Streptococcus equisimilis hyaluronan synthase gene and expression thereof in Bacillus subtilis

• US 20020160489A1
• Filed: 09/12/2001
• Published: 10/31/2002
• Est. Priority Date: 07/23/1997
• Status: Abandoned Application

• Current Assignee: Kshama Kumari, Paul Deangelis, Paul H. Weigel
• Original Assignee: Kshama Kumari, Paul Deangelis, Paul H. Weigel
• Inventors: Weigel, Paul H., Kumari, Kshama, DeAngelis, Paul
• Application Number:
  US09/879,959
• Publication Number:
  US 20020160489A1
• Time in Patent Office: Days
• US Class Current: 435/201
• CPC Class Codes: C08B 37/0072 Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
• 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

Abstract

The present invention relates to a nucleic acid segment having a coding region segment encoding enzymatically active Streptococcus equisimilis hyaluronate synthase (seHAS), and to the use of this nucleic acid segment in the preparation of recombinant cells which produce hyaluronate synthase and its hyaluronic acid product. Hyaluronate is also known as hyaluronic acid or hyaluronan.

Claims

1. A purified nucleic acid segment comprising a coding region encoding enzymatically active hyaluronate synthase.

2. The purified nucleic acid segment of claim 1, wherein the purified nucleic acid segment encodes the Streptococcus equisimilis hyaluronate synthase of SEQ ID NO:

2.

3. The purified nucleic acid segment of claim 1, wherein the purified nucleic acid segment comprises a nucleotide sequence in accordance with SEQ ID NO:

1.

50. A procaryotic or eucaryotic host cell transformed or transfected with an isolated nucleic acid segment according to claim 1, 2, or 3 in a manner allowing the host cell to express hyaluronic acid.

4. A purified nucleic acid segment having a coding region encoding enzymatically active hyaluronate synthase, wherein the purified nucleic acid segment is capable of hybridizing to the nucleotide sequence of SEQ ID NO:

1.

5. A purified nucleic acid segment having a coding region encoding enzymatically active hyaluronate synthase, wherein the purified nucleic acid segment has semiconservative or conservative amino codon acid changes when compared to the nucleotide sequence of SEQ ID NO:

1.

7. The recombinant vector of claim 5, wherein the purified nucleic acid segment encodes the Streptococcus equisimilis hyaluronan synthase of SEQ ID NO:

2.

6. A recombinant vector selected from the group consisting of a plasmid, cosmid, phage, or virus vector and wherein the recombinant vector further comprises a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase.

8. The recombinant vector of claim 6, wherein the purified nucleic acid segment comprises a nucleotide sequence in accordance with SEQ ID NO:

1.

9. The recombinant vector of claim 6, wherein the plasmid further comprises an expression vector.

10. The recombinant vector of claim 9, wherein the expression vector comprises a promoter operatively linked to the enzymatically active Streptococcus equisimilis hyaluronan synthase coding region.

11. A recombinant host cell, wherein the recombinant host cell is a prokaryotic cell transformed with a recombinant vector comprising a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase.

12. The recombinant host cell of claim 11, wherein the purified nucleic acid segment encodes the Streptococcus equisimilis hyaluronan synthase of SEQ ID NO:

2.

13. The recombinant host cell of claim 11, wherein the purified nucleic acid segment comprises a nucleotide sequence in accordance with SEQ ID NO:

1.

14. The recombinant host cell of claim 13, wherein the host cell produces hyaluronic acid.

15. The recombinant host cell of claim 11, wherein the enzymatically active hyaluronan synthase is capable of producing a hyaluronic acid polymer having a modified structure.

16. The recombinant host cell of claim 11, wherein the enzymatically active hyaluronan synthase is capable of producing a hyaluronic acid polymer having a modified size distribution.

18. The recombinant host cell of claim 17, wherein the purified nucleic acid segment encodes the Streptococcus equisimilis hyaluronan synthase of SEQ ID NO:

2.

