Top > Search of International Patents > METHOD FOR INDUCING PROTEOLYSIS IN EUKARYOTIC CELL

METHOD FOR INDUCING PROTEOLYSIS IN EUKARYOTIC CELL

Foreign code F130007381
File No. S2012-0021-N0
Posted date Jun 5, 2013
Country WIPO
International application number 2012JP079744
International publication number WO 2013073653
Date of international filing Nov 16, 2012
Date of international publication May 23, 2013
Priority data
  • P2011-252147 (Nov 18, 2011) JP
Title METHOD FOR INDUCING PROTEOLYSIS IN EUKARYOTIC CELL
Abstract Provided are: a novel protein-inducible eukaryotic cell which does not kill a host such as budding yeast and has a satisfactory decomposition ability; and a method for inducing proteolysis using the protein-inducible eukaryotic cell. A proteolysis-inducible non-plant eukaryotic cell, which has a plant-derived TIR1 family protein gene and a chimeric gene capable of expressing a desired protein labeled with a partial sequence of a plant-derived Aux/IAA family protein, and in which the degradation of the desired protein can be induced by an auxin, said non-plant eukaryotic cell being characterized in that the partial sequence is a sequence comprising a region containing at least two Lys residues on each of the N-terminal side and the C-terminal side of a domain II region of an Aux/IAA family protein or a sequence composed of a linked product of two or more of the aforementioned sequences.
Outline of related art and contending technology BACKGROUND ART
Specific cells control the expression level of the protein, the function of the protein in the cell and its protein involved in analyzing a biological phenomenon on the very useful. For this purpose, a plurality of separately to based on the principle of this method has been developed.
For example, E. coli using tetracycline-binding transcriptional repressors, transcription of a gene are introduced into the cell culture expression of tetracycline has been known a method of making the (non-patent document 1, 2). Based on this principle are commercially available and transfer system will, many researchers have actually used results as hereinbefore has been increased. However, this method is a method in which the transcriptional control of the gene of interest for, the actual protein suppression may be performed until it is removed from within the cell often takes a long time, in some cases, a partial expression of the protein of interest is the reduction of secondary cellular responses that activate is also sometimes. Suppressing expression in cells cultured similarly in a method of performing siRNA and shRNA to be introduced into a cell, utilizing the reaction with the interference RNA in a cell of the gene product of interest is known to decompose mRNA. This method based on the actual product commercially available from a number of companies. However, this method also mRNA level for suppression of expression of the is carried out, generally to inhibiting the expression of 24-48 requires a long time to time. The expression inhibition degree, independent of the sequence of the target RNA and the target factors, can be realized by suppressing expression of 90% or more is not so high.
In recent years, inducing the target protein is introduced into the tag, the tag can be controlled by controlling the decomposition of a method for controlling the expression of the protein has been begun to be put into practical use. FKBP12 Protein is modified rapamycin binding protein, rapamycin analog compound Shield 1 to bind to the stabilized, is not coupled destabilization from being degraded within the cells. Using this property, this target protein is fuzed to the protein derived from FKBP12, Shield 1-dependent expression control method in which a (non-patent document 3) published. Shield 1 The methods generally involve C. and about 4 by the removal of the target protein and is resolved, and the like are the following commercially available plasmid set required. In this method in order to stabilize the protein expression is always Shield 1 needs to be added to the medium, the rapamycin analog is Shield 1 for the cytotoxic compound is in doubt. In addition, also necessary for suppressing the expression for about 4 in terms of degradation time, the more rapid cell cycle studies such as the systems which can be decomposed and removed is have been desired.
The inventor considers such circumstances, the plant hormone auxin induced by focusing on plant-specific proteolysis, this degradation pathway for the budding yeast and introduced into a cell or mammalian, auxin can be added to the medium a very short time 15-30 min the target protein expression by the decomposition of the developed a method of inhibiting (Patent Document 1, 2).
Scope of claims (In Japanese)請求の範囲 [請求項1]
 植物由来のTIR1ファミリータンパク質遺伝子と、植物由来Aux/IAAファミリータンパク質の部分配列で標識された目的タンパク質を発現するキメラ遺伝子とを有する、オーキシン類により目的タンパク質の分解が誘導されるタンパク質分解誘導性の非植物真核細胞であって、該部分配列が、Aux/IAAファミリータンパク質のドメインII領域のN末端側及びC末端側に少なくとも2個ずつのLys残基を含む領域からなる配列、又は該配列を2個以上連結してなる配列であることを特徴とするタンパク質分解誘導性の非植物真核細胞。

