Thermostable polymerase

• US 6,130,045 A
• Filed: 06/11/1998
• Issued: 10/10/2000
• Est. Priority Date: 06/11/1998
• Status: Expired due to Term

• Current Assignee: Takara Bio Usa, Inc. (Ningbo Junsheng Investment Group Co., Ltd.)
• Original Assignee: Clontech Laboratories Incorporated
• Inventors: Qiu, Zhi-Hao, Wurst, Helmut
• Primary Examiner(s): Houtteman, Scott W.
• Application Number:
  US09/096,399
• Time in Patent Office: 852 Days
• Field of Search: 435/6, 435/183, 536/23.1
• US Class Current: 435/6.12
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Abstract

A thermostable enzyme having polymerase activity and substantially no nuclease activity is provided. The thermostable enzyme is characterized by comprising a sequence of nine amino acid residues at least proximal to the N-terminus that has less than 50% but greater than 40% amino acid sequence identity with residues 280 to 288 of the naturally occurring Thermus aquaticus polymerase. The subject enzyme finds use in a variety of different application in which polynucleotides are enzymatically produced, particularly in PCR based applications.

Claims

1. A thermostable enzyme having polymerase activity and substantially no nuclease activity, where the amino acid sequence of said enzyme comprises a sequence of nine amino acid residues at least proximal to the N-terminus of the enzyme that has less than 50% but greater than 40% amino acid identity with residues 280 to 288 of naturally occurring Thermus aquaticus polymerase.

2. The thermostable enzyme according to claim 1, wherein the number of amino acid residues in said sequence of nine amino acid residues that are identical to the residues of said residues 280 to 288 of naturally occurring Thermus aquaticus polymerase is four.

3. The thermostable enzyme according to claim 1, wherein said nine amino acid residues are:

space="preserve" listing-type="equation">MRGHEX.sub.1 GLX.sub.2

wherein X₁ and X₂ are hydrophilic residues.

4. The thermostable enzyme according to claim 1, wherein said enzyme has a molecular weight ranging from about 60 to 70 kDal as measured by SDS-PAGE.

5. The thermostable enzyme according to claim 1, wherein the C-terminal portion of said enzyme has an amino acid sequence that is substantially the same as the amino acid sequence of naturally occurring Thermus aquaticus polymerase.

21. A nucleic acid having a nucleotide sequence encoding the thermostable enzyme according to claim 1.

22. A nucleic acid according to claim 21, wherein said nucleic acid has a nucleic acid sequence that is substantially identical to the nucleotide sequence of SEQ ID NO:

01 or SEQ ID NO;

03.

23. A fragment of the nucleic acid according to claim 21.

24. An isolated nucleic acid or mimetic thereof that hybridizes under stringent conditions to the nucleic acid according to claim 21 or its complementary sequence.

25. An expression cassette comprising a transcriptional initiation region functional in an expression host, a nucleic acid having a nucleotide sequence found in the nucleic acid according to claim 21 under the transcriptional regulation of said transcriptional initiation region, and a transcriptional termination region functional in said expression host.

26. A cell comprising an expression cassette according to claim 25 as part of an extrachromosomal element or integrated into the genome of a host cell as a result of introduction of said expression cassette into said host cell.

27. The cellular progeny of the host cell according to claim 26.

28. A method of producing the thermostable enzyme according to claim 1, said method comprising:

growing a cell according to claims 26 or 27, whereby said thermostable enzyme is expressed; and

isolating said thermostable enzyme substantially free of other proteins.

29. A polymerase composition comprising:

the thermostable enzyme according to claim 1; and

at least one additional polymerase.

30. The polymerase composition according to claim 29, wherein said at least one additional polymerase is a Family B polymerase.

31. The polymerase composition according to claim 29, wherein said polymerase is thermostable.

32. The polymerase composition according to claim 29, wherein said polymerase has nuclease activity.

33. The polymerase composition according to claim 29, wherein said polymerase is Deep Vent polymerase.

34. A kit for use in the enzymatic production of polynucleotides, said kit comprising:

the thermostable enzyme according to claim 1.

35. The kit according to claim 34, wherein said kit further comprises dNTPs.

36. The kit according to claim 35, wherein said kit further comprises at least one buffer medium.

37. The kit according to claim 34, wherein said kit further comprises a second polymerase.

6. A thermostable enzyme having polymerase activity and substantially no nuclease activity, wherein the N-terminal domain of said enzyme comprises a sequence of nine amino acid residues at least proximal to the N-terminus, wherein said nine amino acid residues are:

space="preserve" listing-type="equation">MRGHEX.sub.1 GLX.sub.2

wherein X₁ and X₂ are hydrophilic residues.

7. The thermostable enzyme according to claim 6, wherein said hydrophilic residues are polar and uncharged.

8. The thermostable enzyme according to claim 7, wherein said X₁ is selected from the group consisting of threonine and serine.

9. The thermostable enzyme according to claim 7, wherein said X₂ is selected from the group consisting of asparganine and glutamine.

10. The thermostable enzyme according to claim 6, wherein the C-terminal portion of said enzyme has an amino acid sequence that is substantially the same as the amino acid sequence of naturally occurring Thermus aquaticus polymerase.

11. The thermostable enzyme according to claim 10, wherein said enzyme is 553 residues long and the amino acid sequence of residues 10 to 553 is substantially the same as residues 289 to 832 of the naturally occurring Thermus aquaticus polymerase.

12. The thermostable enzyme according to claim 10, wherein said enzyme is 560 residues long and the amino acid sequence of residues 17 to 560 is substantially the same as residues 289 to 832 of the naturally occurring Thermus aquaticus polymerase.

13. A thermostable enzyme having polymerase activity and substantially no nuclease activity, wherein said enzyme is 553 amino acid residues long, wherein the nucleotide sequence of residues 1 to 9 is:

space="preserve" listing-type="equation">MRGHEX.sub.1 GLX.sub.2

wherein X₁ and X₂ are polar uncharged residues; and

the amino acid sequence of residues 10 to 553 is substantially the same as residues 289 to 832 of the naturally occurring Thermus aquaticus polymerase.

14. The thermostable enzyme according to claim 13, wherein X₁ is serine.

15. The thermostable enzyme according to claim 13, wherein X₂ is glutamine.

16. A thermostable polymerase having the amino acid sequence of SEQ ID NO:

02.

17. A thermostable enzyme having polymerase activity and substantially no nuclease activity, wherein said enzyme is 560 amino acid residues long, wherein the nucleotide sequence of residues 1 to 15 is:

space="preserve" listing-type="equation">TMITNSMRGHEX.sub.1 GLX.sub.2

wherein X₁ and X₂ are polar uncharged residues; and

the amino acid sequence of residues 16 to 560 is substantially the same as residues 289 to 832 of the naturally occurring Thermus aquaticus polymerase.

18. The thermostable enzyme according to claim 17, wherein X₁ is serine.

19. The thermostable enzyme according to claim 17, wherein X₂ is glutamine.

20. A thermostable polymerase having the amino acid sequence of SEQ ID NO:

04.