19. The recombinant host cell of claim 17, wherein the purified nucleic acid segment comprises a nucleotide sequence in accordance with SEQ ID NO:

1.

20. The recombinant host cell of claim 19, wherein the host cell produces hyaluronic acid.

21. The recombinant host cell of claim 17, wherein the enzymatically active hyaluronan synthase is capable of producing a hyaluronic acid polymer having a modified structure.

22. The recombinant host cell of claim 17, wherein the enzymatically active hyaluronan synthase is capable of producing a hyaluronic acid polymer having a modified size distribution.

24. The recombinant host cell of claim 23, wherein the purified nucleic acid segment encodes the Streptococcus equisimilis hyaluronan synthase of SEQ ID NO:

2.

25. The recombinant host cell of claim 23, wherein the purified nucleic acid segment comprises a nucleotide sequence in accordance with SEQ ID NO:

1.

26. The recombinant host cell of claim 25, wherein the host cell produces hyaluronic acid.

27. The recombinant host cell of claim 23, wherein the enzymatically active hyaluronan synthase is capable of producing a hyaluronic acid polymer having a modified structure.

28. The recombinant host cell of claim 23, wherein the enzymatically active Streptococcus equisimilis hyaluronan synthase is capable of producing a hyaluronic acid polymer having a modified size distribution.

30. The recombinant host cell of claim 29, wherein the purified nucleic acid segment encodes the Streptococcus equisimilis hyaluronan synthase of SEQ ID NO:

2.

31. The recombinant host cell of claim 29, wherein the purified nucleic acid segment comprises a nucleotide sequence in accordance with SEQ ID NO:

1.

32. The recombinant host cell of claim 31, wherein the host cell produces hyaluronic acid.

33. The recombinant host cell of claim 29, wherein the enzymatically active hyaluronan synthase is capable of producing a hyaluronic acid polymer having a modified structure.

34. The recombinant host cell of claim 29, wherein the enzymatically active hyaluronan synthase is capable of producing a hyaluronic acid polymer having a modified size distribution.

40. The method according to claim 39, wherein the step of recovering the hyaluronic acid comprises extracting the secreted hyaluronic acid from the medium.

41. The method according to claim 40, further comprising the step of purifying the extracted hyaluronic acid.

42. The method according to claim 39, wherein in the step of growing the host organism, the host organism secretes a structurally modified hyaluronic acid.

43. The method according to claim 39, wherein in the step of growing the host organism, the host organism secretes a hyaluronic acid having a modified size.

17. A recombinant host cell, wherein the recombinant host cell is a eukaryotic cell transfected with a recombinant vector comprising a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase.

23. A recombinant host cell, wherein the recombinant host cell is electroporated to introduce a recombinant vector into the recombinant host cell, wherein the recombinant vector comprises a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase.

29. A recombinant host cell, wherein the recombinant host cell is transduced with a recombinant vector comprising a purified nucleic acid segment having a coding region encoding enzymatically active Streptococcus equisimilis hyaluronan synthase.

35. A purified composition, wherein the purified composition comprises an enzymatically active hyaluronan synthase polypeptide.

36. A purified composition, wherein the purified composition comprises a polypeptide having an amino acid sequence in accordance with SEQ ID NO:

2.

37. A method for detecting a DNA species, comprising the steps of:

obtaining a DNA sample;

contacting the DNA sample with a purified nucleic acid segment in accordance with SEQ ID NO;

1;

hybridizing the DNA sample and the purified nucleic acid segment thereby forming a hybridized complex; and

detecting the complex.

38. A method for detecting a bacterial cell that expresses mRNA encoding Streptococcus equisimilis hyaluronan synthase, comprising the steps of:

obtaining a bacterial cell sample;

contacting at least one nucleic acid from the bacterial cell sample with purified nucleic acid segment in accordance with SEQ ID NO;

1;

hybridizing the at least one nucleic acid and the purified nucleic acid segment thereby forming a hybridized complex; and

detecting the hybridized complex, wherein the presence of the hybridized complex is indicative of a bacterial strain that expresses mRNA encoding Streptococcus equisimilis hyaluronan synthase.