[請求項2]
 前記部分配列が、Aux/IAAファミリータンパク質のドメインII領域のN末端側及びC末端側に2~5個ずつのLys残基を含む32~80アミノ酸残基からなる配列、又は該配列を2~4個連結してなる配列である請求項1記載のタンパク質分解誘導性の非植物真核細胞。

[請求項3]
 前記部分配列が、配列番号1~12から選ばれる配列を含む50~80アミノ酸残基からなる配列、又は該配列を2~4個連結してなる配列である請求項1又は2記載のタンパク質分解誘導性の非植物真核細胞。

[請求項4]
 宿主非植物真核細胞に、植物由来のTIR1ファミリータンパク質遺伝子と、植物由来Aux/IAAファミリータンパク質の部分配列で標識された目的タンパク質を発現するキメラ遺伝子を導入する、オーキシン類により目的タンパク質の分解が誘導されるタンパク質分解誘導性の非植物真核細胞の製造法であって、該部分配列が、Aux/IAAファミリータンパク質のドメインII領域のN末端側及びC末端側に少なくとも2個ずつのLys残基を含む領域からなる配列、又は該配列を2個以上連結してなる配列であることを特徴とするタンパク質分解誘導性の非植物真核細胞の製造法。

[請求項5]
 前記部分配列が、Aux/IAAファミリータンパク質のドメインII領域のN末端側及びC末端側に2~5個ずつのLys残基を含む32~80アミノ酸残基からなる配列、又は該配列を2~4個連結してなる配列である請求項4記載のタンパク質分解誘導性の非植物真核細胞の製造法。

[請求項6]
 前記部分配列が、配列番号1~12から選ばれる配列を含む32~80アミノ酸残基からなる配列、又は該配列を2~4個連結してなる配列である請求項4又は5記載のタンパク質分解誘導性の非植物真核細胞の製造法。

[請求項7]
 植物由来のTIR1ファミリータンパク質遺伝子と、植物由来Aux/IAAファミリータンパク質の部分配列で標識された目的タンパク質を発現するキメラ遺伝子とを有する、オーキシン類により目的タンパク質の分解が誘導されるタンパク質分解誘導性の非植物真核細胞用の遺伝子導入用ベクターであって、該部分配列が、Aux/IAAファミリータンパク質のドメインII領域のN末端側及びC末端側に少なくとも2個ずつのLys残基を含む領域からなる配列、又は該配列を2個以上連結してなる配列であることを特徴とするタンパク質分解誘導性の非植物真核細胞用の遺伝子導入用ベクター。

[請求項8]
 前記部分配列が、Aux/IAAファミリータンパク質のドメインII領域のN末端側及びC末端側に2~5個ずつのLys残基を含む32~80アミノ酸残基からなる配列、又は該配列を2~4個連結してなる配列である請求項7記載のタンパク質分解誘導性の非植物真核細胞用の遺伝子導入用ベクター。

[請求項9]
 前記部分配列が、配列番号1~12から選ばれる配列を含む32~80アミノ酸残基からなる配列、又は該配列を2~4個連結してなる配列である請求項7又は8記載のタンパク質分解誘導性の非植物真核細胞用の遺伝子導入用ベクター。

[請求項10]
 請求項1~3のいずれか1項記載の非植物性真核細胞に、オーキシン類を添加することを特徴とする、該非植物真核細胞における目的タンパク質の分解誘導方法。

  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • RESEARCH ORGANIZATION OF INFORMATION AND SYSTEMS
  • OSAKA UNIVERSITY
  • Inventor
  • KANEMAKI, Masato
  • NISHIMURA, Kohei
  • TAKISAWA, Haruhiko
  • MIMURA, Satoru
  • KOBATA, Yui
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 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 PA 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 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

PAGE TOP

close
close
close
close
close
close