Fluorescent separase activity sensor

Patent metadata

Field	Value
Publication number	US-10174359-B2
Application number	US-201314413760-A
Country	US
Kind code	B2
Filing date	Jul 10, 2013
Priority date	Jul 11, 2012
Publication date	Jan 8, 2019
Grant date	Jan 8, 2019

Abstract

Problem to be Solved
An object of the present invention is to provide a separase sensor for visualizing separase activity in a living cell. Another object is to develop a function analysis system of cell division using the separase sensor and further obtain a simple screening method for an anticancer agent.
Solution
The separase sensor of the present invention has two types of fluorescent proteins different in fluorescence wavelength at both ends of an amino acid sequence containing a separase cleavage site and a localization-targeting sequence targeting to a specific site within a cell. Since timing of activating separase and location of separase in a cell can be specified by use of the sensor, cell division can be analyzed and an anticancer agent can be screened.

First claim

The invention claimed is:

1. A biosensor for measuring and/or detecting separase activity in cultured cells, comprising:
a first fluorescent substance at a first end of the fragment of human Scc1, said fragment consisting of the polypeptide of SEQ ID NO. 1;
a second fluorescent substance at the second end of the fragment consisting of the polypeptide of SEQ ID NO. 1: and
a localization-targeting sequence for localizing the biosensor at a localization site within the cultured cells, said localization-targeting sequence at the N- or C-terminus of the biosensor:
wherein the localization-targeting sequence is not a sequence derived from Scc1;
wherein the first fluorescent substance and the second fluorescent substance have non-overlapping emission wavelengths that do not cause excitation of each other; and
wherein the biosensor is a separase sensor configured to detect and/or measure separase activity at the localization site within the cultured cells as a function of a change in emission fluorescence of the biosensor resulting from cleavage of the fragment consisting of the polypeptide of SEQ ID NO. 1 by separase.

2. The biosensor according to claim 1 , wherein the first and the second fluorescent substances are fluorescent proteins.

3. The biosensor according to claim 2 , wherein the localization-targeting sequence specifically localizes the biosensor at centromeres.

4. The biosensor according to claim 3 , wherein the localization-targeting sequence for centromeres is CENP-B.

5. A method to visualize and analyze proteolytic activity of separase in the context of chromosomal segregation in living cells, by transfected vector-derived separase sensor according to claim 4 .

6. A method for screening anticancer agents, comprising contacting test substances to the separase sensor according to claim 1 , visualizing the separase activity, and screening anticancer agents based on separase activity as an index.

7. The biosensor according to claim 2 , wherein the localization-targeting sequence specifically localizes the biosensor at chromosomes.

8. The biosensor according to claim 2 , wherein the localization-targeting sequence specifically localizes the biosensor at cytoplasmic membrane.

9. The biosensor according to claim 2 , wherein the localization-targeting sequence specifically localizes the biosensor at mitochondria.

10. The biosensor according to claim 7 , wherein the localization-targeting sequence for chromosomes is histone H2B.

Assignees

• Japanese Found For Cancer Res

Inventors

• Hirota Toru
• Shindo Norihisa
• Kumada Kazuki

Classifications

• C12Y304/22049

  Separase (3.4.22.49) · CPC title

• C12N9/6472

  Cysteine endopeptidases (3.4.22) · CPC title

• C07K2319/50

  containing protease site · CPC title

• C07K2319/60

  containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP] · CPC title

• G01N2500/04

  Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A) · CPC title