Degron fusion constructs and methods for controlling protein production

Patent metadata

Field	Value
Publication number	US-10550379-B2
Application number	US-201615737712-A
Country	US
Kind code	B2
Filing date	Jun 28, 2016
Priority date	Jun 29, 2015
Publication date	Feb 4, 2020
Grant date	Feb 4, 2020

Abstract

Engineered fusion proteins comprising a self-excising degron for controlling protein production are disclosed. In particular, the inventors have constructed fusion proteins comprising a degron connected to a protein of interest through a cleavable linker comprising a hepatitis C virus (HCV) protease site. The degron can be removed from the protein of interest by a czs-encoded HCV protease such that the protein of interest can be produced with minimal structural modification. Clinically available HCV protease inhibitors can be used to block protease cleavage such that the degron is retained after inhibitor addition on subsequently synthesized protein copies. The degron when attached causes rapid degradation of the linked protein. Such fusions of a degron to a protein of interest will be especially useful when control over protein production with minimal structural modification is desired.

First claim

What is claimed is:

1. A fusion protein comprising:
a) a polypeptide of interest;
b) a degron comprising the amino acid sequence of SEQ ID NO: 1, wherein the degron is operably linked to the polypeptide of interest when the fusion protein is in an uncleaned state, such that the degron promotes degradation of the polypeptide of interest in a cell;
c) a hepatitis C virus (HCV) nonstructural protein 3 (NS3) protease, wherein the protease can be inhibited by contacting said fusion protein with a protease inhibitor; and
d) a cleavable linker that is located between the polypeptide of interest and the degron, wherein the cleavable linker comprises a cleavage site recognized by the protease, wherein cleavage of the cleavable linker by the protease releases the polypeptide of interest from the fusion protein, such that when the fusion protein is in a cleaved state, the degron no longer controls degradation of the polypeptide of interest.

2. The fusion protein of claim 1 , wherein the degron is linked to the N-terminus or the C-terminus of the polypeptide of interest.

3. The fusion protein of claim 2 having the degron linked to the C-terminus of the polypeptide of interest, wherein the fusion protein comprises components arranged from N-terminus to C-terminus in the uncleaved state as follows:
a) the polypeptide of interest;
b) the cleavable linker;
c) the protease; and
d) the degron.

4. The fusion protein of claim 3 comprising a polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:7, wherein the degron is capable of promoting degradation of the polypeptide of interest and the protease is capable of cleaving the fusion protein at the cleavage site.

5. The fusion protein of claim 3 comprising a polypeptide comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:7, wherein the degron is capable of promoting degradation of the polypeptide of interest and the protease is capable of cleaving the fusion protein at the cleavage site.

6. The fusion protein of claim 3 comprising a polypeptide comprising the amino acid sequence of SEQ ID NO:7.

7. The fusion protein of claim 2 wherein the degron is linked to the N-terminus of the polypeptide of interest, the fusion protein comprising components arranged from N-terminus to C-terminus in the uncleaved state as follows:
a) the protease;
b) the degron;
c) the cleavable linker; and
d) the polypeptide of interest.

8. The fusion protein of claim 7 comprising a polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:8, wherein the degron is capable of promoting degradation of the polypeptide of interest and the protease is capable of cleaving the fusion protein at the cleavage site.

9. The fusion protein of claim 7 comprising a polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:8, wherein the degron is capable of promoting degradation of the polypeptide of interest and the protease is capable of cleaving the fusion protein at the cleavage site.

10. The fusion protein of claim 7 comprising a polypeptide comprising the amino acid sequence of SEQ ID NO:8.

11. The fusion protein of claim 1 , further comprising a targeting sequence.

12. The fusion protein of claim 11 , wherein the targeting sequence is selected from the group consisting of a secretory protein signal sequence, a membrane protein signal sequence, a nuclear localization sequence, a nucleolar localization signal sequence, an endoplasmic reticulum localization sequence, a peroxisome localization sequence, a mitochondrial localization sequence, and a protein binding motif sequence.

