# US 7,608,424 B2 · Process for production of a recombinant protein and a fusion protein

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- Number: US 7,608,424 B2
- Document ID: US7608424B2
- Status: Granted
- Kind: B2
- Application number: 11/837,795
- Filed: 2007-08-13
- Published: 2008-04-03
- Granted: 2009-10-27
- Earliest claimed: 2000-12-26
- Assignees: Sekisui Chemical Co., Ltd.
- Inventors: Masahiro Furutani; Junichi Hata; Akiko Togi
- Web page: https://patentgrep.com/patent/US7608424B2

## CPC

- C12N 15/62: DNA sequences coding for fusion proteins
- C07K 19/00: Hybrid peptides {, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes}
- C12N 15/70: Vectors or expression systems specially adapted for E. coli
- C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione
- C07K 2319/21: containing a His-tag
- C07K 2319/35: containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin
- C07K 2319/40: containing a tag for immunodetection, or an epitope for immunisation
- C07K 2319/75: containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
- 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)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}

## Abstract

This invention provides a recombinant protein expression system using a host and cell-free translation system, and is capable of universally expressing a large amount of any protein as soluble protein, while preventing toxicity in hosts, formation of inclusion bodies, and decompositions with proteases. Such may be achieved by expressing the desired protein as a fusion protein with chaperoning, such as about 60 kDa molecular chaperons, 60 kDa heat shock proteins, or thermosomes, and accommodating the desired protein inside of a stereostructure of a chaperonin. The present invention provides a process for producing a protein, which comprises transcribing and translating a gene containing a gene encoding the linked chaperonin subunits and a gene encoding a desired protein thereby synthesizing a fusion protein having the desired protein linked via a peptide linkage to the linked chaperonin subunits.

## Claims

### Claim 1

A fusion protein consisting of 7 chaperonin subunits and at least one desired protein,
wherein the 7 chaperonin subunits assemble into a ring structure, and the desired protein is accommodated inside the ring structure, and
wherein the chaperonin subunit is a bacterial chaperonin subunit.

### Claim 2

A fusion protein consisting of 2, 4, or 8 chaperonin subunits and at least one desired protein,
wherein the 8 chaperonin subunits assemble into a ring structure, and the desired protein is accommodated inside the ring structure,
wherein the chaperonin subunits to desired protein is in a ratio of 2:1, 4:1, or 8:1, and
wherein the chaperonin subunit is an archaeum chaperonin subunit.

### Claim 3

A fusion protein consisting of 3 or 9 chaperonin subunits and at least one desired protein,
wherein the 9 chaperonin subunits assemble into a ring structure, and the desired protein is accommodated inside the ring structure,
wherein the chaperonin subunits to desired protein is in a ratio of 3:1 or 9:1, and
wherein the chaperonin subunit is an archaeum chaperonin subunit.

### Claim 4 (depends on 1)

The fusion protein according to claim 1, wherein the bacterial chaperonin subunits are E. coli GroEL.

### Claim 5 (depends on 2, 3)

The fusion protein according to claim 2 or 3, wherein the archaeum chaperonin subunit is Thermococcus chaperonin β-subunit (TCPβ).

### Claim 6

The fusion protein according to claims or 1, 2, or 3, wherein the chaperonin ring has formed a 2-layer structure associated non-covalently via a ring plane.

### Claim 7

The fusion protein according to claims or 1, 2, or 3, wherein the chaperonin rings have assembled into a fibrous structure linked non-covalently via a ring plane or its side.