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NOVEL PROTEIN IDENTIFICATION METHOD USING YEAST AND FRAGMENTED CDNA LIBRARY

外国特許コード F170009304
整理番号 (S2016-0677-N0)
掲載日 2017年12月20日
出願国 世界知的所有権機関(WIPO)
国際出願番号 2017JP020614
国際公開番号 WO 2017209280
国際出願日 平成29年6月2日(2017.6.2)
国際公開日 平成29年12月7日(2017.12.7)
優先権データ
  • 特願2016-112107 (2016.6.3) JP
発明の名称 (英語) NOVEL PROTEIN IDENTIFICATION METHOD USING YEAST AND FRAGMENTED CDNA LIBRARY
発明の概要(英語) 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.
従来技術、競合技術の概要(英語) 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.
  • 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
  • KAGOSHIMA UNIVERSITY
  • 発明者(英語)
  • KISHIDA Shosei
  • KOYAMA Hirofumi
  • KISHIDA Michiko
  • IIJIMA Mikio
国際特許分類(IPC)
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