Cyan-excitable orange-red fluorescent proteins and bioluminescent resonance energy transfer systems

Patent metadata

Field	Value
Publication number	US-2016376332-A1
Application number	US-201615197772-A
Country	US
Kind code	A1
Filing date	Jun 30, 2016
Priority date	Jun 30, 2015
Publication date	Dec 29, 2016
Grant date	—

Abstract

Engineered orange-red fluorescent proteins with enhanced fluorescent properties, obtained by mutagenesis of mNeptune2, are disclosed. In particular, the invention relates to engineered orange-red fluorescent proteins excitable with cyan light having increased emission intensity and their use in bioluminescent resonance energy transfer systems and fluorescence and bioluminescence imaging.

First claim

What is claimed is:

1 . An orange-red fluorescent protein comprising a polypeptide selected from the group consisting of:
a) a polypeptide comprising the amino acid sequence of SEQ ID NO:1; and b) a polypeptide comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:1, wherein the orange-red fluorescent protein emits light in response to absorption of cyan excitation light.

2 . A fusion protein comprising the orange-red fluorescent protein of claim 1 connected to a polypeptide of interest.

3 . A fusion protein comprising the orange-red fluorescent protein of claim 1 connected to a targeting sequence directing localization to a specific cellular compartment or protein.

4 . The fusion protein of claim 2 , further comprising a targeting sequence directing localization to a specific subcellular compartment or protein.

5 . A bioluminescent fusion protein comprising at least one orange-red fluorescent protein according to claim 1 connected to at least one luciferase, wherein the orange-red fluorescent protein or proteins are operably linked to the luciferase or luciferases to allow bioluminescence resonance energy transfer (BRET) from a luciferase reaction product to the orange-red fluorescent protein or proteins upon reaction of a chemiluminescent substrate by the luciferase or luciferases.

6 . The bioluminescent fusion protein of claim 5 , wherein the luciferase or luciferases comprise a polypeptide selected from the group consisting of:
a) a polypeptide comprising the amino acid sequence of SEQ ID NO:3; and b) a polypeptide comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:3, wherein the orange-red fluorescent protein emits light in response to absorption of cyan excitation light.

7 . The bioluminescent fusion protein of claim 6 comprising a polypeptide selected from the group consisting of:
a) a polypeptide comprising the amino acid sequence of SEQ ID NO:2; and
b) a polypeptide comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:2, wherein the orange-red fluorescent protein emits light in response to absorption of cyan excitation light.

8 . The bioluminescent fusion protein of claim 5 , wherein the targeting sequence directs localization of the fusion protein to a specific tissue, cell-type, cellular compartment, or protein.

9 . A polynucleotide encoding the orange-red fluorescent protein of claim 1 .

10 . A recombinant polynucleotide comprising the polynucleotide of claim 9 operably linked to a transcriptional regulatory region selected from the group consisting of:
a) a promoter with constitutive activity; and
b) a promoter with cell-specific or tissue-specific activity; and
c) a promoter whose activity is regulated by biochemical signals in the cell.

11 . A host cell comprising the recombinant polynucleotide of claim 10 .

12 . A polynucleotide encoding the bioluminescent fusion protein of claim 5 .

13 . A recombinant polynucleotide comprising the polynucleotide of claim 12 operably linked to a transcriptional regulatory region selected from the group consisting of:
a) a promoter with constitutive activity; and
b) a promoter with cell-specific or tissue-specific activity; and
c) a promoter whose activity is regulated by biochemical signals in the cell.

14 . A host cell comprising the recombinant polynucleotide of claim 13 .

15 . A method of using the orange-red fluorescent protein of claim 1 for fluorescence reporting, the method comprising:
a) introducing the orange-red fluorescent protein of claim 1 into a cell or animal by injection of the protein or introduction of a vector expressing the protein; and
b) illuminating the cell or animal with light that produces fluorescence from the protein; and
c) detecting fluorescence emitted by the protein.

16 . The method of claim 15 , wherein a fluorescence image is visualized with an optical microscope, a digital microscope, a charged coupled device (CCD) image sensor, a complementary metal-oxide-semiconductor (CMOS) image sensor, a digital camera, a fiber-optic imaging system, or a medical imaging system.

17 . The method of claim 15 , further comprising:
a) introducing both a green fluorescent protein and the orange-red fluorescent protein of claim 1 into a cell or animal, such that either protein is introduced by injection or expression from a vector; and b) illuminating the cell or animal with light that produces fluorescence from both the orange-red fluorescent protein and the green fluorescent protein simultaneously; and c) recording the orange-red fluorescence emitted by the orange-red fluorescent protein and green fluorescence emitted by the green fluorescent protein.

18 . The method of claim 17 , wherein a fluorescence image is visualized with an optical microscope, a digital microscope, a charged coupled device (CCD) image sensor, a complementary metal-oxide-semiconductor (CMOS) image sensor, a digital camera, a fiber-optic imaging system, or a medical imaging system.

19 . A method for using the bioluminescent fusion protein of claim 5 for bioluminescence reporting, the method comprising:
a) introducing the bioluminescent fusion protein of claim 5 into a cell or animal by injection of the protein or introduction of a vector expressing the protein; and
b) contacting the bioluminescent fusion protein with a chemiluminescent substrate in the cell or animal; and
c) recording bioluminescence from the cell or animal by detecting bioluminescence emitted from the fusion protein.

20 . The method of claim 19 , wherein a bioluminescence image is visualized with an optical microscope, a digital microscope, a charged coupled device (CCD) image sensor, a complementary metal-oxide-semiconductor (CMOS) image sensor, a digital camera, a fiber-optic imaging system, or a medical imaging system.

Assignees

• Lin Michael
• Chu Jun
• Oh Younghee
• Univ Leland Stanford Junior

Inventors

• Lin Michael
• Chu Jun
• Oh Younghee

Classifications

• G01N33/582

  with fluorescent label · CPC title

• G01N21/6458

  Fluorescence microscopy (fluorescence microscopes per se G02B21/0076 and G02B21/16) · CPC title

• G01N21/6428

  Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53) · CPC title

• C12Q1/66

  involving luciferase · CPC title

• G01N21/763

  Bioluminescence · CPC title