CREN7 CHIMERIC PROTEIN

• US 20110020877A1
• Filed: 01/09/2009
• Published: 01/27/2011
• Est. Priority Date: 01/11/2008
• Status: Abandoned Application

• Current Assignee: Genesys Biotech Ltd.
• Original Assignee: Genesys Logic
• Inventors: Morant, Nicholas, Wilkinson, Martin, Clark, Duncan Roy
• Application Number:
  US12/812,519
• Publication Number:
  US 20110020877A1
• Time in Patent Office: Days
• US Class Current: 435/91.2
• CPC Class Codes: C07K 2319/80 containing a DNA binding domain, e.g. Lacl or Tet-repressor
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Abstract

There is provided a chimeric protein comprising a nucleic acid modifying enzyme domain having nucleic acid modifying activity joined with an Cren7 enhancer domain or variant thereof, in which the Cren7 enhancer domain or variant thereof enhances the activity of the nucleic acid modifying enzyme domain compared with a corresponding protein lacking the Cren7 enhancer domain or variant thereof. There is also provided an isolated nucleic acid encoding the chimeric protein of the invention and methods utilising the protein.

Claims

1. A chimeric protein comprising a nucleic acid modifying enzyme domain having nucleic acid modifying activity joined with a Cren7 enhancer domain or variant thereof, in which the Cren7 enhancer domain or variant thereof enhances the activity of the nucleic acid modifying enzyme domain compared with a corresponding protein lacking the Cren7 enhancer domain or variant thereof.

2. A chimeric protein according to claim 1, wherein the Cren7 enhancer domain variant is a functional variant having at least 35% sequence identity with the Cren7 enhancer protein of SEQ ID NO:

1.

3. The protein according to claim 1, in which the nucleic acid modifying enzyme domain comprises a nucleic acid polymerase domain.

6. The protein according to claim 1, in which the Cren7 enhancer domain is one of the group comprising:

Sulfolobus solfataricus Cren7 enhancer protein (SEQ ID NO;

     1);

Sulfolobus acidocaldarius Cren7 enhancer protein (SEQ ID NO;

     2);

Metallosphaera sedula Cren7 enhancer protein (SEQ ID NO;

     3);

Staphylothermus marinus Cren7 enhancer protein (SEQ ID NO;

     4);

Hyperthermus butylicus 0878 Cren7 enhancer protein (SEQ ID NO;

     5);

Hyperthermus butylicus 1128 Cren7 enhancer protein (SEQ ID NO;

     6);

Aeropyrum pernix Cren7 enhancer protein (SEQ ID NO;

     7);

Caldivirga maquilingensis Cren7 enhancer protein (SEQ ID NO;

     8);

Ignicoccus hospitalis Cren7 enhancer protein (SEQ ID NO;

     9);

Pyrobaculum islandicum Cren7 enhancer protein (SEQ ID NO;

     10);

Pyrobaculum arsenaticum Cren7 enhancer protein (SEQ ID NO;

     11);

Pyrobaculum aerophilum Cren7 enhancer protein (SEQ ID NO;

     12);

Pyrobaculum calidifontis Cren7 enhancer protein (SEQ ID NO;

     13);

Thermoproteus neutrophilus Cren7 enhancer protein (SEQ ID NO;

     14), Sulfolobus shibatae Cren7 enhancer protein (SEQ ID NO;

     59); and

Sulfolobus tokodaii Cren7 enhancer protein (SEQ ID NO;

60).

8. The protein according to claim 1, in which the Cren7 enhancer domain or variant thereof comprises the conserved amino acid sequence:

G-X₁-X₂-X₁-X₁-X₃-X₁-P-X₁-K-X₄-W-X₁-L-X₁-P-X₁-G-X₅-X₁-G-V-X₁-X₆-X₇-L-F-X₈-X₁-P-X₉-X₁₀-G-X₁₁X₁-X₁₇ R X₁ X₁ X₁₃ (SEQ ID NO;

     15);

or comprises the conserved amino acid sequence;

X₂-X₁-X₁-X₁-X₁₀-G-X₁-X₁-X₁-X₁-X₃-X₁-P-X₁-K-X₄-W-X₁-L-X₁-P-X₁-G-X₅-X₁-G-V-X₁-X₆-X₇-L-F-X₈-X₁-P-X₉-X₁₀-G-X₁₁-X₁-X₁₂-R-X₁-X₁-X₁₃ (SEQ ID NO;

61) where X₁ is any amino acid, X₂ is K, R or E, X₃ is L or no amino acid, X₄ is A, V or T, X₅ is K or R, X₆ is I or V, X₇ is G or A, X₈ is K, R or Q, X₉ is D, N or E, X₁₀ is any or no amino acid, X₁₁ is K or H, X₁₂ is I, V or F, and X₁₃ is I, V or L.

9. The protein according to claim 1, in which the Cren7 enhancer domain is one of the group comprising:

Sulfolobus solfataricus Cren7 enhancer protein (SEQ ID NO;

     1);

Hyperthermus butylicus 1128 Cren7 enhancer protein (SEQ ID NO;

     6);

Aeropyrum pernix Cren7 enhancer protein (SEQ ID NO;

     7) or in which the Cren7 enhancer domain is a functional variant of any of the Cren7 enhancer proteins of SEQ ID NOs;

1, 6 or 7.

11. The protein according to claim 3, in which the nucleic acid polymerase domain comprises a thermostable DNA polymerase or a functional mutant, variant or derivative thereof, or of a mesophilic DNA polymerase or a functional mutant, variant or derivative thereof, or of an intermediate temperature DNA polymerase or a functional mutant, variant or derivative thereof.

13. The protein according to claim 11, in which the protein is a fusion protein having the sequence of any one of SEQ ID NOs 16-24, or a functional mutant, variant or derivative thereof.

18. A composition comprising the chimeric protein as defined in claim 1.

19. An isolated nucleic acid encoding the chimeric protein as defined in claim 1.

21. A vector comprising the isolated nucleic acid as defined in claim 19.

22. A host cell transformed with the vector of claim 21.

23. A kit comprising the chimeric protein as defined in claim 1, together with packaging materials therefor.

24. A method of modifying a nucleic acid, comprising:

a. contacting the nucleic acid with the chimeric protein as defined in claim 1 under conditions which allow activity of the nucleic acid modifying enzyme domain; and

b. permitting the nucleic acid modifying enzyme domain to modify the nucleic acid.

25. A method of catalysing the synthesis of a polynucleotide from a target nucleic acid, comprising the steps of:

a. providing a chimeric protein as defined in claim 3; and

b. contacting the target nucleic acid with the chimeric protein under conditions which allow the addition by the chimeric protein of nucleotide units to a nucleotide chain using the target nucleic acid, thereby synthesising the polynucleotide.

26. A method of amplifying a sequence of a target nucleic acid using a thermocycling reaction, comprising the steps of:

a. contacting the target nucleic acid with a chimeric protein as defined in claim 3; and

b. incubating the target nucleic acid with the chimeric protein under thermocycling reaction conditions which allow amplification of the target nucleic acid.

28. A kit comprising the composition of claim 18, together with packaging materials therefor.

29. A kit comprising the isolated nucleic acid of claim 19, together with packaging materials therefor.

30. A kit comprising the vector of claim 21, together with packaging materials therefor.

31. A kit comprising the host cell of claim 22, together with packaging materials therefor.

4. (canceled)

5. (canceled)

7. (canceled)

10. (canceled)

12. (canceled)

14-17. -17. (canceled)

20. (canceled)

27. (canceled)