JP2009065925A - Polypeptide having photogenic catalytic activity - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a polypeptide having a photogenic catalytic activity, consisting of less amino acid residues in the polypeptide having the photogenic catalytic activity by using luciferin as a photogenic substrate. <P>SOLUTION: The polypeptide comprises an amino acid sequence consisting of a specific sequence of secretor luciferase originated from a shellfish living in deep sea. The polypeptide has the photogenic catalytic activity, and is useful as a reporter protein, a photogenic marker, etc. Also, a polynucleotide encoding the polypeptide is useful as a reporter gene, etc. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a polypeptide having a luminescent catalytic activity using luciferin (for example, coelenterazines) as a substrate, a polynucleotide encoding the same, a recombinant vector containing the polynucleotide, a transformant containing the recombinant vector, The present invention relates to a method for producing the polypeptide.

  Of the luciferin-luciferase luminescence reaction systems reported to date, a simple luminescence reaction system is a system that is performed only with a luminescent substrate (luciferin), oxygen, and an enzyme (luciferase). Among the luciferases used in such a luminescence reaction system, the genes of the luciferase groups (for example, luciferases described in Table 1) using coelenterazine and Cypridina luciferin, which are imidazopyrazinone compounds, as luminescent substrates have been isolated. With the advancement of luminescence detection devices, higher sensitivity, and the spread of general-purpose machines, they are already recognized and widely used as industrially important enzymes or reporter proteins. For such a luciferase group, various detection methods have been established. However, when the luciferases disclosed so far are used industrially, there are disadvantages and advantages, respectively, and there is no luciferase for all purposes. Therefore, it is necessary to select an appropriate luciferase for each application.

Table 1. Classification of imidazopyrazinone luciferases

The molecular weights of these imidazopyrazinone luciferases vary. For example, when used as a reporter gene or as a fusion protein, the smaller the molecular weight, the easier the gene recombination operation is, which is very advantageous. Examples of the luciferase having a small molecular weight include the secretory Evil luciferase catalytic domain 19 kDa (169 amino acids) derived from the crustacean shrimp and the secreted Gaussia luciferase (168 amino acids) derived from Gaussia belonging to the copepod. However, secreted Gaussia luciferase is the smallest luciferase reported so far. In the primary structure of Gaussia luciferase, an overlap of 71 amino acids presumed to be formed by gene duplication has been found, but the catalytic activity has not been studied at all.
The gene and amino acid sequence (195 amino acids) of Gaussia luciferase have already been reported (for example, Patent Document 1: WO 99/49019). Further, a metric deluciferase gene and an amino acid sequence (219 amino acids) which are included in the same species as Gaussia and show homology with Gaussia luciferase have also been reported (for example, Non-patent Document 1: J. Biol. Chem. 279). , 3212-3217 (2004)).
International Publication No. 99/49019 Pamphlet Markova, SV, Golz, S., Frank, LA, Kalthof, B., and Vysotski, ES (2004) Cloning and expression of cDNA for a luciferase from the marine copepod Metridia longa. J. Biol. Chem. 279, 3212- 3217.

  In the above situation, a polypeptide having a luminescence catalytic activity using luciferin as a luminescent substrate and having an amino acid sequence consisting of a smaller number of amino acid residues has been demanded. If such a polypeptide can be obtained, it will be an important step for creating an artificial catalyst, and a polypeptide (enzyme) that is a molecule having a novel luminescent catalytic activity will be created based on the polypeptide (domain). Is also possible.

