Purified thermostable DNA polymerase obtainable from thermococcus litoralis

• US 5,322,785 A
• Filed: 04/17/1991
• Issued: 06/21/1994
• Est. Priority Date: 04/26/1990
• Status: Expired due to Term

• Current Assignee: New England Biolabs Incorporated
• Original Assignee: New England Biolabs Incorporated
• Inventors: Kucera, Rebecca, Jack, William E., Perler, Francine, Comb, Donald G.
• Primary Examiner(s): Wax, Robert A.
• Assistant Examiner(s): Moore, William W.
• Application Number:
  US07/686,340
• Time in Patent Office: 1,161 Days
• Field of Search: 435/194, 435/193, 435/69.1, 435/252.33, 435/320.1, 435/252.31, 536/27, 536/23.2
• US Class Current: 435/194
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Abstract

There is provided an extremely thermostable enzyme obtainable from Thermococcus litoralis. The thermostable enzyme has a molecular weight of about 90,000-95,000 daltons, a half-life of about 60 minutes at 100° C. in the absence of stabilizer, and a half-life of about 95 minutes at 100° C. in the presence of stabilizer, such as octoxynol (TRITON X-100) or bovine serum albumin. The thermostable enzyme possesses a 3'"'"'-5'"'"' proofreading exonuclease activity. The thermostable enzyme may be native or recombinant and may be used for second-strand cDNA synthesis in cDNA cloning, DNA sequencing, and DNA amplification.

Claims

1. A thermostable DNA polymerase endogenous to Thermococcus litoralis, wherein said polymerase is substantially free of 3'"'"'-5'"'"' exonuclease activity and is substantially free of T. litoralis nucleic acids.

2. The thermostable enzyme of claim 1, wherein said enzyme is expressed by E. coli NEB 681.

3. An isolated DNA fragment consisting essentially of an about 1.3 kb DNA region encoding the amino-terminal portion of a thermostable DNA polymerase endogenous to Thermococcus litoralis.

4. The isolated DNA fragment of claim 3, wherein the isolated DNA comprises nucleotides 1 to 1274 of SEQ ID NO:

1.

5. A vector containing the isolated DNA fragment of claim 3.

6. The vector of claim 5, wherein such vector is bacteriophage NEB 618.

7. An isolated DNA fragment consisting essentially of about a 1.6 kb DNA region encoding the intermediate portion of a thermostable DNA polymerase endogenous to Thermococcus litoralis exclusive of about a 5'"'"' terminal 1.3 kb and about a 3'"'"' terminal 1.9 kb portions of the coding region for the DNA polymerase.

8. The isolated DNA fragment of claim 7, wherein the isolated DNA comprises nucleotides 1269 to 2856 of SEQ ID NO:

1.

9. A vector containing the isolated DNA fragment of claim 8.

10. The vector of claim 9, wherein such vector is bacteriophage NEB 620.

11. An isolated DNA fragment consisting essentially of a 1.9 kb DNA region encoding a portion of the carboxyl terminal of a thermostable DNA polymerase endogenous to Thermococcus litoralis.

12. The isolated DNA fragment of claim 11, wherein the isolated DNA comprises nucleotides 2851 to 4771 of SEQ ID NO:

1.

13. A vector containing the isolated DNA fragment of claim 11.

14. The vector of claim 13, wherein such vector is plasmid V153-2.

16. The vector of claim 15, further comprising the isolated DNA fragment of claim 11 operably linked in the proper reading frame to produce Thermococcus litoralis DNA polymerase or a portion thereof.

17. An isolated DNA fragment consisting essentially of a DNA region encoding a thermostable DNA polymerase endogenous to Thermococcus litoralis.

15. A vector comprising the isolated DNA fragment of claim 3 operably linked in the proper reading frame with the isolated DNA fragment of claim 17 to product Thermococcus litoralis DNA polymerase or a portion thereof.

18. A vector containing the DNA sequence of claim 17.

19. A microbial host transformed by the vector of claim 18.

20. An isolated DNA fragment according to claim 17 contained within an approximately 14 kb BamHI restriction fragment of bacteriophage NEB 619.

21. The isolated DNA fragment according to claim 17, comprising an approximately 4 kb BamHI/NdeI restriction fragment of plasmid pPR969.

22. The isolated DNA fragment of claim 17, comprising the DNA fragment of SEQ ID NO:

1.

23. The isolated DNA fragment of claim 22, wherein nucleotides 1776 to 3389 have been deleted.

24. A vector containing the DNA of claim 22.

25. A vector containing the DNA of claim 23.

26. The vector of claim 25, wherein said vector is plasmid V174-1B1.

27. The vector of claim 25, wherein said vector is plasmid pPR969.

28. A microbial host transformed with the vector of claim 26 or claim 27.

29. The transformant of claim 28, wherein said transformant is E. coli NEB 671.

30. The transformant of claim 28, wherein said transformant is E. coli NEB687.

31. A process for the preparation of Thermococcus litoralis DNA polymerase comprising culturing the transformed microbial host of any of claims 28, 29, or 30 under conditions suitable for the expression of Thermococcus litoralis DNA polymerase and recovering Thermococcus litoralis DNA polymerase.

38. The isolated DNA of claim 22, which further codes for the thermostable endonuclease of claim 36.

39. The isolated DNA of claim 38, wherein the coding sequence for the endonuclease starts at nucleotide 3534.

32. A method for producing Thermococcus litoralis DNA polymerase comprising the steps of

(a) purifying total DNA from Thermococcus litoralis;

(b) isolating DNA from the total DNA of step (a) which codes for the DNA polymerase;

(c) removing an about 1.6 kb intervening DNA from said isolated DNA of step (b) wherein said intervening DNA corresponds to a region from about nucleotides 1761-1775 to about nucleotides 3384-3392 as depicted in SEQ ID NO;

1, whereby upon removal a consensus region corresponding to consensus region III of FIG. No. 7 is formed;

(d) ligating the DNA of step (c) into an appropriate vector;

(e) transforming a host with the vector of step (d);

(f) cultivating the transformed host of step (e) under conditions suitable for expression of the T. litoralis DNA polymerase;

(g) recovering the Thermococcus litoralis DNA polymerase.

33. The method of claim 32, wherein the isolated DNA consists essentially of the DNA of SEQ ID NO:

1.

34. The method of claim 32, wherein said intervening DNA corresponds to a region from nucleotide 1773-1775 to nucleotide 3386-3389 as depicted in SEQ ID NO:

1.

35. The method of claim 32, wherein the intervening DNA comprises nucleotides 1776 to 3389 of FIG. No. 6.

36. A substantially pure thermostable endonuclease endogenous to Thermococcus litoralis which cleaves double-stranded deoxynucleotide acid pBR322 at position 164 and positions 2411.

37. The thermostable endonuclease of claim 36, having a molecular weight of about 33,000-37,000 daltons.