Hyaluronate synthase gene and uses thereof

• US 20030104415A1
• Filed: 04/15/2002
• Published: 06/05/2003
• Est. Priority Date: 07/01/1994
• Status: Active Grant

• Current Assignee: John Papaconstantinou, Paul H. Weigel, Paul L. Deangelis
• Original Assignee: John Papaconstantinou, Paul H. Weigel, Paul L. Deangelis
• Inventors: DeAngelis, Paul L., Weigel, Paul H., Papaconstantinou, John
• Granted Patent: US 6,855,502 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 9/1051 Hexosyltransferases (2.4.1)
• CPC Class Codes: C12Y 204/01212 Hyaluronan synthase (2.4.1.212)

Abstract

Disclosed are DNA sequences encoding hyaluronic acid synthase that are employed to construct recombinant cells useful in the production of hyaluronate synthase and hyaluronic acid (HA). In preferred aspects, chromosomal DNA encoding the HA synthase gene, hasA, was cloned from a Streptococcus pyogenes genomic library. These vectors were used to transform host cells such as E. coli and acapsular Streptococci to produce hyaluronic acid. Resultant transformants were screened to identify colonies which have incorporated HA synthase DNA in a form that is being actively transcribed into the corresponding HA synthase enzyme. These colonies were selected and employed in the production of hyaluronic acid.

Claims

1. A purified nucleic acid segment encoding HA synthase, wherein the segment:

(a) encodes the HA synthase of SEQ ID NO;

2;

or (b) is capable of hybridizing to the nucleotide sequence of SEQ ID NO;

1 under standard hybridization conditions.

2. The nucleic acid segment of claim 1, further defined as including a nucleotide sequence in accordance with SEQ ID NO:

1.

3. The nucleic acid segment of claim 1, further defined as a recombinant vector.

4. The nucleic acid segment of claim 3, wherein said recombinant vector is a plasmid.

5. The nucleic acid segment of claim 4, further defined a s an expression vector comprising a promoter operatively linked to the HA synthase coding region.

25. A recombinant host cell comprising the recombinant vector having a DNA segment in accordance with claim 1 or claim 10.

26. The recombinant host cell of claim 25, wherein the host cell is a prokaryotic cell.

27. The recombinant host cell of claim 26, wherein the host cell is a eukaryotic cell.

6. A nucleic acid segment which comprises at least a 14 nucleotide long contiguous stretch which corresponds to, or is complementary to, a nucleic acid sequence of SEQ ID NO:

1, and is capable of hybridizing to said nucleic acid segment under standard hybridization conditions.

7. The nucleic acid segment of claim 6, further defined as comprising at least a 20 nucleotide long contiguous stretch which corresponds to, or is complementary to, the nucleic acid sequence of SEQ ID NO:

1.

8. The nucleic acid segment of claim 7, further defined as comprising at least a 30 nucleotide long contiguous stretch which corresponds to, or is complementary to, the nucleic acid sequence of SEQ ID NO:

1.

9. The nucleic acid segment of claim 8, further defined as comprising at least a 50 nucleotide long contiguous stretch which corresponds to, or is complementary to, the nucleic acid sequence of SEQ ID NO:

1.

10. The nucleic acid segment of claim 9, further defined as comprising at least a 100 nucleotide long contiguous stretch which corresponds to, or is complementary to, the nucleic acid sequence of SEQ ID NO:

1.

11. The nucleic acid segment of claim 10, further defined as comprising at least a 200 nucleotide long contiguous stretch which corresponds to, or is complementary to, the nucleic acid sequence of SEQ ID NO:

1.

12. The nucleic acid segment of claim 11, further defined as comprising at least a 300 nucleotide long contiguous stretch which corresponds to, or is complementary to, the nucleic acid sequence of SEQ ID NO:

1.

13. The nucleic acid segment of claim 12, further defined as comprising at least a 500 nucleotide long contiguous stretch which corresponds to, or is complementary to, the nucleic acid sequence of SEQ ID NO:

1.

