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Novel polypeptide exhibiting fluorescent properties and use thereof achieved

Foreign code F190009896
File No. 08118-EP
Posted date Aug 26, 2019
Country EPO
Application number 14757001
Gazette No. 2963115
Gazette No. 2963115
Date of filing Feb 28, 2014
Gazette Date Jan 6, 2016
Gazette Date Apr 1, 2020
International application number JP2014055160
International publication number WO2014133158
Date of international filing Feb 28, 2014
Date of international publication Sep 4, 2014
Priority data
  • P2013-040097 (Feb 28, 2013) JP
  • 2014JP55160 (Feb 28, 2014) WO
Title Novel polypeptide exhibiting fluorescent properties and use thereof achieved
Abstract In order to provide a novel fluorescent protein and use thereof, the polypeptide according to the present invention has fluorescent properties in the presence of bilirubin and includes (1) the amino acid sequence of SEQ ID NO: 1, (2) an amino acid sequence having, for example, substitution of 1 to 21 amino acids in the amino acid sequence of SEQ ID NO: 1, (3) an amino acid sequence having 85% or more sequence identity to the amino acid sequence of SEQ ID NO: 1, or (4) the amino acid sequence encoded by a polynucleotide that hybridizes with a polynucleotide consisting of a sequence complementary to the polynucleotide encoding the polypeptide according to the amino acid sequence in (1) under a stringent condition.
Outline of related art and contending technology Background Art
Fluorescent proteins, such as green fluorescent protein (GFP), are indispensable as a tool for visualizing cells, tissue, biological individuals, and so on.
Most of fluorescent proteins have been isolated from invertebrates such as coral, sea anemones, and arthropods. However, for example, Non Patent Literatures 1 and 2 and Patent Literatures 1 and 2 report that a vertebrate, Japanese eel (Anguilla japonica), has a fluorescent protein.
Citation List
Patent Literature
Patent Literature 1: JP Patent Publication (Kokai) No. 2007-254371 A (Date of publication: October 4, 2007)
Patent Literature 2: JP Patent Publication (Kokai) No. 2008-141988 A (Date of publication: June 26, 2008)

Non Patent Literature
Non Patent Literature 1: Masao HONDA, Jinsuke KISHINO, Miyuki IMAMURA, Seiichi HAYASHI: Abstracts of the Fiscal Year Heisei 16 meeting of the Japanese Society of Fisheries Science (April 2, 2004, p. 203, 1101)
Non Patent Literature 2: Hayashi et al., Fisheries Science, 75, 1461-1469, 2009
Scope of claims [claim1]
1. A polypeptide-bilirubin complex constituted by bringing bilirubin into contact with a polypeptide or a fusion polypeptide in a form free from bilirubin, wherein the polypeptide is selected from the group consisting of the polypeptides of the following (1) to (3) having fluorescent properties in the presence of bilirubin, and the fusion polypeptide comprises the polypeptide selected from the group consisting of the polypeptides of the following (1) to (3) having fluorescent properties in the presence of bilirubin and another polypeptide:
(1) a polypeptide including the amino acid sequence of SEQ ID NO: 1;
(2) a polypeptide including an amino acid sequence having substitution, deletion, insertion, and/or addition of 1 to 21 amino acids in the amino acid sequence of SEQ ID NO: 1; and
(3) a polypeptide having 85% or more sequence identity to the amino acid sequence of SEQ ID NO: 1.

[claim2]
2. An in vitro method for detecting bilirubin in a biological or non-biological sample, the method comprising:
a contact step of bringing a polypeptide or a fusion polypeptide into contact with the biological or non-biological sample; and
a detection step of detecting fluorescence emitted by the polypeptide or the fusion polypeptide, after the contact step, wherein the polypeptide is selected from the group consisting of the polypeptides of the following (1) to (3) having fluorescent properties in the presence of bilirubin, and the fusion polypeptide comprises the polypeptide selected from the group consisting of the polypeptides of the following (1) to (3) having fluorescent properties in the presence of bilirubin and another polypeptide:
(1) a polypeptide including the amino acid sequence of SEQ ID NO: 1;
(2) a polypeptide including an amino acid sequence having substitution, deletion, insertion, and/or addition of 1 to 21 amino acids in the amino acid sequence of SEQ ID NO: 1; and
(3) a polypeptide having 85% or more sequence identity to the amino acid sequence of SEQ ID NO: 1.

[claim3]
3. The method according to claim 2, wherein
the biological or non-biological sample is a sample derived from blood or urine collected from a living body.

[claim4]
4. An in vitro method for inspecting the presence or absence of a predisposing factor or onset of liver disease or hemolytic disease in a biological or non-biological sample, the method comprising:
a contact step of bringing a polypeptide or a fusion polypeptide into contact with the biological or non-biological sample;
a detection step of detecting fluorescence emitted by the polypeptide or the fusion polypeptide, after the contact step, wherein the polypeptide is selected from the group consisting of the polypeptides of the following (1) to (3) having fluorescent properties in the presence of bilirubin, and the fusion polypeptide comprises the polypeptide selected from the group consisting of the polypeptides of the following (1) to (3) having fluorescent properties in the presence of bilirubin and another polypeptide:
(1) a polypeptide including the amino acid sequence of SEQ ID NO: 1;
(2) a polypeptide including an amino acid sequence having substitution, deletion, insertion, and/or addition of 1 to 21 amino acids in the amino acid sequence of SEQ ID NO: 1; and
(3) a polypeptide having 85% or more sequence identity to the amino acid sequence of SEQ ID NO: 1;
and
a testing step of inspecting the presence or absence of the predisposing factor or onset of liver disease or hemolytic disease based on the detection result in the detection step.

[claim5]
5. The method according to claim 4, wherein the biological or non-biological sample is a sample derived from blood or urine collected from a living body.

[claim6]
6. A bilirubin detecting kit comprising at least one selected from a polypeptide, a polynucleotide encoding the polypeptide, a recombinant vector comprising the polynucleotide, a transformant comprising the polynucleotide or the recombinant vector, and a fusion polypeptide; and at least one selected from a reagent and/or a tool for detecting bilirubin, a reagent and/or a tool for preparing a sample for detection, a manual of the detection kit, a sample for a control in detection, and reference data for analyzing a detection result,
wherein the polypeptide is selected from the group consisting of the polypeptides of the following (1) to (3) having fluorescent properties in the presence of bilirubin, and the fusion polypeptide comprises the polypeptide selected from the group consisting of the polypeptides of the following (1) to (3) having fluorescent properties in the presence of bilirubin and another polypeptide:
(1) a polypeptide including the amino acid sequence of SEQ ID NO: 1;
(2) a polypeptide including an amino acid sequence having substitution, deletion, insertion, and/or addition of 1 to 21 amino acids in the amino acid sequence of SEQ ID NO: 1; and
(3) a polypeptide having 85% or more sequence identity to the amino acid sequence of SEQ ID NO: 1.
  • Applicant
  • RIKEN
  • Inventor
  • MIYAWAKI, Atsushi
  • KUMAGAI, Akiko
IPC(International Patent Classification)
Specified countries Contracting States: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

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