# US 5,702,921 A · Expression of biologically active human C-reactive protein in escherichia coli

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- Number: US 5,702,921 A
- Document ID: US5702921A
- Status: Granted
- Kind: A
- Application number: 08/621,897
- Filed: 1996-03-26
- Granted: 1997-12-30
- Earliest claimed: 1993-04-27
- Assignees: Orienta Yeast Co., Ltd.
- Inventors: Toshio Tanaka
- Web page: https://patentgrep.com/patent/US5702921A

## CPC

- C07K 14/4737: {C-reactive protein}
- C12N 15/70: Vectors or expression systems specially adapted for E. coli
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention relates to a plasmid possessing the restriction enzyme map shown in FIG. 1 , and this plasmid is largely characterized in that the kil gene is linked downstream from the human CRP (C-reactive protein) gene. Using this plasmid, it is possible to produce large amounts of human CRP by extracellular secretion.

## Claims

### Claim 1

A recombinant plasmid comprising an inducible bacterial promoter which is operably linked to a gene encoding a signal peptide operably linked to the gene encoding human C-reactive protein (CRP).

### Claim 2 (depends on 1)

The recombinant plasmid according to claim 1, wherein said inducible bacterial promoter is the tryptophan promoter and said signal peptide is the Escherichia coli alkaline phosphatase signal peptide.

### Claim 3 (depends on 1)

The recombinant plasmid according to claim 1, further comprising the kil gene.

### Claim 4

The recombinant plasmid pTRPCRPS-kil having the pTRPCRPS-kil restriction map set forth in FIG. 1.

### Claim 5 (depends on 1)

An Escherichia coli cell which is transformed with the recombinant plasmid of claim 1.

### Claim 6 (depends on 2)

An Escherichia coli cell which is transformed with the recombinant plasmid of claim 2.

### Claim 7 (depends on 3)

An Escherichia coli cell which is transformed with the recombinant plasmid of claim 3.

### Claim 8

An Escherichia coli cell which is transformed with the recombinant plasmid pTRPCRPS-kil.

### Claim 9 (depends on 5)

A method for producing recombinant human CRP which has the biological activity of non-recombinant human CRP, comprising culturing the Escherichia coli cell of claim 5 whereby said Escherichia coli cell expresses and secretes said recombinant human CRP which has the biological activity of non-recombinant human CRP, and recovering the secreted recombinant human CRP.

### Claim 10 (depends on 6)

A method for producing recombinant human CRP which has the biological activity of non-recombinant human CRP, comprising culturing the Escherichia coli cell of claim 6 whereby said Escherichia coli cell expresses and secretes said recombinant human CRP which has the biological activity of non-recombinant human CRP, and recovering the secreted recombinant human CRP.

### Claim 11 (depends on 7)

A method for producing recombinant human CRP which has the biological activity of non-recombinant human CRP, comprising culturing the Escherichia coli cell of claim 7 whereby said Escherichia coli cell expresses and secretes said recombinant human CRP which has the biological activity of non-recombinant human CRP, and recovering the secreted recombinant human CRP.

### Claim 12 (depends on 8)

A method for producing recombinant human CRP which has the biological activity of non-recombinant human CRP, comprising culturing the Escherichia coli cell of claim 8 whereby said Escherichia coli cell expresses and secretes said recombinant human CRP which has the biological activity of non-recombinant human CRP, and recovering the secreted recombinant human CRP.

### Claim 13 (depends on 9)

The method of claim 9, wherein said recombinant human CRP has the identical conformation as non-recombinant human CRP.

### Claim 14 (depends on 10)

The method of claim 10, wherein said recombinant human CRP has the identical conformation as non-recombinant human CRP.

### Claim 15 (depends on 11)

The method of claim 11, wherein said recombinant human CRP has the identical conformation as non-recombinant human CRP.

### Claim 16 (depends on 12)

The method of claim 12, wherein said recombinant human CRP has the identical conformation as non-recombinant human CRP.