Chimeric genes for the catalytic protein of
A novel luciferase that distinct from conventional luciferase has been desired. A luciferase mutant comprising the amino acid sequence of SEQ ID NO: 2 substituted at tyrosine at the position of 138, and at least 3 positions selected from the group consisting of isoleucine at the position of 90, proline at the position of 115, glutamine at the position of 124, and asparagine at the position of 166.
1. A luciferase mutant selected from (a) or (b) below:
(a) a luciferase mutant comprising the amino acid sequence of SEQ ID NO: 2 substituted at the positions consisting of:
(i) tyrosine at the position of 138 and isoleucine at the position of 90; and
(ii) at least 2 positions selected from the group consisting of proline at the position of 115, glutamine at the position of 124, and asparagine at the position of 166; and wherein the luciferase mutant has luciferase activity; and
(b) a luciferase mutant comprising the amino acid sequence of SEQ ID NO: 2 substituted at the positions consisting of:
(i) tyrosine at the position of 138 and isoleucine at the position of 90;
(ii) at least 2 positions selected from the group consisting of proline at the position of 115, glutamine at the position of 124, and asparagine at the position of 166; and
(iii) one to sixteen positions other than the positions of 4, 11, 18, 27, 33, 43, 44, 54, 68, 72, and 75, and having a luciferase activity.
2. The luciferase mutant according to claim 1 , wherein the luciferase mutant comprises the amino acid sequence of SEQ ID NO: 2 substituted at the positions consisting of:
(i) tyrosine at the position of 138 and isoleucine at the position of 90;
(ii) at least 2 positions selected from the group consisting of proline at the position of 115, glutamine at the position of 124, and asparagine at the position of 166; and
(iii) one to ten positions other than the positions of 4, 11, 18, 27, 33, 43, 44, 54, 68, 72, and 75.
3. The luciferase mutant according to claim 1 , wherein the tyrosine at the position 138 is substituted with isoleucine or valine.
4. The luciferase mutant according to claim 1 , wherein the luciferase mutant is selected from (c) or (d) below:
(c) a luciferase mutant comprising the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 16; and,
(d) a luciferase mutant comprising the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 16, substituted at one to sixteen positions other than the positions of 4, 11, 18, 27, 33, 43, 44, 54, 68, 72, and 75, of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 16.
5. The luciferase mutant according to claim 4 , wherein the luciferase mutant comprises the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 16, substituted at one to ten positions other than the positions of 4, 11, 18, 27, 33, 43, 44, 54, 68, 72, and 75, of SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 16.
6. A polynucleotide comprising a polynucleotide encoding the luciferase mutant according to claim 1 .
7. A recombinant vector comprising the polynucleotide according to claim 6 .
8. A transformant transformed with the recombinant vector according to claim 7 .
9. A method for producing the luciferase mutant according to claim 1 , which comprises the steps of culturing a transformant transformed with a recombinant vector comprising a polynucleotide encoding the luciferase mutant according to claim 1 and producing the luciferase mutant.
10. A kit comprising at least one selected from the luciferase mutant according to claim 1 , a polynucleotide encoding the luciferase mutant, a recombinant vector comprising the polynucleotide encoding the luciferase mutant, and a transformant transformed with the recombinant vector comprising the polynucleotide encoding the luciferase mutant.
11. The kit according to claim 10 , further comprising a luciferin.
12. The kit according to claim 11 , wherein the luciferin is a coelenterazine analogue.
13. The kit according to claim 12 , wherein the coelenterazine analogue is coelenterazine or h-coelenterazine.
14. A method for performing a luminescence reaction, which comprises contacting the luciferase mutant according to claim 1 with a luciferin.
15. The method according to claim 14 , wherein the luciferin is a coelenterazine analogue.
16. The method according to claim 15 , wherein the coelenterazine analogue is coelenterazine or h-coelenterazine.
17. A method for assaying the transcription activity of a promoter-containing polynucleotide sequence, which comprises using a polynucleotide encoding the luciferase mutant according to claim 1 as a reporter gene, wherein the polynucleotide encoding the luciferase mutant is operably fused to the promoter-containing polynucleotide sequence, wherein the luciferase mutant expressed is contacted with a luciferin.
18. The method according to claim 17 , wherein the luciferin is a coelenterazine analogue.
19. The method according to claim 18 , wherein the coelenterazine analogue is coelenterazine or h-coelenterazine.