A protein having luciferase activity and at least 60% similarity to luciferase from Photinus pyralis, Luciola mingrelica, Luciola cruciata or Luciola lateralis. Hotaria paroula, Pyrophorus plagiophtalamus, Lampyris noctiluca, Pyrocoelia nayako or Photinus pennsylanvanica wherein in the sequence of the enzyme, at least one of (a) the amino acid residue corresponding to residue 214 in Photinus pyralis luciferase; (b) the amino acid residue corresponding to residue 232 in Photinus pyralis luciferase; (c) the amino acid residue corresponding to residue 295 in Photinus pyralis luciferase; (d) the amino acid residue corresponding to acid 14 of Photinus pyralis luciferase; (e) the amino acid residue corresponding to amino acid 35 of Photinus pyralis luciferase; (f) the amino acid residue corresponding to amino acid residue 105 of Photinus pyralis luciferase; (g) the amino acid residue corresponding to amino acid residue 234 of Photinus pyralis luciferase; (h) the amino acid residue corresponding to amino acid residue 420 of Photinus pyralis luciferase; (i) the amino acid residue corresponding to amino acid residue 310 of Photinus pyralis luciferase; is different to the amino acid which appears in the corresponding wild type sequence and wherein the luciferase enzyme has increased thermostability as compared to an enzyme having the amino acid of the corresponding wild-type luciferase at this position.
1. An isolated recombinant protein comprising a variant of wild-type Photinus pyralis luciferase of SEQ ID NO:37, wherein the amino acid sequence of said recombinant protein has no more than 30 amino acid differences as compared to the amino acid sequence of SEQ ID NO:37, wherein the recombinant protein has alanine at each residue corresponding to positions 214 and 232 of SEQ ID NO:37, and wherein the recombinant protein has luciferase activity and increased thermostability as compared to the wild-type Photinus pyralis luciferase of SEQ ID NO:37.
2. The isolated recombinant protein of claim 1 , wherein the recombinant protein further comprises an amino acid other than glutamic acid at the residue corresponding to position 354 of SEQ ID NO:37.
3. An isolated nucleic acid which encodes the recombinant protein according to claim 1 .
4. A vector comprising the nucleic acid according to claim 3 .
5. An isolated cell transformed with the vector according to claim 4 .
6. The cell according to claim 5 which is a prokaryotic cell.
7. The cell according to claim 5 which is a plant cell.
8. A plant comprising the cell according to claim 7 .
9. A bioluminescent assay comprising the steps of:
contacting the recombinant protein of claim 1 with luciferin and detecting bioluminescence.
10. A kit comprising the recombinant protein according to claim 1 .
11. The kit according to claim 10 which further comprises luciferin.
12. An isolated recombinant protein comprising the amino acid sequence of SEQ ID NO:41, wherein the recombinant protein has luciferase activity and increased thermostability as compared to wild-type Photinus pyralis luciferase.
13. An isolated recombinant protein comprising the amino acid sequence of SEQ ID NO:42, wherein the recombinant protein has luciferase activity and increased thermostability as compared to wild-type Photinus pyralis luciferase.
14. An isolated nucleic acid which encodes the recombinant protein according to claim 12 .
15. An isolated nucleic acid which encodes the recombinant protein according to claim 13 .
16. A vector comprising the nucleic acid according to claim 14 .
17. A vector comprising the nucleic acid according to claim 15 .
18. An isolated cell transformed with the vector according to claim 16 .
19. An isolated cell transformed with the vector according to claim 17 .
20. The cell according to claim 18 which is a prokaryotic cell.
21. The cell according to claim 19 which is a prokaryotic cell.
22. The cell according to claim 18 which is a plant cell.
23. The cell according to claim 19 which is a plant cell.
24. A plant comprising the cell according to claim 22 .
25. A plant comprising the cell according to claim 23 .
26. A bioluminescent assay comprising the steps of:
contacting the recombinant protein of claim 12 with luciferin and detecting bioluminescence.
27. A bioluminescent assay comprising the steps of: contacting the recombinant protein of claim 13 with luciferin and detecting bioluminescence.
28. A kit comprising the recombinant protein according to claim 12 .
29. A kit comprising the recombinant protein according to claim 13 .
30. The kit according to claim 28 which further comprises luciferin.
31. The kit according to claim 29 which further comprises luciferin.
32. An isolated recombinant protein comprising a variant of wild-type Photinus pyralis luciferase of SEQ ID NO:37, wherein the amino acid sequence of said recombinant protein has no more than 30 amino acid differences as compared to the amino acid sequence of SEQ ID NO:37, wherein the recombinant protein has alanine, leucine, and alanine at residues corresponding to positions 214, 215, and 232, respectively, of SEQ ID NO:37, and wherein the recombinant protein has luciferase activity and increased thermostability as compared to the wild-type Photinus pyralis luciferase of SEQ ID NO:37.
33. The isolated recombinant protein of claim 32 , wherein the recombinant protein further comprises an amino acid other than glutamic acid at the residue corresponding to position 354 of SEQ ID NO: 37.
34. An isolated nucleic acid which encodes the recombinant protein according to claim 32 .
35. A vector comprising the nucleic acid according to claim 34 .
36. An isolated cell transformed with the vector according to claim 35 .
37. The cell according to claim 36 which is a prokaryotic cell.
38. The cell according to claim 36 which is a plant cell.
39. A plant comprising the cell according to claim 38 .
40. A bioluminescent assay comprising the steps of:
contacting the recombinant protein of claim 32 with luciferin and detecting bioluminescence.
41. A kit comprising the recombinant protein according to claim 32 .
42. The kit according to claim 41 which further comprises luciferin.