Disordered protein-based seeds for molecular clustering

Patent metadata

Field	Value
Publication number	US-10538756-B2
Application number	US-201715618361-A
Country	US
Kind code	B2
Filing date	Jun 9, 2017
Priority date	Mar 6, 2017
Publication date	Jan 21, 2020
Grant date	Jan 21, 2020

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

What is claimed is:

1. A system, comprising:
a first protein construct comprising at least one self-assembling protein subunit fused to at least one light-sensitive receptor protein capable of binding to a cognate partner; and
a second protein construct comprising the cognate partner of the light-sensitive receptor protein fused to a full length or truncated low complexity or intrinsically-disordered protein region.

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

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

4. The system according to claim 1 , wherein the at least one light-sensitive receptor protein capable of binding to a cognate partner is an engineered protein.

5. The system according to claim 4 , wherein the engineered protein is iLID.

6. The system according to claim 4 , wherein the self-assembling protein subunit is fused to two iLID proteins.

7. The system according to claim 1 , wherein the light-sensitive receptor protein capable of binding to a cognate partner is ssrA and the cognate partner of the light-sensitive receptor protein is sspB.

8. The system according to claim 1 , wherein the intrinsically-disordered protein region is selected from the group consisting of FUS or FUSn.

9. The system of claim 1 , wherein the light-sensitive receptor protein capable of binding to a cognate partner is sensitive to at least one visible wavelength of light.

10. The system according to claim 1 , wherein the second protein construct further comprises a fluorescent tag.

11. A cell line that produces the system of claim 1 .

12. A method for forming an assembled phase, comprising the steps of:
producing at least one self-assembling protein subunit fused to at least one light-sensitive receptor protein capable of binding to a cognate partner;
producing a cognate partner of the at least one light-sensitive receptor protein fused to a full length or truncated intrinsically-disordered protein region and a target protein;
forming at least one assembled phase comprising the at least one self-assembling protein subunit fused to the at least one light-sensitive receptor protein and the cognate partner of the at least one light-sensitive receptor protein fused to the full length or truncated intrinsically-disordered protein region and the target protein, wherein the at least one light sensitive receptor protein is bound to the cognate partner by exposing the at least one light sensitive receptor protein to at least one predetermined wavelength of light.

13. The method according to claim 12 , wherein the formation of the assembled phase occurs within a living cell comprising pathological protein aggregates.

14. The method according to claim 13 , wherein the pathological protein aggregates comprise amyloid fibers.

15. The method according to claim 12 , further comprising separating the at least one self-assembling protein subunit fused to the at least one light-sensitive receptor protein bound to the cognate partner of the at least one light-sensitive receptor protein fused to the full length or truncated intrinsically-disordered protein region and the target protein via at least one of a centrifuge or a magnetic field.

16. The method according to claim 12 , wherein the cognate partner of the at least one light-sensitive receptor protein fused to a full length or truncated intrinsically-disordered protein region and a target protein includes a cleavage tag between the target protein and the intrinsically disordered protein region.

17. The method according to claim 16 , 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).

18. The method according to claim 16 , wherein the cleavage tag is a self cleaving tag.

19. The method according to claim 12 , further comprising the steps of:
cleaving the target protein from the intrinsically-disordered protein region.

20. A kit for inducing in vivo liquid-liquid phase separation, comprising:
a plasmid encoding for a first and second protein construct; and
at least one light emitting device configured to activate at least a portion of the first protein construct,
wherein the first protein construct comprising at least one self-assembling protein subunit fused to at least one light-sensitive receptor protein capable of binding to a cognate partner, and
wherein the second protein construct comprises the cognate partner of the light-sensitive receptor protein fused to a full length or truncated intrinsically disordered protein.

Assignees

• Univ Princeton

Inventors

• Brangwynne Cliff
• Bracha Dan

Classifications

• 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

• C07K1/14 Primary

  Extraction; Separation; Purification · CPC title

• C12N11/02 Primary

  Enzymes or microbial cells immobilised on or in an organic carrier · CPC title

• C07K2319/60

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