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MODIFIED Β-GLUCURONIDASE

Foreign code F150008484
File No. E102P07WO
Posted date Oct 26, 2015
Country WIPO
International application number 2014JP069539
International publication number WO 2015016124
Date of international filing Jul 24, 2014
Date of international publication Feb 5, 2015
Priority data
  • P2013-156615 (Jul 29, 2013) JP
Title MODIFIED Β-GLUCURONIDASE
Abstract Provided is a modified β-glucuronidase in which a mutation has been introduced to the amino acid sequence of a β-glucuronidase monomer, and wherein the modified β-glucuronidase is mutated and novel and has methionine or serine at the 561st position from the N terminal side of the amino acid sequence, and due to being a modified β-glucuronidase, has high enzyme activity when compared to the wild form, and is easily detected.
Outline of related art and contending technology BACKGROUND ART
Such as within a cell to express the gene of interest in for analyzing, quantifying the transcription of the gene product, is a need to identify. However, in the case of directly analyzing the transcripts, purified mRNA, analyzing the hybridization of the electrophoresis takes time and, further, the internalized by the cell needs to be distinguished from other mRNA is also a careful manipulation is required. As a useful technique in such a case, using a reporter gene encoding an enzyme, which can be analyzed to indirectly known.
Β - glucuronidase gene, a plant cell that does not have a particularly useful are hitherto used as a reporter gene. In the case of using β - glucuronidase gene as a reporter gene, the expression of the gene from the β - glucuronidase. Β - glucuronidase is, in the state of monomer activity not shown, tetramer only in its activity, its detection is enabled. As an example of the method of assembling a conventional, that are assembled to the plate perpendicular to one another by screwing a screw. By previous studies, the activity of the β - glucuronidase expression, depending on the size of cells and requires considerable time proved to be point (for example, t. Matsuura et al., "Kinetic Analysis of β-Galactosidase and β-Glucuronidase Tetramerization Coupled with Protein Translation", Journal of Biological Chemistry ., (2011) 286, 22028 reference (non-patent document 1). ).
Scope of claims (In Japanese)請求の範囲 [請求項1]
 β-グルクロニダーゼ単量体のアミノ酸配列に変異が導入された改良型のβ-グルクロニダーゼであって、
 前記アミノ酸配列のN末端側から561番目の位置にメチオニンまたはセリンを有する、改良型のβ-グルクロニダーゼ。

[請求項2]
 前記アミノ酸配列のN末端側から561番目の位置にメチオニンを有し、かつ、前記アミノ酸配列のN末端側から520番目の位置にロイシンを有する、請求項1に記載の改良型のβ-グルクロニダーゼ。

[請求項3]
 前記アミノ酸配列のN末端側から561番目の位置にセリンを有し、かつ、前記アミノ酸配列のN末端側から444番目の位置にアラニンを有する、請求項1に記載の改良型のβ-グルクロニダーゼ。

[請求項4]
 配列番号10、12、14、16、18または20のアミノ酸配列を有する、請求項1に記載の改良型のβ-グルクロニダーゼ。

[請求項5]
 請求項1~4のいずれか1項に記載の改良型のβ-グルクロニダーゼにさらに変異が導入された改良型のβ-グルクロニダーゼであって、以下の(a)~(d)の少なくともいずれかを有する、改良型のβ-グルクロニダーゼ。
 (a)前記β-グルクロニダーゼ単量体のアミノ酸配列のN末端側から64番目の位置にバリンを有する、
 (b)前記β-グルクロニダーゼ単量体のアミノ酸配列のN末端側から123番目の位置にバリンを有する、
 (c)前記β-グルクロニダーゼ単量体のアミノ酸配列のN末端側から145番目の位置にロイシンを有する、
 (d)前記β-グルクロニダーゼ単量体のアミノ酸配列のN末端側から451番目の位置にバリンを有する。

[請求項6]
 前記(a)~(d)を全て有する、請求項5に記載の改良型のβ-グルクロニダーゼ。

[請求項7]
 配列番号27または29のアミノ酸配列を有する、請求項5に記載の改良型のβ-グルクロニダーゼ。

  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • JAPAN SCIENCE AND TECHNOLOGY AGENCY
  • Inventor
  • YOMO, Tetsuya
  • SUNAMI, Takeshi
  • NISHIKAWA, Takehiro
IPC(International Patent Classification)
Specified countries National States: AE AG AL AM AO AT AU AZ BA BB BG BH BN 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 IR IS JP KE KG 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 PA PE PG PH PL PT QA RO RS RU RW SA 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 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 KM ML MR NE SN TD TG
Reference ( R and D project ) ERATO YOMO Dynamical Micro-scale Reaction Environment AREA
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