Recombinant obese (ob) proteins

• US 5,968,779 A
• Filed: 01/05/1998
• Issued: 10/19/1999
• Est. Priority Date: 05/05/1995
• Status: Expired due to Fees

• Current Assignee: Hoffmann-La Roche Incorporated (Roche Holding AG)
• Original Assignee: Hoffmann-La Roche Incorporated (Roche Holding AG)
• Inventors: Devos, Rene, Guisez, Yves, Campfield, Arthur
• Primary Examiner(s): Prouty, Rebecca E.
• Application Number:
  US09/003,081
• Time in Patent Office: 652 Days
• Field of Search: 435/69.4, 435/69.8, 435/71.2, 435/252.33, 435/320.1, 536/23.5, 536/23.51
• US Class Current: 435/69.4
• CPC Class Codes: 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
• CPC Class Codes: C07K 14/5759 Products of obesity genes, e.g. leptin, obese (OB), tub, fat
• CPC Class Codes: C07K 2319/00 Fusion polypeptide

Abstract

Proteins which modulate body weight of animals and humans for the treatment, prevention and control of obesity and associated diseases or conditions, and the recombinant expression of these biologically active proteins in purified and homogeneous forms.

Claims

1. An expression vector comprising:

a) a promoter sequence, and

b) a DNA sequence encoding a fusion protein, which fusion protein comprises the murine ob protein of SEQ ID NO;

3 or the human ob protein of SEQ ID NO;

6, and the signal peptide for the outer membrane protein A of E. coli,

which expression vector is capable of expressing the fusion protein in Escherichia coli host cells.

2. The expression vector of claim 1, wherein the promoter sequence consists of both a lac-operator promoter and a lipoprotein promoter.

6. An Escherichia coli host organism transformed with the expression vector of claim 1.

3. A DNA sequence comprising a first and second part, wherein:

(a) the first part is the sOmpA gene sequence of SEQ ID NO;

7 encoding the sOmpA peptide; and

(b) the second part is the nucleotide sequence encoding the murine ob protein or the nucleotide sequence encoding the human ob protein.

4. The DNA sequence of claim 3, wherein the murine ob protein comprises SEQ ID NO:

3.

5. The DNA sequence of claim 3, wherein the human ob protein comprises SEQ ID NO:

6.

7. A method of producing biologically active recombinant human or murine obese protein free of other mammalian proteins comprising the steps of:

a) constructing an expression vector having a promoter sequence, and a DNA sequence encoding a fusion protein, which fusion protein comprises SEQ ID NO;

3 or SEQ ID NO;

6, and the signal peptide for the outer membrane protein A of E. coli;

b) inserting the expression vector into an E. coli host cell to transform the E. coli host cell;

c) expressing the fusion protein in the E. coli host cell; and

d) treating the E. coli host cell with cold osmotic shock buffer to liberate the murine or human ob protein free of other mammalian proteins and free of the signal peptide.