As a result of diligent research, the present inventor has found that a polypeptide containing the 27th to 97th amino acid sequence or the 98th to 168th amino acid sequence of a secreted luciferase derived from a crustacean living in the deep sea is luciferin (for example, coelenterazines). It has been found that it has a luminescent catalytic activity using as a substrate.
In addition, the inventors have found that the luminescent catalytic activity of Gaussia luciferase or a polypeptide containing the amino acid sequence of 27-97 or 98-168 of Gaussia luciferase is activated by halide ions.
As a result of further studies based on these findings, the present invention has been completed.
That is, the present invention
(1) The polypeptide according to any one of the following (a) to (d):
(A) a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3;
(B) a luminescent catalytic activity comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 and luciferin as a substrate; A polypeptide having;
(C) a polypeptide having an amino acid sequence having 80% or more identity to SEQ ID NO: 1 or SEQ ID NO: 3 and having a luminescent catalytic activity using luciferin as a substrate; and (d) SEQ ID NO: Luminescent catalytic activity comprising an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence 2 or SEQ ID NO: 4 and using luciferin as a substrate A polypeptide having:
However, the protein containing the amino acid sequence represented by SEQ ID NO: 6 is excluded.
(1a) The polypeptide according to any one of the following (a) to (d):
(A) a polypeptide comprising the amino acid sequence of SEQ ID NO: 1;
(B) a polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 1, and having a luminescent catalytic activity using luciferin as a substrate;
(C) a polypeptide having an amino acid sequence having 80% or more identity to SEQ ID NO: 1 and having a luminescence catalytic activity using luciferin as a substrate; and (d) a nucleotide sequence of SEQ ID NO: 2. A polypeptide containing an amino acid sequence encoded by a polynucleotide that hybridizes with a polynucleotide comprising a complementary base sequence under stringent conditions and having a luminescent catalytic activity using luciferin as a substrate;
However, the protein containing the amino acid sequence represented by SEQ ID NO: 6 is excluded.
(1b) The polypeptide according to any one of the following (a) to (d):
(A) a polypeptide comprising the amino acid sequence of SEQ ID NO: 3;
(B) a polypeptide having an amino acid sequence in which one to a plurality of amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 3 and having a luminescence catalytic activity using luciferin as a substrate;
(C) a polypeptide having an amino acid sequence having 80% or more identity to SEQ ID NO: 3 and having a luminescent catalytic activity using luciferin as a substrate; and (d) a nucleotide sequence of SEQ ID NO: 4 A polypeptide containing an amino acid sequence encoded by a polynucleotide that hybridizes with a polynucleotide comprising a complementary base sequence under stringent conditions and having a luminescent catalytic activity using luciferin as a substrate;
However, the protein containing the amino acid sequence represented by SEQ ID NO: 6 is excluded.
(2) The polypeptide according to (1) above, which is any of the following (a) to (d):
(A) a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3;
(B) a luminescence catalytic activity comprising an amino acid sequence in which 1 to 25 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 and luciferin as a substrate; A polypeptide having;
(C) a polypeptide having an amino acid sequence having 90% or more identity to SEQ ID NO: 1 or SEQ ID NO: 3 and having a luminescent catalytic activity using luciferin as a substrate; and (d) SEQ ID NO: A luminescent catalyst comprising an amino acid sequence encoded by a polynucleotide that hybridizes under high stringency conditions with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of 2 or SEQ ID NO: 4, and using luciferin as a substrate A polypeptide having activity;
(2a) The polypeptide according to (1a) above, which is any of the following (a) to (d):
(A) a polypeptide comprising the amino acid sequence of SEQ ID NO: 1;
(B) a polypeptide having an amino acid sequence in which 1 to 25 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 1 and having a luminescence catalytic activity using luciferin as a substrate;
(C) a polypeptide having an amino acid sequence having 90% or more identity to SEQ ID NO: 1 and having a luminescence catalytic activity using luciferin as a substrate; and (d) a base sequence of SEQ ID NO: 2. A polypeptide comprising an amino acid sequence encoded by a polynucleotide that hybridizes under high stringency conditions with a polynucleotide comprising a complementary base sequence, and having a luminescent catalytic activity using luciferin as a substrate;
(2b) The polypeptide according to (1b) above, which is any of the following (a) to (d):
(A) a polypeptide comprising the amino acid sequence of SEQ ID NO: 3;
(B) a polypeptide having an amino acid sequence in which 1 to 25 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 3 and having a luminescent catalytic activity using luciferin as a substrate;
(C) a polypeptide having an amino acid sequence having 90% or more identity to SEQ ID NO: 3 and having a luminescence catalytic activity using luciferin as a substrate; and (d) a nucleotide sequence of SEQ ID NO: 4 A polypeptide comprising an amino acid sequence encoded by a polynucleotide that hybridizes under high stringency conditions with a polynucleotide comprising a complementary base sequence, and having a luminescent catalytic activity using luciferin as a substrate;
(3) The polypeptide according to (1) above, which is a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
(3a) The polypeptide according to (1a) above, which is a polypeptide containing the amino acid sequence of SEQ ID NO: 1,
(3b) The polypeptide according to (1b) above, which is a polypeptide containing the amino acid sequence of SEQ ID NO: 3.
(4) a polynucleotide containing a polynucleotide encoding the polypeptide according to any one of (1) to (3) above,
(5) The polynucleotide according to (4) above, which is any of the following (e) to (i):
(E) a polynucleotide containing a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4;
(F) a polypeptide having a luminescent catalytic activity that hybridizes with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4 under stringent conditions and has luciferin as a substrate. A polynucleotide containing the encoding polynucleotide;
(G) a polynucleotide comprising a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3;
(H) a luminescent catalytic activity comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 and luciferin as a substrate; A polynucleotide containing a polynucleotide encoding the polypeptide having; and (i) an amino acid sequence having 80% or more identity to SEQ ID NO: 1 or SEQ ID NO: 3, and using luciferin as a substrate A polynucleotide comprising a polynucleotide encoding a polypeptide having a luminescent catalytic activity,
(5a) The polynucleotide according to (4) above, which is any of the following (e) to (i):
(E) a polynucleotide containing a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2;
(F) a polynucleotide that encodes a polypeptide that hybridizes with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 2 under stringent conditions and has a luminescence catalytic activity using luciferin as a substrate; Containing polynucleotides;
(G) a polynucleotide comprising a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1;
(H) a polypeptide containing an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 1 and having a luminescent catalytic activity using luciferin as a substrate A polynucleotide containing a polynucleotide that encodes; and (i) an amino acid sequence having at least 80% identity to SEQ ID NO: 1 and encoding a polypeptide having a luminescent catalytic activity using luciferin as a substrate A polynucleotide containing the polynucleotide;
