that the protein to be decomposed is a protein capable of being decomposed with a ubiquitin-proteasome system and the probe reagent is decomposed from the C-terminal side but the decomposition is terminated at the decomposition termination peptide; a nucleic acid encoding the probe reagent; and use of the probe reagent or the nucleic acid.
従来技術、競合技術の概要(英語) BACKGROUND ART
1 Cells as one of the protein with a pathway, ubiquitin - proteasome system is well known. In this reaction system, the denatured protein or abnormal protein folding and a plurality of small protein called ubiquitin, added to the chain. This ubiquitin chains as a marker of the protein should be decomposed and, degradation of the protein recognized by the proteasome device destruction. This system, the removal of the abnormal protein in a cell is performed. However, the ubiquitin - proteasome system, within the cell not only for the purpose of quality control of the system is not adopted. Structurally, functionally normal protein receptor protein, at that time and to degrade in response to the status of a cell, a decrease in the activity of the control is performed in the various cell functions. As a result of the large protein ubiquitin - proteasome system is controlled by the amount present is known. As the function of these proteins, cell cycle control, regulation of gene expression, stress response, in a wide variety restoration of DNA. In this way, a biological phenomenon in many cells is controlled by this system can be made, ubiquitin - proteasome system maintenance of the normal activities of the cells being essential. Therefore, deterioration of the function of this proteolytic system affect the critical to the cell. Alzheimer's disease is a neurodegenerative disease is Parkinson's disease, intracellular accumulation of abnormal protein aggregation is observed, as the cause of the onset of these diseases of the ubiquitin - proteasome system dysfunction has been suggested. The ubiquitin - proteasome system is, to be involved in the restoration of cell cycle or from DNA, canceration of cells from the failure of this system is induced also known. In this way, the ubiquitin - proteasome system a number of important biological phenomenon from the responsible for the control, the reaction was allowed to precisely and easily develop a method for measuring, life phenomena exhibited by elucidation of mechanism as well as, methods of treatment for disease induced by the abnormality in the development of chemical agents and the like greatly contribute to appear.
Ubiquitin - proteasome system performed in a cell of the measurement of the degradation of the protein, conventional, of the biochemical techniques such as Western blotting has been used. In this method a large number of cells are collectively destroyed, to recover components thereof. An object to be measured contained in the protein, depending on the molecular weight by electrophoresis separation, using antibody specific for further detected by staining. As a result of protein abundance and degradation activity is measured. However, this method is troublesome and time-consuming operation and then, the measurement of living in individual cells cannot be performed.
In recent years, and the like related to the firefly bioluminescent reaction luciferin, luciferase, fluorescent protein that is obtained from the aequorea victoria, for monitoring the kinetics of intracellular molecules of probe reagent as application techniques greatly developed. This is in combination with the advances in the techniques of microscopic imaging, a particular physiological activity within the cell to visualize the space-time, a method for measuring have been generalized. Activity of the proteasome for degradation of the protein, in a living cell by this method has been measured. In this method, a measuring object and luciferase or a protein fuzed to the fluorescent protein and the probe reagent (patent document 1). When the protein is present in cells, light emitting label or a fluorescence is observed, when proteasome degradation of the luciferase or fluorescent protein fuzed together and also can be decomposed into fellow traveler, not the luminescence or fluorescence was observed. Therefore, these variations in light intensity monitor of the cracking activity of this protein can be measured. So far by this method, IκB α, p27, p53, degradation of the protein such as HIF-1α measurement is being performed (patent document 2, non-patent document 1-3). However, these to be used as markers of probe reagent is luciferase or fluorescent protein is from the two types of 1, only the luminescence or fluorescence 1 measured by the wavelength. For this reason, the intensity of light, of the expression amount of non-uniform distribution of the probe reagent, in the form of cells and tissues, such as quenching by fading, such as unevenness of the excitation light does not depend on the degradation of the protein activity can be affected by factors such as, quantitative measurement is difficult. Increases the proteolytic activities that also decreases when the amount of the light intensity, of the short of the sensitivity of the detector resulting in a reduction in S/N, a precise measure is also a problem that it is difficult to.
1 The photometric method of wavelength only becomes a problem in degradation of the protein does not depend on the adverse effects of the factors is, the luminescence or fluorescence measured at a wavelength 2, that the mechanical strength of the second fuel can be cancelled by setting the ratio. In the method of Davis et al., protein as the object of measurement, the light emission of a click beetle green IκB α derived from fuzed to luciferase (CBG68) and expressed in a cell. At the same time click beetle-derived luciferase (CBR) red light emission of the expressed in the same cell. CBG68 Emits light in a quantity, according to the activity of the proteasome degradation IκB α showing the increase or decrease. On the other hand, the emission will be affected not CBR was used as control. They are cultured in multi-well plates at a cell population was measured and, due to a difference between the number of cells between wells in the amount of the light emitted in order to correct, red/green IκB α of the cracking activity measured as the ratio of the emission intensity of the (non-patent document 4). In this approach, the photometric method compared with the wavelength 1 is improved when it is quantitative, one 2 for the expression of probe molecules to individual, between cells of the expression amount of these ratio, that is the ratio of the difficult to have the same problem. Also be used as markers depending on the nature of the fluorescent protein luciferase, which are not uniformly distributed within the cells of the localized difference might be the performance of computer systems. These are, in particular at the single cell level of degradation of the protein activity was measured and if it is desired to become factors that reduce the accuracy.
  • 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
  • JAPAN SCIENCE AND TECHNOLOGY AGENCY
  • RIKEN
  • 発明者(英語)
  • MIYAWAKI Atsushi
  • HIRANO Masahiko
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 RO RS RU 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 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
参考情報 (研究プロジェクト等) ERATO MIYAWAKI Life Function Dynamics AREA
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