13. The fusion protein of claim 1 , further comprising a tag.

14. The fusion protein of claim 13 , wherein the tag is selected from the group consisting of a His-tag, a Strep-tag, a TAP-tag, an S-tag, an SBP-tag, an Arg-tag, a calmodulin-binding peptide tag, a cellulose-binding domain tag, a DsbA tag, a c-myc tag, a glutathione S-transferase tag, a FLAG tag, a HAT-tag, a maltose-binding protein tag, a NusA tag, and a thioredoxin tag.

15. The fusion protein of claim 1 , further comprising a detectable label.

16. The fusion protein of claim 15 , wherein the detectable label is a fluorescent label, a bioluminescent label, a chemiluminescent label, a colorimetric label, or an isotopic label.

17. The fusion protein of claim 16 , wherein the detectable label is a fluorescent protein or a bioluminescent protein.

18. The fusion protein of claim 1 , wherein the polypeptide of interest is selected from the group consisting of a membrane protein, a receptor, a hormone, a transport protein, a transcription factor, a cytoskeletal protein, an extracellular matrix protein, a signal-transduction protein, and an enzyme.

19. The fusion protein of claim 1 , wherein the polypeptide of interest comprises a biologically active domain of a protein.

20. The fusion protein of claim 19 , wherein the biologically active domain is a catalytic domain, a ligand binding domain, or a protein-protein interaction domain.

21. An isolated polynucleotide encoding the fusion protein of claim 1 .

22. A recombinant polynucleotide comprising the polynucleotide of claim 21 operably linked to a promoter.

23. The recombinant polynucleotide of claim 22 , wherein the promoter is an endogenous promoter or exogenous promoter.

24. The recombinant polynucleotide of claim 22 comprising a polynucleotide selected from the group consisting of:
a) a polynucleotide encoding a fusion protein comprising a sequence selected from the group consisting of SEQ ID NO:7 and SEQ ID NO:8; and
b) a polynucleotide encoding a fusion protein comprising a sequence at least 80% identical to a sequence selected from the group consisting of SEQ ID NO:7 and SEQ, ID NO:8, wherein the degron is capable of promoting degradation of the polypeptide of interest, and the protease is capable of cleaving the fusion protein at a cleavage site.

25. The recombinant polynucleotide of claim 22 , further comprising a multiple cloning site.

26. The recombinant polynucleotide of claim 22 , further comprising a nucleotide sequence encoding a tag, a detectable label, a targeting sequence, or a linker.

27. The recombinant polynucleotide of claim 26 , wherein the detectable label is a fluorescent protein or a bioluminescent protein.

28. A host cell transformed with the recombinant polynucleotide of claim 22 .

29. The host cell of claim 28 , wherein the host cell is a eukaryotic cell.

30. An organoid comprising the host cell of claim 28 .

31. A kit comprising the recombinant polynucleotide of claim 22 .

32. A kit comprising the host cell of claim 29 .

33. A method for producing a fusion protein, the method comprising:
a) transforming a host cell with the recombinant polynucleotide of claim 22 ;
b) culturing the transformed host cell under conditions whereby the fusion protein is expressed; and
c) isolating the fusion protein from the host cell.

34. A method of controlling production of a polypeptide of interest, the method comprising:
a) transforming a host cell with the recombinant polynucleotide of claim 22 ;
b) cu

Assignees

• Univ Leland Stanford Junior

Inventors

• Lin Michael Z
• Chung Hokyung
• Jacobs Conor

Classifications

• C12N15/86

  Viral vectors · CPC title

• C12N9/48

  acting on peptide bonds (3.4) · CPC title

• C12N2760/18432

  Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent · CPC title

• C12Y304/21098

  Hepacivirin (3.4.21.98) · CPC title

• C07K2319/95

  containing a motif/fusion for degradation (ubiquitin fusions, PEST sequence) · CPC title