(5b) The polynucleotide according to (4) above, which is any of the following (e) to (i):
(E) a polynucleotide containing a polynucleotide consisting of the base sequence of SEQ ID NO: 4;
(F) a polynucleotide that hybridizes with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 4 under a stringent condition and encodes a polypeptide having a luminescence catalytic activity using luciferin as a substrate; Containing polynucleotides;
(G) a polynucleotide comprising a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 3;
(H) a polypeptide containing an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 3 and having a luminescent catalytic activity using luciferin as a substrate And (i) encoding a polypeptide having an amino acid sequence having 80% or more identity to SEQ ID NO: 3 and having a luminescent catalytic activity using luciferin as a substrate A polynucleotide containing the polynucleotide;
(6) The polynucleotide according to (4), which is any of the following (e) to (i):
(E) a polynucleotide containing a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4;
(F) a polypeptide having a luminescence catalytic activity that hybridizes with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4 under a highly stringent condition and uses luciferin as a substrate. A polynucleotide comprising a polynucleotide encoding
(G) a polynucleotide comprising a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3;
(H) a luminescent catalytic activity comprising an amino acid sequence in which 1 to 25 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 and luciferin as a substrate; A polynucleotide containing a polynucleotide encoding the polypeptide having; and (i) an amino acid sequence having 90% or more identity to SEQ ID NO: 1 or SEQ ID NO: 3, and using luciferin as a substrate A polynucleotide comprising a polynucleotide encoding a polypeptide having a luminescent catalytic activity,
(6a) The polynucleotide according to (4), which is any of the following (e) to (i):
(E) a polynucleotide containing a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2;
(F) a polynucleotide that hybridizes with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 2 under a highly stringent condition and encodes a polypeptide having a luminescence catalytic activity using luciferin as a substrate A polynucleotide comprising:
(G) a polynucleotide comprising a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1;
(H) A polypeptide containing an amino acid sequence in which 1 to 25 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 1 and having a luminescent catalytic activity using luciferin as a substrate And (i) encoding a polypeptide having an amino acid sequence having 90% or more identity to SEQ ID NO: 1 and having a luminescence catalytic activity using luciferin as a substrate A polynucleotide containing the polynucleotide;
(6b) The polynucleotide according to (4) above, which is any of the following (e) to (i):
(E) a polynucleotide containing a polynucleotide consisting of the base sequence of SEQ ID NO: 4;
(F) a polynucleotide that hybridizes with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 4 under a highly stringent condition and encodes a polypeptide having a luminescence catalytic activity using luciferin as a substrate A polynucleotide comprising:
(G) a polynucleotide comprising a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 3;
(H) A polypeptide containing an amino acid sequence in which 1 to 25 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 3 and having a luminescent catalytic activity using luciferin as a substrate And (i) encoding a polypeptide having an amino acid sequence having 90% or more identity to SEQ ID NO: 3 and having a luminescence catalytic activity using luciferin as a substrate A polynucleotide containing the polynucleotide;
(7) A recombinant vector containing the polynucleotide according to any one of (4) to (6b) above,
(8) A transformant introduced with the recombinant vector according to (7) above,
(9) Any of the above (1) to (3b), comprising a step of culturing the transformant according to (8) and producing the polypeptide according to any of (1) to (3b) above. A process for producing the polypeptide according to claim 1,
(10) A kit comprising the polypeptide according to any one of (1) to (3b) above,
(11) A kit comprising the polynucleotide according to any one of (4) to (6b) above, the recombinant vector according to (7) above, or the transformant according to (8) above;
(12) The kit according to (10) or (11), further comprising luciferin,
(13) The kit according to (12) above, wherein the luciferin is coelenterazines,
(14) The kit according to (13), wherein the coelenterazine is coelenterazine,
(15) The polypeptide according to any one of the following (a) to (d):
(A) a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3;
(B) a luminescence catalytic activity comprising an amino acid sequence in which 1 to 25 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 and luciferin as a substrate; A polypeptide having;
(C) a polypeptide having an amino acid sequence having 90% or more identity to SEQ ID NO: 1 or SEQ ID NO: 3 and having a luminescent catalytic activity using luciferin as a substrate; and (d) SEQ ID NO: A luminescent catalyst comprising an amino acid sequence encoded by a polynucleotide that hybridizes under high stringency conditions with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of 2 or SEQ ID NO: 4, and using luciferin as a substrate A polypeptide having activity,
And a method of performing a luminescent reaction, comprising contacting luciferin with
(16) The method according to (15) above, wherein the luminescence reaction is performed in the presence of halide ions,
(17) The method according to (16) above, wherein the halide ion is a fluorine ion, a bromine ion or an iodine ion,
(18) The polypeptide according to any one of (a) to (d) below, using a halide ion:
(A) a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3;
(B) a luminescence catalytic activity comprising an amino acid sequence in which 1 to 25 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 and luciferin as a substrate; A polypeptide having;
(C) a polypeptide having an amino acid sequence having 90% or more identity to SEQ ID NO: 1 or SEQ ID NO: 3 and having a luminescent catalytic activity using luciferin as a substrate; and (d) SEQ ID NO: A luminescent catalyst comprising an amino acid sequence encoded by a polynucleotide that hybridizes under high stringency conditions with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of 2 or SEQ ID NO: 4, and using luciferin as a substrate A polypeptide having activity; or a protein according to any one of the following (a) to (d):
(A) a protein containing the amino acid sequence of SEQ ID NO: 6 and having a luminescence catalytic activity using luciferin as a substrate;
(B) a protein having an amino acid sequence in which 1 to 25 amino acids are deleted, substituted, inserted or added in the amino acid sequence of SEQ ID NO: 6 and having a luminescence catalytic activity using luciferin as a substrate;
(C) a protein having an amino acid sequence having about 90% or more identity with the amino acid sequence of SEQ ID NO: 6 and having a luminescence catalytic activity using luciferin as a substrate; and (d) a base sequence of SEQ ID NO: 5. A protein having an amino acid sequence encoded by a polynucleotide that hybridizes under high stringency conditions with a polynucleotide comprising a complementary base sequence, and having a luminescent catalytic activity using luciferin as a substrate,
A method for activating the luminescence activity of
(19) The method according to (18) above, wherein the halide ion is a fluorine ion, a bromine ion or an iodine ion.