14. The nucleic acid segment of claim 13, further defined as comprising at least a 1000 nucleotide long contiguous stretch which corresponds to, or is complementary to, the nucleic acid sequence of SEQ ID NO:

1.

15. The nucleic acid segment of claim 14, further defined as comprising at least a 1441 nucleotide long contiguous stretch which corresponds to, or is complementary to, the nucleic acid sequence of SEQ ID NO:

1.

16. The nucleic acid segment of claim 15, further defined as having the nucleic acid sequence of SEQ ID NO:

1.

17. The nucleic acid segment of claim 6, further defined as comprising a nucleic acid fragment of up to 10,000 basepairs in length.

18. The nucleic acid segment of claim 17, further defined as comprising a nucleic acid fragment of up to 5,000 basepairs in length.

19. The nucleic acid segment of claim 18, further defined as comprising a nucleic acid fragment of up to 3,000 basepairs in length.

20. The nucleic acid segment of claim 19, further defined as comprising a nucleic acid fragment of up to 1,000 basepairs in length.

21. The nucleic acid segment of claim 20, further defined as comprising a nucleic acid fragment of up to 500 basepairs in length.

22. The nucleic acid segment of claim 21, further defined as comprising a nucleic acid fragment of up to 100 basepairs in length.

23. The nucleic acid segment of claim 6, further defined as a DNA segment.

24. The nucleic acid segment of claim 6, further defined as including a detectable label.

28. A purified composition comprising a polypeptide having an amino acid sequence in accordance with SEQ ID NO:

2.

29. A method of detecting a DNA species comprising the steps of:

(a) obtaining a DNA sample;

(b) contacting said DNA sample with a nucleic acid segment in accordance with SEQ ID NO;

1 under conditions effective to allow hybridization to form a complex; and

(c) detecting said complex.

31. The method of claim 29, wherein the nucleic acids contacted are located within said cell.

32. The method of claim 29, wherein the nucleic acids are separated from said cell prior to contact.

33. The method of claim 29, wherein the nucleic acid segment comprises a detectable label and hybridized complexes are detected by detecting said label.

30. A method for detecting a bacterial cell that expresses hyaluronic acid synthase, comprising the steps of:

(a) obtaining a bacterial cell sample suspected of expressing hyaluronan synthase;

(b) contacting nucleic acids from said cell sample with a nucleic acid segment in accordance with SEQ ID NO;

1 under conditions effective to allow hybridization of substantially complementary nucleic acid sequences; and

(c) identifying the presence of hybridized complexes containing substantially complementary nucleic acid sequences within said sample;

wherein the presence of increased levels of said substantially complementary nucleic acid sequence is indicative of a bacterial strain that expresses hyaluronan synthase.

34. The method of claim 30, wherein said nucleic acid segment comprises a radio-, an enzymatic-, a fluorescent-, a biotinyl-, or a chemiluminescent-label.

35. A method of preparing hyaluronic acid comprising:

(a) providing a recombinant host cell bearing a vector that encodes a protein that includes an amino acid sequence in accordance with SEQ ID NO;

2;

(b) culturing said cells under conditions permitting nucleic acid expression and protein production; and

(c) recovering hyaluronic acid so produced.

36. The method of claim 35, wherein said host cell is a prokaryote.

37. The method of claim 36, wherein said host cell is a Streptococcal, Lactococcal, Bacilli, Salmonella, Enterococcus, or E. coli host.

38. The method of claim 35, wherein said host cell is a eukaryote.

39. The method of claim 38, wherein said host cell is a Saccharomyces cell.

40. The method of claim 38, wherein said host cell is a HeLa, Vero, NIH 3T3, Chinese Hamster Ovary, or a COS cell.

41. The method of claim 38, wherein said host cell is an insect cell.

42. The method of claim 41, wherein said host cell is infected with a recombinant baculovirus.