Methods of expressing gram-negative glycosaminoglycan synthase genes in gram-positive hosts

• US 7,642,071 B2
• Filed: 02/01/2007
• Issued: 01/05/2010
• Est. Priority Date: 04/01/1999
• Status: Expired due to Fees

• Current Assignee: The Board of Regents of the University of Colorado (University of Colorado System)
• Original Assignee: The Board of Regents of the University of Colorado (University of Colorado System)
• Inventors: DeAngelis, Paul L.
• Primary Examiner(s): Bertoglio; Valarie
• Assistant Examiner(s): MONTANARI, DAVID A
• Application Number:
  US11/701,262
• Publication Number:
  US 20070281342A1
• Time in Patent Office: 1,069 Days
• Field of Search: 435/71.1, 424/93.21
• US Class Current: 435/71.1
• CPC Class Codes: C12N 9/1048 Glycosyltransferases (2.4)
• CPC Class Codes: C12N 9/1051 Hexosyltransferases (2.4.1)
• CPC Class Codes: C12P 19/04 Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
• CPC Class Codes: C12P 19/26 Preparation of nitrogen-containing carbohydrates
• CPC Class Codes: C12Y 204/01175 Glucuronosyl-N-acetylgalactosaminyl-proteoglycan 4-beta-N-acetylgalactosaminyltransferase II (2.4.1.175)

Abstract

The present invention relates to a Gram-negative glycosaminoglycan gene and methods of making and using same. The present invention relates to recombinant Gram-positive host cells containing a Gram-negative glycosaminoglycan synthase gene, and methods of producing glycosaminoglycans using such recombinant host cells.

Claims

1. A method for producing a chondroitin polymer in vivo, comprising the steps of:

providing a purified nucleic acid segment encoding an enzymatically active chondroitin synthase, wherein the chondroitin synthase is a single protein that is a dual-action transferase that catalyzes the polymerization of UDP-GIcUA and UDP-GaINAc to form chondroitin, and wherein the purified nucleic acid segment comprises at least one of;

(A) the nucleic acid sequence of SEQ ID NO;

1;

(B) a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO;

2;

(C) a nucleic acid sequence that is at least 80% identical to SEQ ID NO;

1; and

(D) a nucleic acid sequence that encodes an amino acid sequence that is at least 90% identical to the entirety of SEQ ID NO;

2;

providing a Gram-positive host cell;

placing the purified nucleic acid segment encoding the enzymatically active chondroitin synthase in the Gram-positive host cell, thereby providing a recombinant Gram-positive host cell having a purified nucleic acid segment encoding an enzymatically active chondroitin synthase therein;

placing the recombinant Gram-positive host cell in a medium suitable for the expression of the enzymatically active chondroitin synthase, whereby a chondroitin polymer is produced; and

isolating the chondroitin polymer.

2. The method of claim 1 wherein, in the step of providing a purified nucleic acid segment encoding an enzymatically active chondroitin synthase, the purified nucleic acid segment comprises (A).

3. The method of claim 1 wherein, in the step of providing a purified nucleic acid segment encoding an enzymatically active chondroitin synthase, the purified nucleic acid segment comprises (B).

4. The method of claim 1 wherein, in the step of providing a purified nucleic acid segment encoding an enzymatically active chondroitin synthase, the purified nucleic acid segment comprises (C).

5. The method of claim 1 wherein, in the step of providing a purified nucleic acid segment encoding an enzymatically active chondroitin synthase, the purified nucleic acid segment comprises (D).

6. The method of claim 1 wherein, in the step of providing a Gram-positive host cell, the Gram-positive host cell is selected from the group consisting of a Bacillus cell, Staphylococcus cell, Peptococcus cell, Lactobacillus cell, Lactococcus cell, Actinomyces cell, and Streptomyces cell.

7. The method of claim 1 wherein, in the step of providing a Gram-positive host cell, the Gram-positive host cell comprises nucleic acid segments encoding enzymes which produce UDP-GIcUA and UDP-GaINAc.

8. The method of claim 1 wherein, in the step of isolating the chondroitin polymer, the chondroitin polymer is isolated from at least one of the medium and the recombinant Gram-positive host cell.

9. The method of claim 8, further comprising the step of purifying the isolated chondroitin polymer.

10. The method of claim 1, further comprising the step of sulfating the isolated chondroitin polymer.

11. The method of claim 1, further comprising the step of epimerizing the isolated chondroitin polymer.

12. A method for producing a chondroitin polymer in vivo, comprising the steps of:

providing a purified nucleic acid segment encoding an enzymatically active chondroitin synthase, wherein the chondroitin synthase is a single protein that is a dual-action transferase that catalyzes the polymerization of UDP-GIcUA and UDP-GaINAc to form chondroitin, and wherein the purified nucleic acid segment comprises at least one of;

(A) the nucleic acid sequence of SEQ ID NO;

1;

(B) a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO;

2;

(C) a nucleic acid sequence that is at least 800/a identical to SEQ ID NO;

1; and

(D) a nucleic acid sequence that encodes an amino acid sequence that is at least 900/a identical to the entirety of SEQ ID NO;

2;

providing a Bacillus host cell, wherein the Bacillus host cell comprises nucleic acid segments encoding enzymes which produce UDP-GIcUA and UDP-GaINAc;

placing the purified nucleic acid segment encoding the enzymatically active chondroitin synthase in the Bacillus host cell, thereby providing a recombinant Bacillus host cell having a purified nucleic acid segment encoding an enzymatically active chondroitin synthase therein;

placing the recombinant Bacillus host cell in a medium suitable for the expression of the enzymatically active chondroitin synthase, whereby a chondroitin polymer is produced; and

isolating the chondroitin polymer.

13. The method of claim 12 wherein, in the step of providing a purified nucleic acid segment encoding an enzymatically active chondroitin synthase, the purified nucleic acid segment comprises (A).

14. The method of claim 12 wherein, in the step of providing a purified nucleic acid segment encoding an enzymatically active chondroitin synthase, the purified nucleic acid segment comprises (B).

15. The method of claim 12 wherein, in the step of providing a purified nucleic acid segment encoding an enzymatically active chondroitin synthase, the purified nucleic acid segment comprises (C).

16. The method of claim 12 wherein, in the step of providing a purified nucleic acid segment encoding an enzymatically active chondroitin synthase, the purified nucleic acid segment comprises (D).

17. The method of claim 12, further comprising the step of purifying the isolated chondroitin polymer.

18. The method of claim 12, further comprising the step of sulfating the isolated chondroitin polymer.

19. The method of claim 12, further comprising the step of epimerizing the isolated chondroitin polymer.