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LIGAND FLUORESCENT SENSOR PROTEIN AND USE THEREOF

外国特許コード F170009106
整理番号 (S2016-0169-N0)
掲載日 2017年6月15日
出願国 世界知的所有権機関(WIPO)
国際出願番号 2016JP085902
国際公開番号 WO 2017094885
国際出願日 平成28年12月2日(2016.12.2)
国際公開日 平成29年6月8日(2017.6.8)
優先権データ
  • 特願2015-237524 (2015.12.4) JP
  • 2016US-62340533 (2016.5.24) US
発明の名称 (英語) LIGAND FLUORESCENT SENSOR PROTEIN AND USE THEREOF
発明の概要(英語) The present invention involves a ligand fluorescent sensor protein that specifically responds to ligands, leading to a change in fluorescence properties. The ligand fluorescent sensor protein includes a first fluorescent protein domain, an N terminal-side linker, a ligand-bonding domain, a C terminal-side linker, and a second fluorescent protein domain. The fluorescent protein used in the ligand fluorescent sensor protein has a β-barrel structure. The first fluorescent protein domain includes a β1–β3 β-sheet region from the N-terminal of the fluorescent protein, an α-helix region continuing therefrom, and a β4–β6 β-sheet region. The second fluorescent protein domain includes a β7–β11 β-sheet region of the same fluorescent protein as the first fluorescent protein domain. The N-terminal-side linker and the C-terminal-side linker are each independently a polypeptide comprising one or a plurality of amino acids.
特許請求の範囲(英語) [claim1]
1. Responding to the ligand uniquely, being the ligand fluorescent sensor protein where fluorescent quality changes,
As for the aforementioned ligand fluorescent sensor protein, 1st fluorescent protein domain and N terminal side linker and ligand connection domain and C terminal side linker and 2nd fluorescent protein domain implication,
It is something where the fluorescent protein which is used for the aforementioned ligand fluorescent sensor protein has I barrel structure,
The aforementioned 1st fluorescent protein domain follows to this with the I seat territory of .beta.1-.beta.3, from N end of the aforementioned fluorescent protein, to I the ritsukusu territory and the I seat territory of .beta.4-.beta.6 implication,
Implication the I seat territory of .beta.7-.beta.11 of the aforementioned fluorescent protein where the aforementioned 2nd fluorescent protein domain is identical with the aforementioned 1st fluorescent protein domain,
As for the aforementioned N terminal side linker and the aforementioned C terminal side linker, becoming independent respectively, the ligand fluorescent sensor protein which features that it is the polypeptide which consists of 1 or several amino acids.
[claim2]
2. In the claim 1 where the aforementioned fluorescent protein is BFP, GFP, Citrine or mApple the ligand fluorescent sensor protein of statement.
[claim3]
3. As for the aforementioned ligand fluorescent sensor protein, 1st fluorescent protein domain and N terminal side linker and ligand connection domain and C terminal side linker and 2nd fluorescent protein domain, connecting to this order directly with peptide bond from N end facing toward C end, in the claim 1 which includes the polypeptide which becomes or 2 the ligand fluorescent sensor protein of statement.
[claim4]
4. As for the aforementioned ligand fluorescent sensor protein, implication two ligand connection domains,
From N 1st ligand connection domain and N terminal side linker and, 2nd fluorescent protein domain and, 1st fluorescent protein domain and C terminal side linker and, 2nd ligand connection domain and, connecting to this order directly with peptide bond end facing toward C end, in the claim 1 which includes the polypeptide which becomes or 2 the ligand fluorescent sensor protein of statement.
[claim5]
5. As for the aforementioned 1st fluorescent protein domain, (B1) below - (B3) implication no polypeptide,
As for the aforementioned 2nd fluorescent protein domain, (C1) below - (C3) implication no polypeptide,
-description above 1 fluorescence protein domain and 2 fluorescence protein domain with identical fluorescence protein to be derived do claim 1 or 2 to statement ligand fluorescence sensor protein.