Since the polypeptide of the present invention has a luminescent catalytic activity using luciferin (for example, coelenterazines) as a substrate and has a low molecular weight, the production efficiency of a polypeptide having a luminescent catalytic activity can be improved. .
According to the method for activating the luminescence activity of the present invention, the luminescence efficiency of the polypeptide of the present invention or the protein used in the present invention can be improved.

Hereinafter, embodiments of the present invention will be described in detail.
1. Polypeptide of the Present Invention Polypeptide I of the present invention is a polypeptide having the amino acid sequence of SEQ ID NO: 1 and a polypeptide having substantially the same activity or function as the polypeptide having the amino acid sequence of SEQ ID NO: 1. Means. In the present specification, a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 and a polypeptide having substantially the same activity or function as the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 are referred to as “carboxy terminal domain-deleted Gaussian”. Sometimes referred to as “luciferase”.
The polypeptide II of the present invention means a polypeptide having the amino acid sequence of SEQ ID NO: 3 and a polypeptide having substantially the same activity or function as the polypeptide having the amino acid sequence of SEQ ID NO: 3. In the present specification, a polypeptide having the amino acid sequence of SEQ ID NO: 3 and a polypeptide having substantially the same activity or function as the polypeptide having the amino acid sequence of SEQ ID NO: 3 are referred to as “amino-terminal domain-deleted Gaussia”. Sometimes referred to as “luciferase”.
The polypeptide I of the present invention and the polypeptide II of the present invention may be collectively referred to as “the polypeptide of the present invention”.
The protein used in the present invention means a protein consisting of the amino acid sequence of SEQ ID NO: 6 and a protein having substantially the same activity or function as the protein consisting of the amino acid sequence of SEQ ID NO: 6. The protein used in the present invention includes “Gaussia luciferase”.

