Flavobacterium heparinum expression system

• US 20010034043A1
• Filed: 01/19/2001
• Published: 10/25/2001
• Est. Priority Date: 01/21/2000
• Status: Active Grant

• Current Assignee: Biomarin Pharmaceutical Incorporated (Amgen Inc.)
• Original Assignee: Biomarin Pharmaceutical Incorporated (Amgen Inc.)
• Inventors: Zimmerman, Joseph, Su, Hongsheng, Shao, Zhongqi, Blain, Francoise, Tkalec, Ana Lydia
• Granted Patent: US 6,841,375 B2
• Time in Patent Office: Days
• US Class Current: 435/69.1
• CPC Class Codes: C12N 15/74 Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora

Abstract

This invention is directed to Flavobacterium heparinum for use as a host cell organism for the expression of homologous and heterologous genes.

Claims

1. A host cell for recombinant DNA expression comprising Flavobacterium heparinum.

2. The host cell of

claim 1

further comprising a vector.

3. The host cell of

claim 2

wherein said vector is a plasmid system.

4. The host cell of

claim 3

wherein said plasmid system is selected from the group consisting of a modified broad-host plasmid.

5. The host cell of

claim 1

wherein said recombinant DNA is integrated into the Flavobacterium heparinum chromosome.

6. The host cell of

claim 5

wherein said recombinant DNA is integrated through homologous recombination.

7. The host cell of

claim 6

wherein a gene encoded by said integrated DNA is expressed at high levels.

8. The host cell of

claim 5

wherein said recombinant DNA is integrated through any of bacteriophage integration, transposition of a transposon and transposition of an insertion sequence element.

9. The host cell of

claim 1

further comprising a selective marker for selection of host cells expressing a desired recombinant DNA.

10. The host cell of

claim 9

wherein said selective marker comprises one or more of a gene encoding antibiotic resistance, heavy metal resistance, a physiological growth inhibitory factor, and an amino acid requirement factor.

11. The host cell of

claim 10

wherein said selective marker expression is regulated by a regulatory region from Flavobacterium heparinum.

12. The host cell of

claim 11

wherein said regulatory region is the heparinase I gene regulatory region.

13. The host cell of

claim 1

wherein said recombinant DNA is expressed under the control of a regulatory region from Flavobacterium heparinum.

14. The host cell of

claim 13

wherein said regulatory region is the heparinase I gene regulatory region.

15. The host cell of

claim 1

wherein said recombinant DNA is introduced into said cell by conjugation.

16. The host cell of

claim 1

wherein said recombinant DNA is introduced into said cell by electroporation.

17. The host cell of

claim 1

wherein said recombinant DNA is introduced into said cell by bacterial phage transfection.

18. The host cell of

claim 1

wherein said cell glycosylates glycoproteins encoded by said recombinant DNA.

19. The host cell of

claim 1

wherein said cell expresses recombinant DNA containing a homologous gene.

20. The host cell of

claim 1

wherein said cell expresses recombinant DNA containing a heterologous gene.

21. A Flavobacterium heparinum host organism transformed with recombinant DNA comprising a homologous or a heterologous gene placed under the control of a gene promoter derived from a protein endogenous to the F. heparinum host and operably linked to the coding sequence for the homologous or heterologous gene.

22. The F. heparinum host organism of

claim 21

, wherein said gene promoter is hepA.

23. A method for producing a desired polypeptide or protein comprising expressing recombinant DNA comprising a coding sequence for the desired polypeptide or protein in a F. heparinum host organism;

24. The method of

claim 23

, wherein the expressed polypeptide or protein is glycosylated.

25. The method of

claim 23

, wherein the expressed polypeptide or protein is biologically active.

26. An expression system for expressing a desired polypeptide or protein comprising:

(1) a F. heparinum host organism

(2) nucleotide sequences encoding a desired polypeptide or protein, and

(3) a vector for expressing the nucleotide sequences capable of expressing the desired polypeptide or protein.

27. A vector which upon introduction into a F. heparinum host cell effects expression of DNA encoding a desired polypeptide or protein, the vector comprising (a) a functional origin of replication (OriC) region;

(b) replication (rep) genes; and

(c) a gene promoter derived from a protein endogenous to the F. heparinum host.

28. The vector depicted in FIG. 1.

29. The vector depicted in FIG. 2.