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NOVEL PEPTIDE COMPLEX AND HYBRID COMPLEX THEREOF, AND USE OF SAID HYBRID COMPLEX

Foreign code F130007366
File No. S2012-0062-N0
Posted date May 15, 2013
Country WIPO
International application number 2012JP076654
International publication number WO 2013061818
Date of international filing Oct 16, 2012
Date of international publication May 2, 2013
Priority data
  • P2011-233812 (Oct 25, 2011) JP
Title NOVEL PEPTIDE COMPLEX AND HYBRID COMPLEX THEREOF, AND USE OF SAID HYBRID COMPLEX
Abstract The present invention provides a means for delivering a substance of interest into a cell, which, even when the substance of interest is a protein, can introduce the protein into a cell with high efficiency regardless of the type of the protein, enables a given function of the protein to be exerted without inhibiting the function after the introduction of the protein into the cell, and does not use any metal that may affect a living body. A peptide complex [A] according to the present invention is characterized by comprising a leucine zipper peptide [LZ(K)] comprising the amino acid sequence represented by SEQ ID NO: 1: N'-EYQALKKKVAQLKAKNQALKKKVAQLKHK-C' or the like or a leucine zipper peptide [LZ(E)] comprising the amino acid sequence represented by SEQ ID NO: 2: N'-EYQALEKEVAQLEAENQALEKEVAQLEHE-C' or the like and an intracellular invasion peptide [CPP] linked to the leucine zipper peptide.
Outline of related art and contending technology BACKGROUND ART
Conventional, the desired protein as a technique for carrying into a cell, the cell entry peptide (cell-penetrating peptide protein; CPP) introducing technologies are the most commonly widely used.
However, the techniques described above CPP fusion protein is used, a protein of interest CPP are connected is vary significantly depending on the type of the production efficiency, can be made not most. In addition, CPP fusion protein in the cell after the introduction, the CPP is sometimes inhibited the function of the protein (CPP is generally positively charged and in many cases, this is the negatively charged nucleic acid or a biological material that interacts with the adversely affected by the possibility has been created).
Such as a technique to deal with the problem, Dowdy et al.'s group is non-patent document 1, histidine 6 to a protein of interest is introduced into the (so-called histidine tag), this modified metal ligands of the metal (nickel) via CPP (without using a covalent bond) by linking the, there is a method of intracellular delivery and confer the ability to described.
Non-patent document 1 is described in the art, are widely used in protein purification generally histidine tag and confer the ability to transport into a cell is used as is, by the use of a metal, a full-fledged for the treatment of intracellular transportation technique, so-called drug delivery system (DDS) in view of application to the anxiety remains.
On the other hand, non-patent document 2-5 Bosshard et al.'s group is, on the basis of the naturally-occurring Fos artificially developed, the mutant-type Fos is disclosed. Is a leucine zipper (coiled coil), a transcription factor DNA-binding motifs found in the dimerization domain of in. To the direction of the dimer peptide 7 amino acid residues and by the repetition of the α helix is included, the position of the 4 th leucine (d) that are arranged, a hydrophobic amino acid residue of a dimer by interaction of a characteristic of the formation of aggregates. Non-patent document disclosed variants Fos, at a predetermined position in the amino acid sequence of amino acids and basic or acidic amino acids (glutamic acid) (lysine) designed so as to include, the natural Fos binding stability than with being improved.
However, disclosed in non-patent document 2-5 mutants Fos, Fos thermodynamic behavior for the purpose of analyzing only those fabricated. On the other hand the Fos dimer linking the peptide to the cell entry and, by utilizing such Fos (such as peptides) of the desired material efficiently can be introduced into cells and the like are to be expressed, not describe or suggest.
Scope of claims (In Japanese)請求の範囲 [請求項1]
 配列番号1のアミノ酸配列からなるロイシンジッパーペプチド[LZ(K)]もしくは当該アミノ酸配列中の1~8個のアミノ酸に対する変異を有するアミノ酸配列からなるロイシンジッパーペプチド[LZ(K)']または配列番号2のアミノ酸配列からなるロイシンジッパーペプチド[LZ(E)]もしくは当該アミノ酸配列中の1~8個のアミノ酸に対する変異を有するアミノ酸配列で表されるロイシンジッパーペプチド[LZ(E)']と、これに連結された細胞内侵入ペプチド[CPP]とを含むことを特徴とする、ペプチド複合体[A]。
N'-EYQALKKKVAQLKAKNQALKKKVAQLKHK-C' 配列番号1
N'-EYQALEKEVAQLEAENQALEKEVAQLEHE-C' 配列番号2

[請求項2]
 さらに、前記ロイシンジッパーペプチドおよび/または細胞内侵入ペプチドに連結された蛍光色素を含む、請求項1に記載のペプチド複合体[A]。

[請求項3]
 前記蛍光色素が蛍光性アミノ酸である、請求項2に記載のペプチド複合体[A]。

[請求項4]
 請求項1~3のいずれかに記載のペプチド複合体[A]と、
 当該ペプチド複合体[A]に含まれる第一のロイシンジッパーペプチドと会合可能な第二のロイシンジッパーペプチドと、これに連結された細胞内への被運搬物質とを含むペプチド複合体[B]と
 から形成されることを特徴とする、ハイブリッド複合体;
 ここで、前記第一のロイシンジッパーペプチドが前記ロイシンジッパーペプチド[LZ(K)]もしくは[LZ(K)']である場合、前記第二のロイシンジッパーペプチドは前記ロイシンジッパーペプチド[LZ(E)]もしくは[LZ(E)']であり、前記第一のロイシンジッパーペプチドが前記ロイシンジッパーペプチド[LZ(E)]もしくは[LZ(E)']である場合、前記第二のロイシンジッパーペプチドは前記ロイシンジッパーペプチド[LZ(K)]もしくは[LZ(K)']である。

[請求項5]
 前記被運搬物質がタンパク質またはペプチドである、請求項4に記載のハイブリッド複合体。

[請求項6]
 請求項4または5に記載のハイブリッド複合体を含有する治療薬または診断薬。

  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • NATIONAL UNIVERSITY CORPORATION OKAYAMA UNIVERSITY
  • Inventor
  • KITAMATSU, Mizuki
  • MICHIUE, Hiroyuki
  • WANG, Feifei
  • NAKASHIMA, Mami
  • OHTSUKI, Takashi
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

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