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NOVEL PROTEIN IDENTIFICATION METHOD USING YEAST AND FRAGMENTED CDNA LIBRARY

Foreign code F170009304
File No. (S2016-0677-N0)
Posted date Dec 20, 2017
Country WIPO
International application number 2017JP020614
International publication number WO 2017209280
Date of international filing Jun 2, 2017
Date of international publication Dec 7, 2017
Priority data
  • P2016-112107 (Jun 3, 2016) JP
Title NOVEL PROTEIN IDENTIFICATION METHOD USING YEAST AND FRAGMENTED CDNA LIBRARY
Abstract The purpose of the present invention is to provide a novel method for identifying a protein in a yeast nucleus. More specifically, the present invention relates to a protein identification method which comprises: in a yeast nucleus, expressing a fusion protein comprising a nuclear localization signal, a DNA-binding protein and a bait protein, and another fusion protein comprising a nuclear localization signal, a transcriptional activation protein and a prey protein encoded by a fragmented partial cDNA, said prey protein lacking a signal peptide, a cell membrane or intracellular organelle localization sequence or a transmembrane domain; and then detecting the binding of the bait protein to the prey protein using the activity, luminescence, etc. of a reporter gene as an indication.
Outline of related art and contending technology BACKGROUND ART
Out of the cells associated with the plurality of molecules or binding, cell proliferation and differentiation, adhesion and the like are known to act as biological signal. In particular, various hormone and the like in order to reveal the physiological activity of the ligand molecule is, the identification of the receptor molecule on the cell membrane is useful. For the purpose of these, immunoprecipitation and concentration of a protein complex comprising a receptor molecule, or a method of mass spectrometry using a cell culture is the panning or the like is becoming common. However, still unknown receptors' orphan ligand ' number is known. The receptor is identified, is likely to be associated with a variety of disease conditions are of interest, the complexity of the screening method, does not proceed from the lack of detection sensitivity.
On the other hand, in the cells (cytoplasm) of the molecule as the identification method, yeast two-hybrid method has been known (Non-Patent Document 1-3). Yeast two-hybrid method, a nuclear localization signal using a genetic vector is added with the nuclear localization signal is added to the known gene full-length chimeric gene cDNA (commercially available is normally transferred to the reverse primer oligo dT) the type of the expression 2, the binding molecules are formed in the nucleus, the induction of a reporter gene activity such as detecting the binding between the light emitting molecule.
Yeast two-hybrid method in which, in one of the ligand molecule or receptor gene encoding the protein is inserted into the vector, 5 'end of the signal peptide and 3' near the end of the cells by expression of a transmembrane domain, chimeric gene expression in the nucleus may be difficult to, yeast two-hybrid method of the conventional ligand - receptor binding is not suitable for analysis.
In addition, the yeast two-hybrid method has been reported a number of modifications. For example, organisms other than yeast cells used may be changed to, the reporter gene by aiming at improvement in the development of the like. However, in the related art, a partial region of the protein (membrane) expression in the nucleus as a library, the original interaction occurs outside the cell nucleus detected in yeast two-hybrid intended to be of the known method has not been modified.
Scope of claims (In Japanese)[請求項1]
第1及び第2のベクターを導入した酵母形質転換体を培養する工程を含む、タンパク質の同定方法であって、
第1のベクターは、核内移行シグナルをコードする遺伝子とDNA結合タンパク質をコードする遺伝子とおとりタンパク質をコードする遺伝子とを含み、
第2のベクターは、核内移行シグナルをコードする遺伝子と転写活性化タンパク質をコードする遺伝子と断片化した部分cDNAとを含み、該断片化した部分cDNAは、シグナルペプチド、細胞膜若しくは細胞内小器官局在化配列又は細胞膜貫通領域が欠損した獲物タンパク質をコードするものであり、且つ、
前記酵母形質転換体の核内において、前記核内移行シグナルとDNA結合タンパク質とおとりタンパク質とを含む融合タンパク質、及び前記核内移行シグナルと転写活性化タンパク質と断片化した部分cDNAによりコードされる獲物タンパク質とを含む融合タンパク質が発現し、該おとりタンパク質と該獲物タンパク質との結合を、レポーター遺伝子の活性を指標に検出する、
前記方法。
[請求項2]
おとりタンパク質がリガンドであり、且つ獲物タンパク質が受容体タンパク質である、請求項1記載の方法。
[請求項3]
おとりタンパク質が各種局在シグナル配列を除いた部分cDNAによりコードされるタンパク質である、請求項1記載の方法。
[請求項4]
ランダムプライム逆転写によって得られた、長さ100~1800bpを有するcDNA。
[請求項5]
長さが100~1000bpである、請求項4記載のcDNA。
[請求項6]
核内移行シグナルをコードする遺伝子と転写活性化タンパク質をコードする遺伝子と断片化した部分cDNAとを含むベクターであって、該断片化した部分cDNAが請求項4又は5記載のcDNAである、前記ベクター。
[請求項7]
請求項4若しくは5記載のcDNA又は請求項6記載のベクターを含む、請求項1~3のいずれか1項記載のタンパク質の同定方法用試薬キット。
  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • KAGOSHIMA UNIVERSITY
  • Inventor
  • KISHIDA Shosei
  • KOYAMA Hirofumi
  • KISHIDA Michiko
  • IIJIMA Mikio
IPC(International Patent Classification)
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