  The substantially homogeneous activity or function is, for example, a luminescent catalytic activity using luciferin (for example, coelenterazines) as a substrate, that is, luciferin (for example, coelenterazines) is oxidized by oxygen molecules and oxyluciferin is in an excited state. It means the activity of catalyzing the reaction to be generated. Note that oxyluciferin generated in an excited state emits visible light and becomes a ground state.

The luciferin used in the present invention may be luciferin that can serve as a substrate for the polypeptide of the present invention or the protein used in the present invention. Specific examples of luciferin used in the present invention include coelenterazines.
In the present specification, coelenterazines mean coelenterazine and coelenterazine derivatives. Examples of coelenterazine derivatives include h-coelenterazine, hcp-coelenterazine, cp-coelenterazine, f-coelenterazine, fcp-coelenterazine, n-coelenterazine, Bis-coelenterazine, MeO-coelenterazine, e-coelenterazine, cl-coelenterazine, etc. Can be given.

More specifically, the polypeptide of the present invention includes, for example, (a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, and comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3. A polypeptide having substantially the same activity or function as a peptide; (b) an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added in the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3; A polypeptide comprising and having substantially the same activity or function as a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3; (a ′) the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 Or (b ′) one to a plurality of amino acids deleted, substituted, or inserted in the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 Properly consists of the amino acid sequence added, and SEQ ID NO: 1 or SEQ ID NO: like polypeptide may be mentioned having a polypeptide activity or ability substantially similar comprising the amino acid sequence of 3.
More specifically, the protein used in the present invention includes, for example; (a) an activity containing substantially the same quality as a protein containing the amino acid sequence of SEQ ID NO: 6 and consisting of the amino acid sequence of SEQ ID NO: 6. A protein having a function; (b) a protein comprising an amino acid sequence in which one to a plurality of amino acids are deleted, substituted, inserted or added in the amino acid sequence of SEQ ID NO: 6 and consisting of the amino acid sequence of SEQ ID NO: 6; A protein having substantially the same activity or function; (a ′) a protein comprising the amino acid sequence of SEQ ID NO: 6; or (b ′) one to a plurality of amino acids are deleted from the amino acid sequence of SEQ ID NO: 6. A protein consisting of a substituted, inserted or added amino acid sequence and having substantially the same activity or function as the protein consisting of the amino acid sequence set forth in SEQ ID NO: 6 And the like.

  In the present specification, the range of “1 to plural” in “an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added” is, for example, 1 to 50, 1 to 30, 1-25, 1-22, 1-20, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6 (1-several) 1-5, 1-4, 1-3, 1-2, and 1. In general, the smaller the number of amino acids deleted, substituted, inserted or added, the better. Two or more of the amino acid residue deletions, substitutions, insertions and additions may occur simultaneously. Such proteins are described in “Molecular Cloning 3rd Edition”, “Current Protocols in Molecular Biology”, “Nuc. Acids. Res., 10, 6487 (1982)”, “Proc. Natl. Acad. Sci. USA, 79, 6409 (1982) ”,“ Gene, 34, 315 (1985) ”,“ Nuc. Acids. Res., 13, 4431 (1985) ”,“ Proc. Natl. Acad. Sci. USA , 82, 488 (1985) ”and the like.

In the amino acid sequence deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, the fifth, sixth and eighth amino acid sequences described in SEQ ID NO: 1 or SEQ ID NO: 3 26th, 27th, 29th, 30th, 31st, 34th, 39th, 44th, 45th, 48th, 50th, 51st, 53rd, 65th, 68th and 71st Among amino acids, for example, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 Preferably, one or more, 17 or more, 18 or more, or all 19 amino acids are not deleted or substituted. In general, the greater the number of amino acids that are not deleted or substituted, the better.
Specific examples of the polypeptide I of the present invention include, for example, a polypeptide containing the amino acid sequence of SEQ ID NO: 1, a polypeptide containing the amino acid sequence of SEQ ID NO: 13, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 1. Peptide, polypeptide having the amino acid sequence of SEQ ID NO: 13 and the like can be mentioned.
Specific examples of the polypeptide II of the present invention include, for example, a polypeptide containing the amino acid sequence of SEQ ID NO: 3, a polypeptide containing the amino acid sequence of SEQ ID NO: 17, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 3. Peptide, polypeptide having the amino acid sequence of SEQ ID NO: 17, and the like can be mentioned.
Specific examples of the protein used in the present invention include, for example, a protein containing the amino acid sequence of SEQ ID NO: 6, a protein containing the amino acid sequence of SEQ ID NO: 8, a protein comprising the amino acid sequence of SEQ ID NO: 6, and a sequence A protein having the amino acid sequence of No. 8;

