Methods for obtaining thermostable enzymes, DNA polymerase I variants from Thermus aquaticus having new catalytic activities, methods for obtaining the same, and applications of the same

• US 7,488,816 B2
• Filed: 02/25/2005
• Issued: 02/10/2009
• Est. Priority Date: 02/27/2004
• Status: Active Grant

• Current Assignee: Institut Pasteur
• Original Assignee: Institut Pasteur
• Inventors: Ferris, Stephane, Jestin, Jean-Luc, Vichier-Guerre, Sophie
• Primary Examiner(s): Benzion; Gary
• Assistant Examiner(s): WILDER, CYNTHIA B
• Application Number:
  US11/065,943
• Publication Number:
  US 20050250131A1
• Time in Patent Office: 1,446 Days
• Field of Search: 536/23.1, 530/350
• US Class Current: 536/24.3
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07K 14/43563 from insects
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The present invention provides a method for obtaining thermostable enzymes. The present invention also provides variants of DNA polymerase I from Thermus aquaticus. The present invention further provides methods of identifying mutant DNA polymerases having enhanced catalytic activity. The present invention also provides polynucleotides, expression systems, and host cells encoding the mutant DNA polymerases. Still further, the present invention provides a method to carry out reverse transcriptase-polymerase chain reaction (RT-PCR) and kits to facilitate the same.

Claims

1. A purified polynucleotide which encodes a thermostable polypeptide comprising an amino acid sequence having at least 85% identity to residues 13-555 of SEQ ID NO:

26, wherein said polypeptide has (a) a mutation at E465, (b) a mutation at M470 wherein said mutation at M470 is not M470R, or (c) a mutation at M470 and a mutation at E465.

2. The purified polynucleotide of claim 1, wherein said polypeptide further comprises at least one mutation selected from the group consisting of A331T, S335N, F472Y, M484V, M484T, R518, and W550R.

3. The purified polynucleotide of claim 1, wherein said polypeptide has at least 90% identity to residues 13-555 of SEQ ID NO:

26.

4. The purified polynucleotide of claim 1, wherein said polypeptide has at least 95% identity to residues 13-555 of SEQ ID NO:

26.

5. The purified polynucleotide of claim 1, wherein said polypeptide has at least 97.5% identity to residues 13-555 of SEQ ID NO:

26.

6. The purified polynucleotide of claim 1, wherein said polypeptide comprises a mutation at M470 and E465.

7. The purified polynueleotide of claim 1, wherein said polypeptide further comprises a mutation at R518.

8. The purified polynucleotide of claim 7, wherein said mutation at R518 is R518G.

9. The purified polynucleotide of claim 1, wherein said polypeptide further comprises at least one mutation selected from the group consisting of H203R, F205L, T232S, E253G, Q257R, D274G, L275H, L275P, I276F, V309I, I322N, A331V, S335N, I361F, R374Q, A384T, T387A, Y419C, Y419N, F472Y, F472S, A487T, K490E, P493T, M498T, G499E, M502K, L503P, V506I, R518G, A523V, A526V, P539S, E543K, and W550R.

10. The purified polynucleotide of claim 9, wherein said polypeptide comprises an amino acid sequence corresponding to residues 1-543 of SEQ ID NO:

79.

11. The purified polynucleotide of claim 1, wherein said polynucleotide comprises a sequence corresponding to nucleotides 1-1629 of SEQ ID NO:

78.

12. A purified polynucleotide that is fully complementary to the polynucleotide of claim 1.

13. A purified polynucleotide which hybridizes under stringent conditions to the polynucleotide of claim 1;

wherein said stringent conditions comprise washing in 5×

SSC at a temperature from 50 to 68°

C.

14. A vector comprising the purified polynucleotide of claim 1.

15. The vector of claim 14, wherein said polynucleotide is operably linked to a heterologous expression sequence.

16. An isolated host cell comprising the purified polynucleotide of claim 1.

17. The purified polynucleotide of claim 1, wherein said polypeptide further comprises at least one mutation in amino acids 461 to 490 of SEQ ID NO:

26.

18. The purified polynucleotide of claim 1, wherein said polypeptide further comprises at least one mutation at a position selected from the group consisting of H203, F205, T232, E253, Q257, D274, L275, I276, V309, I322, A331, L332, D333, Y334, S335, I361, R374, A384, T387, Y419, P493, M498, G499, M502, L503, V506, R518, A523, A526, P539, E543, and W550.

19. The purified polynucleotide of claim 1, said polypeptide comprises a mutation at E465.

20. The purified polynucleotide of claim 19, wherein said mutation is at E465 and said mutation is E465K.

21. The purified polynucleotide of claim 1, wherein said mutation is a mutation at M470 wherein said mutation at M470 is not M470R.

22. The purified polynucleotide of claim 21, wherein said mutation is at M470and said mutation is M470K.

23. The purified polynucleotide of claim 1, wherein said polypeptide comprises an amino acid sequence corresponding to residues 1-543 of a sequence selected from the group consisting of SEQ ID NO:

67 and SEQ ID NO;

87.

24. The purified polynucleotide of claim 1, wherein said polynucleotide comprises a sequence corresponding to nucleotides 39-1667 of SEQ ID NO:

33.

25. The purified polynucleotide of claim 1, wherein said polynucleotide comprises a sequence corresponding to nucleotides 1-1629 of a sequence selected from the group consisting of SEQ ID NO:

66 and SEQ ID NO;

86.

26. The purified polynucleotide of claim 1, wherein said polypeptide comprises an amino acid sequence corresponding to residues 13-555 of SEQ ID NO:

34.

27. The purified polynucleotide of claim 6, wherein said mutation at E465 is E465K and said mutation at M470 is M470K or M470R.

28. A purified polynucleotide which encodes a thermostable polypeptide comprising an amino acid sequence having at least 85% identity to residues 13-555 of SEQ ID NO:

26, wherein said polypeptide has a mutation at M470 and further comprises at least one mutation selected from the group consisting of H203R, F205L, T232S, E253G, Q257R, D274G, L275H, L275P, I276F, V309I, I322N, A331V, S335N, I361F, R374Q, A384T, T387A, Y419C, Y419N, F472Y, F472S, A487T, K490E, P493T, M498T, G499E, M502K, L503P, V506I, R518G, A523V, A526V, P5395, E543K, and W550R.

29. The purified polynucleotide of claim 28, wherein said mutation at M470 is M470K or M470R.

30. The purified polynucleotide of claim 28, wherein said polypeptide comprises an amino acid sequence corresponding to residues 1-543 of SEQ ID NO:

81.

31. The purified polynucleotide of claim 28, wherein said polynucleotide comprises a sequence corresponding to nucleotides 1-1629 of SEQ ID NO:

80.