Mutant luciferase
View Patent ↗An isolated recombinant luciferase having luciferase activity. The recombinant luciferase has an amino acid sequence which differs from the wild-type luciferase from Photinus pyralis, Luciola mingrelica, Luciola cruciata, Luciola lateralis, Hotaria parvula, Pyrophorus plagiophthalamus, Lampyris noctiluca, Pyrocoelia miyako or Photinus pennsylvanica . In the sequence of the recombinant luciferase, the amino acid residue corresponding to phenylalanine 295 in Photinus pyralis wild-type luciferase or to leucine 297 in Luciola mingrelica, Luciola cruciata or Luciola lateralis wild-type luciferases, is mutated compared to the corresponding amino acid which appears in the corresponding wild-type luciferase sequence. The recombinant luciferase has increased thermostability compared to the corresponding wild-type luciferase.
1. An isolated recombinant luciferase having luciferase activity and comprising a variant of wild-type Photinus pyralis luciferase of SEQ ID NO: 37, wherein the amino acid sequence of said recombinant luciferase has no more than 50 amino acid differences as compared to the amino acid sequence of SEQ ID NO: 37, wherein the amino acid residue of the recombinant luciferase corresponding to position 295 of SEQ ID NO:37 is leucine, the amino acid residue of the recombinant luciferase corresponding to position 214 of SEQ ID NO:37 is an amino acid residue other than threonine, the amino acid residue of the recombinant luciferase corresponding to position 232 of SEQ ID NO:37 is an amino acid residue other than isoleucine, and the amino acid residue of the recombinant luciferase corresponding to position 354 of SEQ ID NO:37 is an amino acid residue other than glutamic acid, and wherein the recombinant luciferase has increased thermostability as compared to the wild-type Photinus pyralis luciferase of SEQ ID NO:37.
2. The recombinant luciferase of claim 1 , further comprising an amino acid other than phenylalanine at the amino acid residue corresponding to position 14 of SEQ ID NO:37.
3. The recombinant luciferase of claim 2 , wherein the amino acid residue corresponding to position 14 of SEQ ID NO:37 is alanine.
4. The recombinant luciferase of claim 1 , further comprising an amino acid other than leucine at the amino acid residue corresponding to position 35 of SEQ ID NO:37.
5. The recombinant luciferase of claim 4 , wherein the amino acid residue corresponding to position 35 of SEQ ID NO:37 is alanine.
6. The recombinant luciferase of claim 1 , wherein the amino acid at the residue corresponding to position 354 of SEQ ID NO:37 is lysine.
7. The recombinant luciferase of claim 1 , further comprising an amino acid other than alanine at the amino acid residue corresponding to position 215 of SEQ ID NO:37.
8. The recombinant luciferase of claim 7 , wherein the amino acid residue corresponding to position 215 of SEQ ID NO:37 is lysine.
9. The recombinant luciferase of claim 1 , wherein the amino acid residue corresponding to position 214 of SEQ ID NO:37 is alanine, the amino acid residue corresponding to position 232 of SEQ ID NO:37 is alanine and the amino acid residue corresponding to position 354 of SEQ ID NO:37 is lysine.
10. The recombinant luciferase of claim 1 , further comprising an amino acid other than phenylalanine at the amino acid residue corresponding to position 14 of SEQ ID NO:37 and an amino acid other than leucine at the amino acid residue corresponding to position 35 of SEQ ID NO:37.
11. The recombinant luciferase of claim 10 , wherein the amino acid residue corresponding to position 14 of of SEQ ID NO:37 is alanine and the amino acid residue corresponding to position 35 of SEQ ID NO:37 is alanine.
12. The recombinant luciferase of claim 10 , further comprising an amino acid other than alanine at the amino acid residue corresponding to position 215 of SEQ ID NO:37.
13. The recombinant luciferase of claim 12 , wherein the amino acid residue corresponding to position 215 of SEQ ID NO:37 is leucine.
14. A bioluminescent assay comprising the steps of contacting the recombinant luciferase of claim 1 with luciferin and detecting bioluminescence.
15. A kit comprising the recombinant luciferase of claim 1 .