Methods of expressing gram-negative glycosaminoglycan synthase genes in gram-positive hosts
View Patent ↗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.
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.