(B1) The polypeptide which includes the amino acid arrangement which is displayed with arrangement number 1, 3, 5 or 7,
(B2) The amino acid arrangement where 1 or several amino acids is deficient at the time of amino acid arranging which is displayed with arrangement number 1, 3, 5 or 7, insertion, is substituted or is added is included, at the same time, the aforementioned 2nd fluorescent protein domain I barrel structure is formed, the polypeptide which fluorescence is given out,
(B3) The amino acid arrangement to which amino acid arrangement and the identity which are displayed with arrangement number 1, 3, 5 or 7 are 80% or more is included, at the same time, the aforementioned 2nd fluorescent protein domain I barrel structure is formed, the polypeptide which fluorescence is given out,
(C1) The polypeptide which includes the amino acid arrangement which is displayed with arrangement number 2, 4, 6 or 8,
(C2) The amino acid arrangement where 1 or several amino acids is deficient at the time of amino acid arranging which is displayed with arrangement number 2, 4, 6 or 8, insertion, is substituted or is added is included, at the same time, the aforementioned 1st fluorescent protein domain I barrel structure is formed, the polypeptide which fluorescence is given out,
(C3) The amino acid arrangement to which amino acid arrangement and the identity which are displayed with arrangement number 2, 4, 6 or 8 are 80% or more is included, at the same time, the aforementioned 1st fluorescent protein domain I barrel structure is formed, the polypeptide which fluorescence is given out
[claim6]
6. The aforementioned ligand, the nucleotide or that derivative, nuclear acid and the sugar chain, the protein, either of the claim 1-5 which is the lipid mixture, or low-molecular chemical compound in one section the ligand fluorescent sensor protein of statement.
[claim7]
7. The aforementioned nucleotide or that derivative, in the claim 6 which is ATP, cAMP or cGMP the ligand fluorescent sensor protein of statement.
[claim8]
8. In the claim 6 where the aforementioned protein is the antigen or the antibody the ligand fluorescent sensor protein of statement.
[claim9]
9. (D1) below the - (D3) either of the claim 1-8 which includes each polypeptide in one section the ligand fluorescent sensor protein of statement.
(D1) The polypeptide which includes the amino acid arrangement which is displayed with either of the arrangement number 9-14,
(D2) The polypeptide which include the amino acid arrangement where 1 or several amino acids is deficient at the time of amino acid arranging which is displayed with either of the arrangement number 9-14, insertion, is substituted or is added, at the same time, the aforementioned polypeptide (D1) with equality, connection talent to the ligand and possesses fluorescent quality,
(D3) The polypeptide which include the amino acid arrangement to which amino acid arrangement and the identity which are displayed with either of the arrangement number 9-14 are 80% or more, at the same time, the aforementioned polypeptide (D1) with equality, connection talent to the ligand and possesses fluorescent quality
[claim10]
10. Furthermore, either of the claim 1-9 which includes oruganera localization signal peptide in one section the ligand fluorescent sensor protein of statement.
[claim11]
11. In the claim 10 where the aforementioned oruganera localization signal peptide is mitochondria localization signal peptide the ligand fluorescent sensor protein of statement.
[claim12]
12. In the claim 10 where the aforementioned oruganera localization signal peptide is nuclear localization signal peptide the ligand fluorescent sensor protein of statement.
[claim13]
13. Furthermore, either of the claim 1-12 which includes cell membrane permeable peptide in one section the ligand fluorescent sensor protein of statement.
[claim14]
14. Either of the claim 1-13 in one section the polynucleotide which features that the cord/code it does the ligand fluorescent sensor protein of statement.
[claim15]
15. The manifestation vector which features that the polynucleotide of statement is included in claim 14.
[claim16]
16. Either of the claim 1-13 the cell which features that the ligand fluorescent sensor protein of statement 1 types is included in one section at least.
[claim17]
17. Either of the claim 1-13 in one section the cell which features that it possesses the chromosome which 1 types includes the polynucleotide which the ligand fluorescent sensor protein of statement the cord/code is done at least.
[claim18]
18. The cell which features that the manifestation vector of statement 1 types is included in claim 15 at least.
[claim19]
19. Either of the claim 16-18 the non human living thing which features that the cell of statement is included in one section.
