Disordered protein-based seeds for molecular clustering

Patent metadata

Field	Value
Publication number	US-11053492-B2
Application number	US-201916704115-A
Country	US
Kind code	B2
Filing date	Dec 5, 2019
Priority date	Mar 6, 2017
Publication date	Jul 6, 2021
Grant date	Jul 6, 2021

Abstract

A system and method for reversibly controlling clustering of proteins around an engineered multivalent nucleus is disclosed. The system and method utilize clustering, which may be controlled by light activation or deactivation. The system and method enable the spatiotemporal control of protein supramolecular assemblies, including liquid-like droplets under some conditions, and solid-like gels under other conditions. The system and method can be utilized for segregating or locally concentrating desired proteins and/or RNA in cells or cell lysate, which may be useful for protein purification purposes, or for assembling single or multiple membraneless bodies within specific sub-regions of the cells. These synthetically assembled bodies may recruit both transgenic and endogenic proteins and other biomolecules, thus can be linked to affect and even trigger a plethora of cellular processes, including both physiological and pathological (e.g., protein aggregation) processes.

First claim

The invention claimed is:

1. A composition comprising a plurality of fusion proteins, wherein each fusion protein of the plurality of fusion proteins comprising at least one self-assembling protein, a full-length or truncated low complexity or intrinsically disordered protein region, and a target protein,
wherein the at least one self-assembling protein and the low complexity or intrinsically disordered protein region are heterologous,
wherein the target protein is attached to a C-terminus of the low complexity or intrinsically disordered protein region, and
wherein the plurality of fusion proteins are configured to form phase separated clusters.

2. The composition according to claim 1 , wherein the self-assembling protein is ferritin.

3. The composition according to claim 2 , wherein the ferritin is a ferritin heavy or light chain.

4. The composition according to claim 1 , wherein the low complexity or intrinsically disordered protein region is selected from the group consisting of FUS or FUSn.

5. The composition according to claim 1 , further comprising a fluorescent tag.

6. The composition according to claim 1 , further comprising a cleavage tag.

7. The composition according to claim 6 , wherein the cleavage tag is selected from the group consisting of: Human Rhinovirus 3C Protease (3C/PreScission), Enterokinase (EKT), Factor Xa (FXa), Tobacco Etch Virus Protease (TEV), and Thrombin (Thr).

8. The composition according to claim 6 , wherein the cleavage tag is a self-cleaving tag.

9. A cell line that is capable of expressing a plurality of fusion proteins, wherein each fusion protein of the plurality of fusion proteins comprising at least one self-assembling protein, a full-length or truncated low complexity or intrinsically disordered protein region, and a target protein,
wherein the at least one self-assembling protein and the low complexity or intrinsically disordered protein region are heterologous,
wherein the target protein is attached to a C-terminus of the low complexity or intrinsically disordered protein region.

10. A method for forming phase separated clusters in a living cell, comprising the steps of:
expressing within a living cell, a plurality of fusion proteins, wherein each fusion protein of the plurality of fusion proteins comprising at least one self-assembling protein, a full-length or truncated low complexity or intrinsically disordered protein region, and a target protein,
wherein the at least one self-assembling protein and the low complexity or intrinsically disordered protein region are heterologous,
wherein the target protein is attached to a C-terminus of the low complexity or intrinsically disordered protein region; and
allowing the plurality of fusion proteins to undergo phase separation into at least one condensed phase within the living cell, the at least one condensed phase comprising the plurality of fusion proteins, and form at least one phase separated cluster.

11. The method according to claim 10 , wherein the condensed phase in the living cell further comprises amyloid fibers.

12. The method according to claim 10 , wherein the fusion proteins further comprises a cleavage tag.

13. The method according to claim 12 , wherein the cleavage tag is selected from the group consisting of: Human Rhinovirus 3C Protease (3C/PreScission), Enterokinase (EKT), Factor Xa (FXa), Tobacco Etch Virus Protease (TEV), and Thrombin (Thr).

14. The method according to claim 12 , wherein the cleavage tag is a self-cleaving tag.

15. The method according to claim 10 , wherein the at least one phase separated cluster has a diameter of at least 0.3 micrometer.

16. The method according to claim 10 , wherein the fusion proteins are present in the at least one phase separated cluster in a first concentration, the fusion proteins are present outside the at least one phase separated cluster in a second concentration, the first concentration being at least 100 times greater than the second concentration.

Assignees

• Univ Princeton

Inventors

• Brangwynne Cliff
• Bracha Dan

Classifications

• C07K1/14 Primary

  Extraction; Separation; Purification · CPC title

• C07K2319/00

  Fusion polypeptide · CPC title

• C07K2319/735

  containing a domain for self-assembly, e.g. a viral coat protein (includes phage display) · CPC title

• C07K2319/50

  containing protease site · CPC title

• C07K2319/60

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