Optogenetic tool for rapid and reversible clustering of proteins

Patent metadata

Field	Value
Publication number	US-11053491-B2
Application number	US-201916701399-A
Country	US
Kind code	B2
Filing date	Dec 3, 2019
Priority date	Jun 9, 2016
Publication date	Jul 6, 2021
Grant date	Jul 6, 2021

Abstract

A protein construct including a gene encoding a light-sensitive protein fused to at least one of either a low complexity sequence, an intrinsically disordered protein region (IDR), or a repeating sequence of a linker and another gene encoding a light-sensitive protein. Among the many different possibilities contemplated, the protein construct may also advantageously include cleavage tags. This protein construct may be utilized for a variety of functions, including a method for protein purification, which requires introducing the protein construct into a living cell, and inducing the formation of clusters by irradiating the construct with light. The method may also advantageously include cleaving a target protein from an IDR, and separating the clusters via centrifuge. A kit for practicing in vivo aggregation or liquid-liquid phase separation is also included, the kit including the protein construct and a light source capable of producing a wavelength that the light-sensitive protein will respond to.

First claim

What is claimed is:

1. A method for forming irreversible aggregates within a cell, comprising the steps of:
a) providing a cell with polynucleic acid encoding a protein construct, wherein the protein construct comprises:
a first segment comprising at least one protein sensitive to at least one wavelength of light; and
a second segment fused to the first segment, the second segment comprising at least one intrinsically disordered protein region (IDR), and
wherein said first segment and said second segment are heterologous;
b) culturing the cell under conditions which will result in the expression of the protein construct within the cell; and
c) inducing the protein construct to cluster and form an irreversible aggregate by repeatedly exposing the protein construct within the cell to the at least one wavelength of light.

2. The method according to claim 1 , wherein inducing the protein construct to cluster changes the physiology of the cell by modifying transport or reactivity of molecules with the cell.

3. The method according to claim 1 , wherein inducing the protein construct to cluster changes physiology of the cell by causing intermolecular interactions, protein activation or inactivation, manipulation of signaling pathways, or gene expression clusters within the cell.

4. The method according to claim 1 , further comprising lysing the cell and separating the irreversible aggregate via centrifuge.

5. The method according to claim 1 , wherein the protein construct further comprises a cleavage tag.

6. The method according to claim 5 , 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).

7. The method according to claim 5 , wherein the cleavage tag is a sell-cleaving tag.

8. The method according to claim 1 , further comprising expressing a LacO array or dCas9 in the cell.

9. The method according to claim 1 , wherein the IDR is FUS.

10. The method according to claim 1 , wherein the IDR is Ddx4.

11. The method according to claim 1 , wherein the TDR is hnRNPA4.

12. The method according to claim 1 , wherein the at least one protein sensitive to at least one wavelength of light is Cry2.

13. The method according to claim 1 , wherein the at least one protein sensitive to at least one wavelength of light is Cry2olig.

14. The method according to claim 1 , wherein the at least one protein sensitive to at least one wavelength of light is PhyB.

15. The method according to claim 1 , wherein the at least one protein sensitive to at least one wavelength of light is PIF.

16. The method according to claim 1 , wherein the at least one protein sensitive to at least one wavelength of light is a light oxygen voltage sensing (LOV) domain.

17. The method according to claim 1 , wherein the at least one protein sensitive to at least one wavelength of light is Dronpa.

Assignees

• Univ Princeton

Inventors

• Brangwynne Cliff
• Toettcher Jared
• Shin Yongdae

Classifications

• C07K2319/50

  containing protease site · CPC title

• C12N9/88 Primary

  Lyases (4.) · CPC title

• C12N15/62

  DNA sequences coding for fusion proteins · CPC title

• C12Y401/99003

  Deoxyribodipyrimidine photo-lyase (4.1.99.3) · CPC title

• C07K2319/60

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