[claim20]
20. Features that at least one which is chosen from the group which in one section consists of the non human living thing of statement in the cell, and claim 19 of statement is included the kit for ligand density measurement which either of the claim 1-13 in one section the ligand fluorescent sensor protein of statement, in claim 14 the polynucleotide of statement, in claim 15 the manifestation vector of statement, either of the claim 16-18.
[claim21]
21. Making with the ligand fluorescent sensor protein of statement and the standard solution which includes the ligand of known density one section contact, the analytical curve compilation process which measures fluorescent strength, draws up the analytical curve either of the claim 1-13 and,
Making contact with the aforementioned ligand fluorescent sensor protein and the solution which includes the ligand of unknown density, the fluorometry process which measures fluorescent strength and,
The density decisive process which decides the ligand density for the fluorescent strength which was measured in the aforementioned analytical curve compilation process, on the basis of the analytical curve which was drawn up, in the aforementioned fluorometry process and,
Decisive method of ligand density in the suffering inspection sample which features that it has.
[claim22]
22. In one section either of the claim 16-18 inspection method of time-dependant changing the ligand density in the raw cell which features that the process which measures time-dependant fluorescent strength making use of the cell of statement, is had.
[claim23]
23. Inspection method of time-dependant changing the ligand density in the non human living thing which features that the process which measures time-dependant fluorescent strength making use of the non human living thing of statement, is had in claim and 19 lives.
[claim24]
24. Responding to ATP density uniquely, being the ATP fluorescent sensor protein where fluorescent quality changes, as for the said ATP fluorescent sensor protein, from N end directing to C end, 1st fluorescent protein domain and N terminal side linker and ATP connection domain and C terminal side linker and 2nd fluorescent protein domain, to include the polypeptide which directly is connected to this order with peptide bond, as for the said polypeptide,
1st fluorescent protein domain, fluorescent protein BFP, Citrine or mApple from the N end, follows to this with the I seat territory of .beta.1-.beta.3, the I helix territory and the I seat territory of .beta.4-.beta.6 implication,
As for 2nd fluorescent protein domain, implication the I seat territory of .beta.7-.beta.11 of the fluorescent protein which is identical with 1st fluorescent protein domain,
ATP connection domain F [0] F [1] - consists of the I sub unit of the ATP synthetic enzyme,
N terminal side linker and C terminal side linker respectively, the ATP fluorescent sensor protein which features that it is the polypeptide which consists of 1 or several amino acids.
[claim25]
25. (A11) ATP connection domain is amino acid arrangement of arrangement number 15, 1st and 2nd fluorescent protein domain, respectively, arrangement number is amino acid arrangement of 1 and 2, linker of N end and C terminal side, respectively, arrangement number is amino acid arrangement of 16 and 17, the MaLion B polypeptide and,
(A12) ATP connection domain, 1st fluorescent protein domain, 2nd fluorescent protein domain, as for linker of N terminal side, and linker of C terminal side, in the respective independence, arrangement number amino acid arrangement of 15, 1, 2, 16, and 17?, the amino acid arrangement where arrangement number 1 or several amino acids are deficient in amino acid arrangement of 13, 1, 2, 14, and 15, are substituted, or are added is, at the same time, the MaLion B polypeptide (A11) with it possesses identical ATP connection talent and fluorescent quality, the MaLion B polypeptide and,
(B11) ATP connection domain is amino acid arrangement of arrangement number 15, 1st and 2nd fluorescent protein domain, respectively, arrangement number is amino acid arrangement of 5 and 6, linker of N end and C terminal side, respectively, WRG (Trp-Arg-Gly) and is amino acid arrangement of arrangement number 18, the MaLion G polypeptide and,