Examples of the polypeptide containing the amino acid sequence of SEQ ID NO: 1 include a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1, a polypeptide consisting of the amino acid sequence of SEQ ID NO: 13, and the like.
Examples of the polypeptide containing the amino acid sequence of SEQ ID NO: 3 include a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3, a polypeptide consisting of the amino acid sequence of SEQ ID NO: 17, and the like.
Examples of the protein containing the amino acid sequence of SEQ ID NO: 6 include a protein consisting of the amino acid sequence of SEQ ID NO: 6, a protein consisting of the amino acid sequence of SEQ ID NO: 8.

  Examples of the substitution of one or several amino acids in the amino acid sequence of SEQ ID NO: 1 are: (1) the first Arg in the amino acid sequence of SEQ ID NO: 1 is replaced with Ile, and (2) the second Gly is Pro (3) 3rd Lys replaced with Glu, (4) 4th Leu replaced with Ile, (5) 7th Lys replaced with Phe, (6) 9th Leu replaced with Asp (7) 10th Pro is replaced with Leu, (8) 11th Leu is replaced with Glu, (9) 12th Glu is replaced with Pro, (10) 13th Val is replaced with Met, ( 11) 14th Leu replaced with Glu, (12) 15th Lys replaced with Gln, (13) 16th Glu replaced with Phe, (14) 17th Met replaced with Ile, (15) 18th Glu is Al (16) 19th Ala is replaced with Gln, (17) 20th Asn is replaced with Val, (18) 21st Ala is replaced with Asp, (19) 22nd Arg is replaced with Leu (20) 23rd Lys is replaced with Cys, (21) 24th Ala is replaced with Val, (22) 25th Gly is replaced with Asp, (23) 28th Arg is replaced with Thr, ( 24) 32nd Ile replaced with Lys, (25) 33rd Cys replaced with Gly, (26) 35th Ser replaced with Ala, (27) 36th His replaced with Asn, (28) 37th Ile replaced with Val, (29) 38th Lys replaced with Gln, (30) 40th Thr replaced with Ser, (31) 41st Pro replaced with Asp, (32) 42nd Lys replaced with Leu, (33) 43rd Met replaced with Leu, (34) 46th Phe replaced with Trp, (35) 47th Ile replaced with Leu, (36) 49th Gly replaced with Leu (37) 52nd His is replaced with Ala, (38) 54th Tyr is replaced with Phe, (39) 55th Glu is replaced with Ala, (40) 56th Gly is replaced with Ser (41) 57th Asp is replaced with Lys, (42) 58th Lys is replaced with Ile, (43) 59th Glu is replaced with Gln, (44) 60th Ser is replaced with Gly, (45) 61st Ala is replaced with Gln, (46) 62nd Gln is replaced with Val, (47) 63rd Gly is replaced with Asp, (48) 64th Gly is replaced with Lys, (4 9) 66th Gly replaced with Lys, (50) 67th Glu replaced with Gly, (51) 69th Ile replaced with Gly, (52) 70th Val replaced with Gly, (53) Or the combination of these substitutions etc. is mention | raise | lifted.

