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Method for detecting bilirubin using a fluorescent protein UPDATE_EN achieved

Foreign code F190009911
File No. 08118-US
Posted date Aug 26, 2019
Country United States of America
Application number 201414771060
Gazette No. 20160009771
Gazette No. 9957308
Date of filing Feb 28, 2014
Gazette Date Jan 14, 2016
Gazette Date May 1, 2018
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 Method for detecting bilirubin using a fluorescent protein UPDATE_EN 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 amine 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.
Scope of claims [claim1]
1. A method for detecting bilirubin in a subject, the method comprising:
contacting a sample from the subject with a polypeptide, wherein the polypeptide has fluorescent activity upon binding to bilirubin; and
detecting fluorescence emitted by the polypeptide after the contacting step,
wherein the presence of fluorescence emission detects bilirubin in the subject, and
wherein the polypeptide is selected from the group consisting of;
(1) a polypeptide comprising the amino acid sequence of SEQ ID NO: 1;
(2) a polypeptide comprising an amino acid sequence that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1;
(3) a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, except for an amino substitution selected from the group consisting of Al2E, S80N, R82E, K84E, and combinations thereof in the amino acid sequence of SEQ ID NO: 1; and
(4) a polypeptide comprising the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 29.

[claim2]
2. The method according to claim 1, wherein the sample is derived from blood or urine collected from the subject.

[claim3]
3. The method according to claim 1, further comprising:
a testing step of inspecting the presence or absence of a predisposing factor or onset of liver disease or hemolytic disease based on a detection result in the detecting step.

[claim4]
4. The method according to claim 1, wherein the polypeptide is a fusion polypeptide.

[claim5]
5. The method according to claim 1, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 29, or SEQ ID NO: 1 except for an amino acid substitution selected from the group consisting of Al2E, S80N, R82E K84E and combinations thereof in the amino acid sequence of SEQ ID NO: 1.

[claim6]
6. The method according to claim 1, wherein the subject is a human, or is a nonhuman animal.

[claim7]
7. The method according to claim 1, where the polypeptide comprises the amino acid sequence of SEQ ID NO: 1.

[claim8]
8. The method according to claim 1, where the polypeptide comprises an amino acid sequence that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1.

[claim9]
9. The method according to claim 1, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 1, except for an amino acid substitution selected from the group consisting of Al2E, S80N, R82E, K84E, and combinations thereof in the amino acid sequence of SEQ ID NO: 1.

[claim10]
10. The method according to claim 1, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 29.
  • Inventor, and Inventor/Applicant
  • MIYAWAKI Atsushi
  • KUMAGAI Akiko
  • RIKEN
IPC(International Patent Classification)

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