MAM-specific fluorescence calcium sensor and use thereof

Patent metadata

Field	Value
Publication number	US-11474108-B2
Application number	US-201716316087-A
Country	US
Kind code	B2
Filing date	Nov 22, 2017
Priority date	Nov 25, 2016
Publication date	Oct 18, 2022
Grant date	Oct 18, 2022

Abstract

The present disclosure relates to a Mitochondria-Associated endoplasmic reticulum Membrane (MAM)-specific fluorescence calcium sensor and the use thereof. The present disclosure can surmount the limitations of a conventional technique in that verification of calcium migration through MAM requires separate measurements of calcium ion concentrations within ER and mitochondria and situational explanations of the phenomena, and can directly measure concentrations in the paths through which calcium ions move to exclude influences on calcium ion changes through numerous different calcium ion channels existing in mitochondria, thereby providing a convenient and accurate MAM-specific calcium ion sensor.

First claim

The invention claimed is:

1. A Mitochondria-Associated endoplasmic reticulum Membrane(MAM)-specific fluorescent calcium sensor comprising the following structures: (a) a first fluorescent complementary structure in which a linker peptide and a fragment of a calcium ion-sensitive fluorescent sensor protein sequentially bind to a fragment of an Endoplasmic Reticulum(ER)-targeting protein, and (b) a second fluorescent complementary structure in which a linker peptide and a fragment of a calcium ion-sensitive fluorescent sensor protein sequentially bind to a fragment of a mitochondria-targeting protein,
wherein the calcium ion-sensitive fluorescent sensor protein bound to the fragment of the Endoplasmic Reticulum(ER)-targeting protein is a split GCaMP protein which is encoded by a polynucleotide consisting of a base sequence of SEQ ID NO: 6, and
wherein the calcium ion-sensitive fluorescent sensor protein bound to the fragment of the mitochondria-targeting protein is a split GCaMP protein which is encoded by a polynucleotide consisting of a base sequence of SEQ ID NO: 5.

2. The sensor according to claim 1 , wherein the ER-targeting protein is suppressor of actin 1 (SAC1).

3. The sensor according to claim 2 , wherein a fragment of the SAC1 protein consists of amino acids 521 to 587 of a full-length SAC1 protein.

4. The sensor according to claim 1 , wherein the mitochondria-targeting protein is A Kinase Anchoring Protein 1(AKAP1).

5. The sensor according to claim 4 , wherein a fragment of the AKAP1 protein consists of amino acids 34 to 63 of a full-length AKAP1 protein.

6. The sensor according to claim 1 , wherein the mitochondria-targeting protein is Mitofusin 1(MFN1).

7. The sensor according to claim 3 , wherein the fragment of the SAC1 protein is encoded by a polynucleotide consisting of a base sequence of SEQ ID NO: 1.

8. The sensor according to claim 5 , wherein the fragment of the AKAP1 protein is encoded by a polynucleotide consisting of a base sequence of SEQ ID NO: 2.

9. The sensor according to claim 6 , wherein the MFN1 protein is encoded by a polynucleotide consisting of a base sequence of SEQ ID NO: 3.

10. The sensor according to claim 1 , wherein the linker peptide is encoded by a polynucleotide consisting of 1 to 8 repeats of a base sequence of SEQ ID NO: 4.

11. The sensor according to claim 10 , wherein the linker peptide is encoded by a polynucleotide consisting of 2 to 4 repeats of the base sequence of SEQ ID NO: 4.

12. A method of sensing Mitochondria-Associated endoplasmic reticulum Membrane (MAM)-specific calcium using the sensor of claim 1 .

Assignees

• Postech Acad Ind Found
• Postech Academy—Industry Found

Inventors

• Park Sang Ki
• Suh Yeong Jun
• Kim Seung Hyun

Classifications

• G01N33/84

  involving inorganic compounds or pH · CPC title

• G01N2458/00

  Labels used in chemical analysis of biological material · CPC title

• G01N33/6803

  General methods of protein analysis not limited to specific proteins or families of proteins · 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

• G01N33/68

  involving proteins, peptides or amino acids {(involving lipoproteins G01N33/92)} · CPC title