# US 5,705,361 A · Hybrid transposon with PT operon, plasmids and bacterial strains therefor

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- Number: US 5,705,361 A
- Document ID: US5705361A
- Status: Granted
- Kind: A
- Application number: 08/137,139
- Filed: 1993-10-21
- Granted: 1998-01-06
- Earliest claimed: 1991-04-24
- Assignees: Gesellschaft fur Biotechnologische Forschung mbH (GBF)
- Inventors: Mark Walker; Kenneth Timmis
- Web page: https://patentgrep.com/patent/US5705361A

## CPC

- C12N 15/90: Stable introduction of foreign DNA into chromosome
- A61K 39/099: {Bordetella}
- A61P 31/04: Antibacterial agents
- C07K 14/235: from Bordetella (G)
- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N 15/74: Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- 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 invention relates to a hybrid transposon having a PT operon, to plasmids and to bacterial strains (hosts) therefor.

## Claims

### Claim 1

A plasmid for the stable expression of pertussis toxin in a bacterial microorganism which comprises;
a promoterless hybrid transposon wherein a transposase gene is arranged outside of inverted repeats of the transposon; and
having a pertussis toxin operon inserted between the inverted repeats;
said inverted repeats being in shortened form, thereby permitting a further transposition;
said plasmid permitting a stable insertion of the pertussis toxin operon into a bvg-negative bacterial strain.

### Claim 2 (depends on 1)

A plasmid according to claim 1 wherein the transposon is a mini-transposon.

### Claim 3 (depends on 1)

A plasmid according to claim 1 wherein the transposon is from Tn5 and the inverted repeats each have a length of approximately 19 base pairs.

### Claim 4 (depends on 1)

A plasmid according to claim 1 wherein the operon is selected from the group consisting of a wild-type operon and an operon that encodes a pertussis toxin modification effective against whooping cough.

### Claim 5 (depends on 1)

A plasmid according to claim 1 wherein the hybrid transposon contains a kanamycin resistance gene.

### Claim 6

A plasmid which is promotorless having the following features and is
a suicide vector that has a mini-transposon for pertussis toxin expression for transcriptional fusions with native promoters, having
1. a mob site,
2. an ampicillin resistance gene and
3. in each case, 19 base pairs of the terminal Tn5 end which flank,
4. a kanamycin resistance gene and
5. a promotorless operon; and also having
6. a IS50.sub.R -transposase gene.

### Claim 7 (depends on 1)

A bvg-negative bacterial strain containing a plasmid according to claim 1.

### Claim 8 (depends on 7)

The bacterial strain of claim 7 which is selected from the group consisting of Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica.

### Claim 9 (depends on 1, 2, 3, 4, 5, 6)

A bvg-negative bacterial strain wherein a pertussis toxin operon has been incorporated by means of a plasmid according to one of claims 1, 2 and 3 to 6.

### Claim 10 (depends on 9)

The strain of claim 9 selected from the group of species consisting of Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica.

### Claim 11 (depends on 10)

The strain of claim 10 which is Bordetella bronchiseptica ATCC 10580.

### Claim 12 (depends on 1, 2, 3, 4, 5, 6)

A process for the stable expression of pertussis toxin which comprises;
culturing a bvg-negative bacterial strain containing a plasmid according to one of claims 1, 2 and 3 to 6.