  As an example of substitution of one or several amino acids in the amino acid sequence of SEQ ID NO: 3, (1) the first Ile of the amino acid sequence of SEQ ID NO: 3 is substituted with Arg, and (2) the second Pro is Gly (3) 3rd Glu is replaced with Lys, (4) 4th Ile is replaced with Leu, (5) 7th Phe is replaced with Lys, (6) 9th Asp is replaced with Leu (7) 10th Leu is replaced with Pro, (8) 11th Glu is replaced with Leu, (9) 12th Pro is replaced with Glu, (10) 13th Met is replaced with Val, ( 11) 14th Glu replaced with Leu, (12) 15th Gln replaced with Lys, (13) 16th Phe replaced with Glu, (14) 17th Ile replaced with Met, (15) 18th Ala is Gl (16) 19th Gln is replaced with Ala, (17) 20th Val is replaced with Asn, (18) 21st Asp is replaced with Ala, (19) 22nd Leu is replaced with Arg (20) 23rd Cys is replaced with Lys, (21) 24th Val is replaced with Ala, (22) 25th Asp is replaced with Gly, (23) 28th Thr is replaced with Arg, ( 24) 32nd Lys replaced with Ile, (25) 33rd Gly replaced with Cys, (26) 35th Ala replaced with Ser, (27) 36th Asn replaced with His, (28) 37th Val is replaced with Ile, (29) 38th Gln is replaced with Lys, (30) 40th Ser is replaced with Thr, (31) 41st Asp is replaced with Pro, (32) 42nd Leu is replaced with Lys, (33) 43rd Leu is replaced with Met, (34) 46th Trp is replaced with Phe, (35) 47th Leu is replaced with Ile, (36) 49th Gln is replaced with (37) 52nd Ala replaced with His, (38) 54th Phe replaced with Tyr, (39) 55th Ala replaced with Glu, (40) 56th Ser replaced with Gly (41) 57th Lys replaced with Asp, (42) 58th Ile replaced with Lys, (43) 59th Gln replaced with Glu, (44) 60th Gly replaced with Ser, (45) 61st Gln is replaced with Ala, (46) 62nd Val is replaced with Gln, (47) 63rd Asp is replaced with Gly, (48) 64th Lys is replaced with Gly, (4 9) 66th Lys replaced with Gly, (50) 67th Gly replaced with Glu, (51) 69th Gly replaced with Ile, (52) 70th Gly replaced with Val, (53) Or the combination of these substitutions etc. is mention | raise | lifted.

Examples of the polypeptide having substantially the same activity or function as the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 include, for example, about 80% of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3. Contains amino acid sequences having 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more identity And a polypeptide having substantially the same activity or function as the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3. More specifically, for example, (c) a polypeptide having substantially the same activity or function as the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 includes, for example, SEQ ID NO: 1 or The amino acid sequence of SEQ ID NO: 3 and about 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99 And a polypeptide having a luminescence catalytic activity containing an amino acid sequence having at least% identity and having luciferin (for example, coelenterazines) as a substrate.
Examples of the protein having substantially the same activity or function as the protein consisting of the amino acid sequence of SEQ ID NO: 6 include, for example, about 80% or more, 90% or more, 91% or more, 92% of the amino acid sequence of SEQ ID NO: 6. More than 93%, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more of the amino acid sequence having identity and comprising the amino acid sequence of SEQ ID NO: 6 Examples also include proteins having substantially the same activity or function as proteins. More specifically, for example, (c) as a protein having substantially the same activity or function as the protein consisting of the amino acid sequence of SEQ ID NO: 6, for example, about 80% or more of the amino acid sequence of SEQ ID NO: 6 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, and 99% or more amino acid sequence And a protein having a luminescence catalytic activity using luciferin (for example, coelenterazines) as a substrate.
In general, the larger the numerical value of identity, the better. The identity of amino acid sequences and base sequences is determined by BLAST (see, for example, Altzshul SF et al., J. Mol. Biol. 215, 403 (1990)) or FASTA (Pearson WR, Methods in Enzymology 183, 63 (See (1990), etc.). When using BLAST or FASTA, the default parameters of each program are used.

The polypeptide of the present invention includes an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4. And a polypeptide having substantially the same activity or function as a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, (d) a base sequence of SEQ ID NO: 2 or SEQ ID NO: 4 A polynucleotide having an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide comprising a complementary base sequence and having a luminescent catalytic activity using luciferin (eg, coelenterazines) as a substrate Peptides are also included.
The protein used in the present invention contains an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a base sequence complementary to the base sequence of SEQ ID NO: 5. And a protein having substantially the same activity or function as the protein consisting of the amino acid sequence of SEQ ID NO: 6, (d) a polynucleotide consisting of a base sequence complementary to the base sequence of SEQ ID NO: 5, and stringent conditions Also included are proteins having an amino acid sequence encoded by a polynucleotide that hybridizes with the above and having a luminescent catalytic activity using luciferin (eg, coelenterazines) as a substrate.
The polynucleotide that hybridizes under stringent conditions will be described later.

