# US 5,705,387 A · Heat-inducible N-degron module

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- Number: US 5,705,387 A
- Document ID: US5705387A
- Status: Granted
- Kind: A
- Application number: 08/637,508
- Filed: 1996-04-25
- Granted: 1998-01-06
- Earliest claimed: 1994-02-04
- Assignees: California Institute of Technology
- Inventors: Peipei Wu; Jurgen Dohmen; Alexander Varshavsky
- Web page: https://patentgrep.com/patent/US5705387A

## CPC

- C12N 9/0028: {with NAD or NADP as acceptor (1.5.1)}
- C07K 14/00: Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K 14/001: {by chemical synthesis}
- C07K 14/395: from Saccharomyces
- C12N 9/003: {Dihydrofolate reductase [DHFR] (1.5.1.3)}
- C12N 9/12: transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N 15/11: DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity}
- C12N 15/62: DNA sequences coding for fusion proteins
- C07K 2319/00: Fusion polypeptide
- C07K 2319/42: containing a HA(hemagglutinin)-tag
- C07K 2319/61: containing an enzyme fusion for detection (lacZ, luciferase)
- C07K 2319/95: containing a motif/fusion for degradation (ubiquitin fusions, PEST sequence)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

The N-degron is an intracellular degradation signal whose essential determinant is a specific, destabilizing, N-terminal amino acid residue. A set of N-degrons containing different destabilizing residues is manifested as the N-end rule, which relates the in vivo half-life of a protein to the identity of its N-terminal amino acid residue. Disclosed herein is a heat-inducible N-degron module. A heat-inducible N-degron module is a protein or peptide bearing a destabilizing N-terminal amino acid residue which becomes a substrate of the N-end rule pathway only at a temperature high enough to result in at least partial unfolding of the protein. At this elevated (nonpermissive) temperature, the heat-inducible N-degron module (and any protein or peptide attached at its C-terminus) is rapidly degraded in a cell in which the N-end rule pathway is operative. Also disclosed are DNA and protein fusion constructs, methods for screening for additional heat-inducible N-degron modules and methods for using the disclosed heat-inducible N-degron modules.

## Claims

### Claim 1

A heat-inducible N-degron module comprising:
a) a destabilizing N-terminal amino acid residue;
b) an internal lysine amino acid residue which functions as an attachment site for a multiubiquitin chain at a permissive temperature, but not at a non-permissive temperature in a eukaryotic cell or cell extract; the heat-inducible N-degron module being encoded by a DNA sequence which hybridizes, under stringent hybridization conditions, to the DNA sequence represented in SEQ ID NO: 1.

### Claim 2 (depends on 1)

The heat-inducible N-degron module of claim 1 wherein the DNA sequence encodes a variant of wild-type mouse DHFR in which the amino acid residue corresponding to proline at position 66 of wild-type mouse DHFR is replaced with a non-proline residue.

### Claim 3 (depends on 2)

The heat-inducible N-degron module of claim 2 wherein the non-proline residue is leucine.

### Claim 4

A fusion protein comprising a heat-inducible N-degron module, the heat-inducible N-degron module comprising:
a) a destabilizing N-terminal amino acid residue;
b) an internal lysine amino acid residue which functions as an attachment site for a multiubiquitin chain at a permissive temperature, but not at a non-permissive temperature in a eukaryotic cell or cell extract;
the heat inducible N-degron module being joined covalently at its C-terminus to the N-terminus of a protein of interest, the heat inducible N-degron module being encoded by a DNA sequence which hybridizes, under stringent hybridization conditions, to the DNA sequence represented in SEQ ID NO: 1.

### Claim 5 (depends on 4)

The fusion protein of claim 4 wherein the DNA sequence encodes a variant of wild-type mouse DHFR in which the amino acid residue corresponding to proline at position 66 of wild-type mouse DHFR is replaced with a non-proline residue.

### Claim 6 (depends on 5)

The fusion protein of claim 5 wherein the non-proline residue is leucine.

### Claim 7

A cell containing a fusion protein comprising a heat-inducible N-degron module, the heat-inducible N-degron module comprising:
a) a destabilizing N-terminal amino acid residue;
b) an internal lysine amino acid residue which functions as an attachment site for a multiubiquitin chain at a permissive temperature, but not at a non-permissive temperature in a eukaryotic cell or cell extract;
the heat-inducible N-degron module being joined covalently at its C-terminus to the N-terminus of a protein of interest, the heat inducible N-degron module being encoded by a DNA sequence which hybridizes, under stringent hybridization conditions, to the DNA sequence represented in SEQ ID NO: 1.

### Claim 8 (depends on 7)

The cell of claim 7 wherein the DNA sequence encodes a variant of wild-type mouse DHFR in which the amino acid residue corresponding to proline at position 66 of wild-type mouse DHFR is replaced with a non-proline residue.

### Claim 9 (depends on 8)

The cell of claim 8 wherein the non-proline residue is leucine.