Programmable protein circuits in living cells

Patent metadata

Field	Value
Publication number	US-11965191-B2
Application number	US-202117157880-A
Country	US
Kind code	B2
Filing date	Jan 25, 2021
Priority date	Jan 18, 2018
Publication date	Apr 23, 2024
Grant date	Apr 23, 2024

Abstract

The present disclosure relates to systems, methods and compositions provided herein that include a compound protease. The compound protease can contain a protease domain, a cut site for another enzyme and an association domain. In some embodiments, the compound protease is part of a protein circuit.

First claim

What is claimed is:

1. A synthetic protein circuit, comprising:
a first protease, optionally comprising an association domain of the first protease;
a second protease, optionally comprising a complementary association domain of the second protease; and
a first target protein comprising a first degron of the first target protein that destabilizes the first target protein when present on the first target protein by enhancing degradation of the first target protein; wherein the first target protein is configured to interact with the first protease, the second protease, a third protease and/or a fourth protease to form an OR, AND, NAND, IMPLY, NIMPLY, XOR or XNOR logic gate.

2. The synthetic protein circuit of claim 1 , wherein the first target protein further comprises a first cut site specific for the first protease and a second cut site specific for the second protease between the first degron of the first target protein and a distal part of the first target protein from the first degron, and wherein the first target protein is stabilized by cleavage of either of the first cut site and the second cut site.

3. The synthetic protein circuit of claim 1 , wherein the first target protein further comprises a first cut site of the first target protein specific for the first protease between the first degron of the first target protein and a distal part of the first target protein from the first degron, and a second cut site specific for the second protease connected to a second degron of the first target protein and an optional association domain of the first target protein, and wherein the first target protein is stabilized by cleavage of both of the first cut site and the second cut site.

4. The synthetic protein circuit of claim 1 ,
wherein the third protease comprises:
a first cut site specific for the first protease,
a second cut site specific for the second protease, and
an optional association domain of the third protease,
wherein the third protease is configured to be deactivated by cleavage of either of the first cut site specific for the first protease and the second cut site specific for the second protease;

wherein the first target protein comprises a first cut site specific for the third protease between the first degron of the first target protein and a distal part of the first target protein from the first degron, and wherein the first target protein is stabilized by cleavage of the first cut site specific for the third protease.

5. The synthetic protein circuit of claim 4 , wherein the third protease further comprises a first domain of the third protease and a second domain of the third protease;
wherein the first domain of the third protease comprises third protease first and second cut sites specific for the first and second proteases respectively, and wherein the third protease further comprises an optional association domain;
wherein the second domain of the third protease comprises a third protease third cut site specific for the first protease, a third protease fourth cut site specific for the second protease, and an optional complementary association domain of the third protease; and
wherein the third protease is configured to be deactivated by cleavage of any of the third protease cut sites.

6. The synthetic protein circuit of claim 1 , wherein
the third protease comprises a third protease first cut site specific for the first protease, and configured to be deactivated by cleavage of the third protease first cut site; and
the fourth protease comprises a fourth protease first cut site specific for the second protease, and configured to be deactivated by cleavage of the fourth protease first cut site;
wherein the first target protein comprises a first cut site specific for the third protease and a second cut site specific for the fourth protease between the first degron of the first target protein and a distal part of the first target protein from the first degron, and wherein the first target protein is stabilized by cleavage of the first cut site specific for the third protease and the second cut site specific for the fourth protease.

7. The synthetic protein circuit of claim 6 , wherein the third protease further comprises a first domain of the third protease, a second domain of the third protease, and wherein the third protease comprises an optional complementary association domain;
wherein the first domain of the third protease comprises the third protease first cut site specific for the first protease;
wherein the second domain of the third protease comprises a third protease second cut site specific for the first protease;
wherein the complementary association domain of the third protease optionally comprises a first part of the third protease connected to the third protease first cut site and a second part of the third protease connect to the third protease second cut site; and
wherein the third protease is configured to be deactivated by cleavage of either of the third protease first cut site and the third protease second cut site.

8. The synthetic protein circuit of claim 6 , wherein the fourth protease further comprises a first domain of the fourth protease, a second domain of the fourth protease, and an optional association domain of the fourth protease;
wherein the first domain of the fourth protease comprises the fourth protease first cut site specific for the second protease;
wherein the second domain of the fourth protease comprises a fourth protease second cut site specific for the second protease;
wherein the association domain of the fourth protease optionally comprises a first part of the fourth protease connected to the fourth protease first cut site and a second part of the fourth protease connected to the fourth protease second cut site; and
wherein the fourth protease is configured to be deactivated by cleavage of either of the fourth protease first cut site and the fourth protease second cut site.

9. The synthetic protein circuit of claim 1 wherein
the third protease comprises a third protease first cut site specific for the second protease, and configured to be deactivated by cleavage of the third protease first cut site;
wherein the first target protein further comprises a first cut site specific for the first protease and a second cut site specific for the third protease between the first degron of the first target protein and a distal part of the first target protein from the first degron of the first target protein, and wherein the first target protein is stabilized by cleavage of the first cut site specific for the first protease or the second cut site specific for the third protease.

10. The synthetic protein circuit of claim 9 , wherein the third protease further comprises a first domain, a second domain, and an optional association domain;
wherein the first domain of the third protease comprises the third protease first cut site specific for the second protease;
wherein the second domain of the third protease comprises a third protease second cut site specific for the second protease;
wherein the association domain of the third protease optionally comprises a first part of the third protease connected to the third protease first cut site and a second part of the third protease connected to the third protease second cut site; and
wherein the third protease is configured to be deactivated by cleavage of the third protease first cut site or the third protease second cut site.

11. The synthetic protein circuit of claim 1
wherein the third protease comprises a third protease first cut site specific for the first protease, and configured to be deactivated by cleavage of the third protease first cut site;
wherein the

Assignees

• California Inst Of Techn

Inventors

• Gao Xiaojing
• Chong Lucy S
• Elowitz Michael
• Budde Mark William
• Kim Matthew Sun-Min

Classifications

• C12N9/506 Primary

  derived from RNA viruses · CPC title

• C12N9/003

  Dihydrofolate reductase [DHFR] (1.5.1.3) · CPC title

• C12N9/96

  Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates · CPC title

• C12Y105/01003

  Dihydrofolate reductase (1.5.1.3) · CPC title

• C12Y304/21098

  Hepacivirin (3.4.21.98) · CPC title