Codon-optimized gene for mutated shrimp luciferase and method for use thereof
There has been a demand for a codon-optimized gene for the mutated catalytic domain of Oplophorus luciferase, which is capable of efficiently expressing a protein both in a cultured animal cell and Escherichia coli . There has also been a demand for a substrate coelenterazine analogue showing a higher activity than that of native 19 kDa protein. The invention provides a polynucleotide comprising a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2. According to the invention, bis-coelenterazine is used as a substrate coelenterazine analogue suitable for the photoprotein encoded by the polynucleotide comprising the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2.
1. An isolated polynucleotide selected from the group consisting of (a) to (d) below:
(a) a polynucleotide comprising a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2;
(b) polynucleotide comprising a polynucleotide encoding a protein consisting of a nucleotide sequence in which 1 to 20 nucleotides are deleted, substituted, inserted and/or added in the nucleotide sequence of SEQ ID NO: 2, and having a luminescent catalyst activity by using luciferin as a substrate;
(c) a polynucleotide comprising a polynucleotide encoding a protein consisting of a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 2, and having a luminescent catalyst activity by using lucifeuin as a substrate; and,
(d) a polynucleotide comprising a polynucleotide encoding a protein which hybridizes to a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 2 under high stringency conditions, and has a luminescent catalyst activity by using luciferin as a substrate, wherein the high stringency conditions are 5×SSC, 5×Denhardt's solution, 0.5% (w/v) SDS, 50% (v/v) formamide, and 50° C.
2. The polynucleotide according to claim 1 , which is selected from the group consisting of (a) to (c) below:
(a) a polynucleotide comprising a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2;
(b) a polynucleotide comprising a polynucleotide encoding a protein consisting of a nucleotide sequence in which 1 to 15 nucleotides are deleted, substituted, inserted and/or added in the nucleotide sequence of SEQ ID NO: 2, and having a luminescent catalyst activity by using luciferin as a substrate; and
(c) a polynucleotide comprising a polynucleotide encoding a protein consisting of a nucleotide sequence having at least 95% identity to the nucleotide sequence of SEQ ID NO: 2, and having a luminescent catalyst activity by using luciferin as a substrate.
3. The polynucleotide according to claim 1 , which is selected from the group consisting of (a) to (c) below:
(a) a polynucleotide comprising a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2;
(b) a polynucleotide comprising a polynucleotide encoding a protein consisting of a nucleotide sequence in which 1 to 10 nucleotides are deleted, substituted, inserted and/or added in the nucleotide sequence of SEQ ID NO: 2, and having a luminescent catalyst activity by using luciferin as a substrate; and,
(c) a polynucleotide comprising a polynucleotide encoding a protein consisting of a nucleotide sequence having at least 98% identity to the nucleotide sequence of SEC) ID NO: 2, and having a luminescent catalyst activity by using luciferin as a substrate.
4. An isolated polynucleotide comprising a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2.
5. The polynucleotide according to claim 1 , further comprising a polynucleotide encoding a polypeptide consisting of an amino acid sequence for promoting translation and/or a polynucleotide encoding a polypeptide consisting of an amino acid sequence for purification.
6. An isolated polynucleotide consisting of any one of the nucleotide sequences of SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 14.
7. A recombinant vector comprising the polynucleotide according to claim 1 .
8. An isolated transformed host cell comprising the recombinant vector according to claim 7 .
9. A method of producing a protein, which comprises the step of culturing the transformed host cell according to claim 8 to produce the protein encoded by the polynucleotide.
10. A kit comprising the polynucleotide according to claim 1 , a recombinant vector comprising the polynucleotide or an isolated transformed host cell comprising the vector.
11. The kit according to claim 10 , further comprising bis-coelenterazine.