# US 5,708,149 A · Method for producing purified recombinant Haemophilus influenzae transferrin binding proteins

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- Number: US 5,708,149 A
- Document ID: US5708149A
- Status: Granted
- Kind: A
- Application number: 08/487,890
- Filed: 1995-06-07
- Granted: 1998-01-13
- Earliest claimed: 1993-11-08
- Assignees: Connaught Laboratories Limited
- Inventors: Sheena Loosmore; Robin Harkness; Anthony Schryvers; Pele Chong; Scott Gray-Owen; Yan-Ping Yang; Andrew Murdin; Michel Klein
- Web page: https://patentgrep.com/patent/US5708149A

## CPC

- C07K 14/285: from Pasteurellaceae (F), e.g. Haemophilus influenza
- A61P 31/00: Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P 31/04: Antibacterial agents
- A61P 37/04: Immunostimulants
- C07K 16/2881: {against CD71}
- A61K 39/00: Medicinal preparations containing antigens or antibodies (materials for immunoassay G01N33/53)
- A61K 2039/51: {comprising whole cells, viruses or DNA/RNA}
- C07K 2317/34: Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
- C12N 2799/021: for the expression of a heterologous nucleic acid
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

Purified and isolated nucleic acid is provided which encodes a transferrin receptor protein of a strain of Haemophilus or a fragment or an analog of the transferrin receptor protein. The nucleic acid sequence may be used to produce peptides free of contaminants derived from bacteria normally containing the Tbp1 or Tbp2 proteins for purposes of diagnostics and medical treatment. Furthermore, the nucleic acid molecule may be used in the diagnosis of infection. Also provided are recombinant Tbp1 or Tbp2 and methods for purification of the same. Live vectors expressing epitopes of transferrin receptor protein for vaccination are provided.

## Claims

### Claim 1

A method of producing an isolated and purified Tbp1 or Tbp2 protein of a strain of Haemophilus influenzae, comprising the steps of:
(a) providing a recombinant host expressing, in inclusion bodies, a Tbp1 or Tbp2 protein, but not both, of a strain of Haemophilus influenzae;
(b) growing said host to provide a cell mass wherein said Tbp1 or Tbp2 protein is present in inclusion bodies;
(c) disrupting the cell mass to provide a cell lysate;
(d) fractionating the cell lysate to provide a first supernatant and a first pellet, the first supernatant comprising substantially a large proportion of soluble host proteins;
(e) separating said first supernatant from said first pellet;
(f) selectively extracting the first pellet to remove substantially all soluble host proteins and host membrane proteins therefrom to provide a second supernatant and an extracted pellet containing the inclusion bodies;
(g) separating said second supernatant from said extracted pellet;
(h) solubilizing the extracted pellet to provide a solubilized extract; and
(i) fractionating the solubilized extract to provide a Tbp1 or Tbp2 protein containing fraction.

### Claim 2 (depends on 1)

The method of claim 1 wherein the cell lysate is fractionated by centrifugation thereof.

### Claim 3 (depends on 2)

The method of claim 2 wherein the step of selectively extracting the first pellet comprises at least one detergent extraction.

### Claim 4 (depends on 3)

The method of claim 3 wherein the solubilized extract is fractionated by gel filtration to provide said Tbp1 or Tbp2 protein containing fraction.

### Claim 5 (depends on 4)

The method of claim 4 including subsequently dialyzing the Tbp1 or Tbp2 protein containing fraction to remove at least said detergent to provide a further purified solution of Tbp1 or Tbp2 protein.