NOVEL POLYPEPTIDE EXHIBITING FLUORESCENT PROPERTIES AND USE THEREOF

• US 20160009771A1
• Filed: 02/28/2014
• Published: 01/14/2016
• Est. Priority Date: 02/28/2013
• Status: Active Grant

• Current Assignee: Riken
• Original Assignee: Riken
• Inventors: MIYAWAKI, Atsushi, KUMAGAI, Akiko
• Granted Patent: US 9,957,308 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C07K 14/00 Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• CPC Class Codes: C07K 14/461 from fish
• CPC Class Codes: C07K 2319/60 containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: G01N 2800/085 Liver diseases, e.g. portal hypertension, fibrosis, cirrhosis, bilirubin
• CPC Class Codes: G01N 2800/22 Haematology
• CPC Class Codes: G01N 33/72 involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood

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.

Claims

1. A polypeptide shown in any of the following (1) to (4) and having fluorescent properties in the presence of bilirubin:

(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;

(3) a polypeptide having 85% or more sequence identity to the amino acid sequence of SEQ ID NO;

1; and

(4) a polypeptide including an amino acid sequence encoded by a polynucleotide that hybridizes with a polynucleotide consisting of the sequence complementary to a polynucleotide encoding the polypeptide according to the above (1) under a stringent condition.

3. The polypeptide according to claim 1, wherein the polypeptide has a dissociation constant for bilirubin of 0.1 nM or more and 100 nM or less.

6. A fusion polypeptide comprising the polypeptide according to claim 1 and another polypeptide.

7. A polypeptide-bilirubin complex constituted by bringing bilirubin into contact with the polypeptide according to claim 1 in a form free from bilirubin.

8. A method for detecting bilirubin in a subject, the method comprising:

a contact step of bringing the polypeptide according to claim 1, into contact with the subject; and

a detection step of detecting fluorescence emitted by the polypeptide or the fusion polypeptide, after the contact step.

9. The method according to claim 8, wherein

the subject is a sample derived from blood or urine collected from a living body.

10. The method according to claim 8, 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 detection step.

11. A bilirubin detecting kit comprising a polypeptide according to claim 1.

13. A method for detecting bilirubin in a subject, the method comprising:

a contact step of bringing the fusion polypeptide according to claim 6 into contact with the subject; and

a detection step of detecting fluorescence emitted by the polypeptide or the fusion polypeptide, after the contact step.

15. A bilirubin detecting kit comprising the polypeptide according to claim 3.

18. A bilirubin detecting kit comprising the fusion polypeptide according to claim 6.

2. A polynucleotide shown in any of the following (1) to (4):

(1) a polynucleotide encoding a polypeptide including the amino acid sequence of SEQ ID NO;

1;

(2) a polynucleotide encoding 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 having fluorescent properties in the presence of bilirubin;

(3) a polynucleotide encoding a polypeptide having 85% or more sequence identity to the amino acid sequence of SEQ ID NO;

1 and having fluorescent properties in the presence of bilirubin; and

(4) a polynucleotide hybridizing with a polynucleotide consisting of a sequence complementary to the polynucleotide according to the above (1) under a stringent condition and encoding a polypeptide having fluorescent properties in the presence of bilirubin.

4. A recombinant vector comprising the polynucleotide according to claim 2.

5. A transformant comprising the polynucleotide according to claim 2.

12. A transformant comprising the recombinant vector according to claim 4.

14. A bilirubin detecting kit comprising the polynucleotide according to claim 2.

16. A bilirubin detecting kit comprising the recombinant vector according to claim 4.

17. A bilirubin detecting kit comprising the transformant according to claim 5.