39. A method for producing hyaluronic acid, comprising the steps of:

introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase into a host organism, wherein the host organism contains nucleic acid segments encoding enzymes which produce UDP-GlcNAc and UDP-GlcA;

growing the host organism in a medium to secrete hyaluronic acid; and

recovering the secreted hyaluronic acid.

44. A pharmaceutical composition comprising a preselected pharmaceutical drug and an effective amount of hyaluronic acid produced by hyaluronan synthase.

45. The pharmaceutical composition of claim 44, wherein the hyaluronic acid is produced by the Streptococcus equisimilis hyaluronan synthase of SEQ ID NO:

2.

46. The pharmaceutical composition according to claim 44, wherein the molecular weight of the hyaluronic acid is modified thereby producing a modified molecular weight pharmaceutical composition capable of evading an immune response.

47. The pharmaceutical composition according to claim 44, wherein the molecular weight of the hyaluronic acid is modified thereby producing a modified molecular weight pharmaceutical composition capable of targeting a specific tissue or cell type within the patient having an affinity for the modified molecular weight pharmaceutical composition.

48. A purified and isolated nucleic acid sequence encoding enzymatically active hyaluronan synthase, the nucleic acid sequence selected from the group consisting of:

(a) the nucleic acid sequence in accordance with SEQ ID NO;

1;

(b) complementary nucleic acid sequences to the nucleic acid sequence in accordance with SEQ ID NO;

1;

(c) nucleic acid sequences which will hybridize to the nucleic acid in accordance with SEQ ID NO;

1;

(d) nucleic acid sequences which will hybridize to the complementary nucleic acid sequences of SEQ ID NO;

1; and

(e) nucleic acid sequences which will hybridize to PCR probes selected from the group consisting of PCR probes of SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6.

49. A purified and isolated nucleic acid segment consisting essentially of a nucleic acid segment encoding enzymatically active hyaluronan synthase.

51. An isolated nucleic acid segment consisting essentially of a nucleic acid segment encoding hyaluronan synthase having a nucleic acid segment sufficiently duplicative of the nucleic acid segment in accordance of SEQ ID NO:

1 to allow possession of the biological property of encoding for Streptococcus equisimilis hyaluronan synthase.

52. A cDNA sequence according to claim 51.

53. A procaryotic or eucaryotic host cell transformed or transfected with a nucleic acid segment according to claim 51 in a manner allowing the host cell to express hyaluronic acid.

54. A purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase, wherein the purified nucleic acid segment is capable of hybridizing to the nucleotide sequence in accordance with SEQ ID NO:

1.

55. A purified nucleic acid segment according to SEQ ID NO:

3 capable of hybridizing to SEQ ID NO;

1.

56. A purified nucleic acid segment according to SEQ ID NO:

4 capable of hybridizing to SEQ ID NO;

1.

57. A purified nucleic acid segment according to SEQ ID NO:

5 capable of hybridizing to SEQ ID NO;

1.

58. A purified nucleic acid segment according to SEQ ID NO:

4 capable of hybridizing to SEQ ID NO;

1.

59. A purified nucleic acid segment having a coding region encoding enzymatically active hyaluronate synthase, the purified nucleic acid segment selected from the group consisting of:

(A) the nucleic acid segment according to SEQ ID NO;

2;

(B) the nucleotide sequence in accordance with SEQ ID NO;

1;

(C) nucleic acid segments which hybridize to the nucleic acid segments defined in (A) or (B) or fragments thereof; and

(D) nucleic acid segments which but for the degeneracy of the genetic code, or encoding of functionally equivalent amino acids, would hybridize to the nucleic acid segments defined in (A), (B), and (C).