(B12) ATP connection domain, 1st fluorescent protein domain, 2nd fluorescent protein domain, as for linker of N terminal side and linker of C terminal side, in the respective independence, arrangement number 15, 5 and 6, WRG (Trp-Arg-Gly), and amino acid arrangement of arrangement number 18?, the amino acid arrangement where arrangement number 15, 5 and 6, WRG (Trp-Arg-Gly), and 1 or several amino acids are deficient in amino acid arrangement of arrangement number, 18 are substituted or are added is, at the same time, the MaLion G polypeptide (B11) with it possesses identical ATP connection talent and fluorescent qualityMaLion G polypeptide and,
(C11) ATP connection domain is amino acid arrangement of arrangement number 15, 1st and 2nd fluorescent protein domain, respectively, arrangement number is amino acid arrangement of 7 and 8, linker of N end and C terminal side, respectively, arrangement number 19 and PEE (Pro-Glu-Glu) is amino acid arrangement, the MaLion R polypeptide and,
(C12) ATP connection domain, 1st fluorescent protein domain, 2nd fluorescent protein domain, linker of N terminal side and linker of C terminal side respectively, arrangement number 15, 7, 8, 19, and PEE (Pro-Glu-Glu) amino acid arrangement?, the amino acid arrangement where arrangement number 15, 7, 8, 19, and PEE (Pro-Glu-Glu) 1 or several amino acids are deficient in amino acid arrangement, are substituted or are added is, at the same time, the MaLion R polypeptide (C11) with it possesses identical ATP connection talent and fluorescent quality, from the MaLion R polypeptideIn the claim 24 which at least includes one which is selected from the group which becomes polypeptide the ATP fluorescent sensor protein of statement.
[claim26]
26. (A21) It consists of the amino acid arrangement of arrangement number 9, the MaLion B polypeptide and,
(A22) Among amino acid arrangements of arrangement number 9, arrangement number 1 or several amino acids are deficient in the amino acid arrangement which excludes the amino acid arrangement of 16 and 17, consist of the amino acid arrangement which it is substituted or is added, at the same time, the MaLion B polypeptide (A21) with possess identical ATP connection talent and fluorescent quality, the MaLion B polypeptide and,
(B21) It consists of the amino acid arrangement of arrangement number 10, the MaLion G polypeptide and,
(B22) Among amino acid arrangements of arrangement number 10, WRG of 146-148th rank of arrangement number 10 (Trp-Arg-Gly) 1 or several amino acids are deficient in the amino acid arrangement which excludes with the amino acid arrangement of amino acid arrangement and arrangement number 18, consist of the amino acid arrangement which it is substituted or is added, at the same time, the MaLion G polypeptide (B21) with possess identical ATP connection talent and fluorescent quality, the MaLion G polypeptide and,
(C21) It consists of the amino acid arrangement of arrangement number 11, the MaLion R polypeptide and,
(C22) In the claim 24 which at least includes one which is selected from the group where among amino acid arrangements of arrangement number 11, PEE of 288-290th rank of arrangement number 11 (Pro-Glu-Glu) 1 or several amino acids are deficient in the amino acid arrangement which excludes with the amino acid arrangement of amino acid arrangement and arrangement number 19, consist of the amino acid arrangement which it is substituted or is added, at the same time, the MaLion R polypeptide (C21) with possess identical ATP connection talent and fluorescent quality, consist of with the MaLion R polypeptide polypeptide or 25 the ATP fluorescent sensor protein of statement.
[claim27]
27. Either of the claim 24-26 the fluorescent composition which features that the ATP fluorescent sensor protein of statement is included in one section.
[claim28]
28. As for the aforementioned ATP fluorescent sensor protein in the claim 27 which is firmly converted to the solid backing the fluorescent composition of statement.
[claim29]
29. As for the aforementioned ATP fluorescent sensor protein, in the claim 28 which at least is the ATP fluorescent sensor protein of 2 where opposite of 1st and 2nd fluorescent protein domain differs types the fluorescent composition of statement.
  • 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
  • UNIVERSITY OF TOKYO
  • WASEDA UNIVERSITY
  • 発明者(英語)
  • TSUBOI TAKASHI
  • KITAGUCHI TETSUYA
  • ARAI SATOSHI
  • UEDA HIROSHI
国際特許分類(IPC)
指定国 (WO201794885)
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 DJ 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 KW KZ LA LC LK LR LS 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 ST TD TG

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