  The polypeptide of the present invention or the protein used in the present invention may further contain a peptide sequence for purification and / or a secretory signal peptide. The peptide sequence used in this technical field can be used as the peptide sequence for purification. Examples of the peptide sequence for purification include a histidine tag sequence having an amino acid sequence of 4 or more, preferably 6 or more histidine residues, an amino acid sequence of a binding domain to glutathione of glutathione S-transferase, or Examples include the amino acid sequence of protein A. The secretory signal peptide means a peptide region that plays a role of allowing a protein or polypeptide bound to the secretory signal peptide to permeate the cell membrane. The amino acid sequences of such secretory signal peptides and the nucleic acid sequences encoding them are well known and reported in the art (eg von Heijine G (1988) Biochim. Biohys. Acra 947: 307-333, von Heijine G (1990) J. Membr. Biol. 115: 195-201). More specifically, examples of the secretion signal peptide include a secretion signal peptide (OmpA) derived from outer membrane protein A of E. coli (Ghrayeb, J. et al. (1984) EMBO J. 3: 2437-2442), cholera. Examples include secretory signal peptides derived from fungus-derived cholera toxin.

  There are no particular limitations on the method for obtaining the polypeptide of the present invention or the protein used in the present invention. The polypeptide of the present invention or the protein used in the present invention may be a polypeptide synthesized by chemical synthesis or a recombinant protein produced by a gene recombination technique. When the polypeptide of the present invention or the protein used in the present invention is chemically synthesized, for example, it can be synthesized by Fmoc method (fluorenylmethyloxycarbonyl method), tBoc method (t-butyloxycarbonyl method) or the like. it can. Chemical synthesis using peptide synthesizers such as Advanced Chemtech, PerkinElmer, Pharmacia, Protein Technology Instrument, Syntheselvega, Perceptive, Shimadzu, etc. You can also. When the polypeptide of the present invention or the protein used in the present invention is produced by a gene recombination technique, it can be produced by an ordinary gene recombination technique. More specifically, the polypeptide of the present invention or the present invention can be used by introducing a polynucleotide (eg, DNA) encoding the polypeptide of the present invention or the protein used in the present invention into an appropriate expression system. Proteins can be made. The expression of the polypeptide of the present invention or the polynucleotide encoding the protein used in the present invention, the expression of the polypeptide of the present invention or the protein used in the present invention will be described later.

2. Polynucleotide of the Present Invention The present invention also provides a polynucleotide encoding the above-described polypeptide of the present invention or the protein used in the present invention. The polynucleotide of the present invention may be any polynucleotide as long as it contains a base sequence encoding the polypeptide of the present invention or the protein used in the present invention, but is preferably DNA. Examples of DNA include genomic DNA, genomic DNA library, cell / tissue-derived cDNA, cell / tissue-derived cDNA library, and synthetic DNA. The vector used for the library is not particularly limited and may be any of bacteriophage, plasmid, cosmid, phagemid and the like. Moreover, it can also be amplified directly by reverse transcription polymerase chain reaction (hereinafter abbreviated as RT-PCR method) using a total RNA or mRNA fraction prepared from the cells / tissues described above.

The polynucleotide of the present invention includes (e) a polynucleotide containing a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4; (f) complementary to the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4 A polypeptide that hybridizes with a polynucleotide comprising a basic nucleotide sequence under stringent conditions and has substantially the same activity or function as a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 A polynucleotide comprising a polynucleotide encoding; (g) a polynucleotide comprising a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3; (h) SEQ ID NO: 1 or sequence In the amino acid sequence of No. 3, one to a plurality of amino acids are deleted, substituted, inserted and And / or a polynucleotide containing a polynucleotide that encodes a polypeptide containing an added amino acid sequence and having substantially the same activity or function as a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 Or (i) a polypeptide substantially comprising an amino acid sequence having 80% or more identity to SEQ ID NO: 1 or SEQ ID NO: 3 and consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3; In particular, a polynucleotide containing a polynucleotide encoding a polypeptide having the same activity or function is included.
Furthermore, the polynucleotide of the present invention includes (e) a polynucleotide containing a polynucleotide consisting of the base sequence of SEQ ID NO: 5; (f) a polynucleotide consisting of a base sequence complementary to the base sequence of SEQ ID NO: 5 A polynucleotide containing a polynucleotide encoding a polypeptide that hybridizes with the polypeptide under stringent conditions and has substantially the same activity or function as the polypeptide consisting of the amino acid sequence of SEQ ID NO: 6; (g) A polynucleotide comprising a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 6; (h) one to a plurality of amino acids in the amino acid sequence of SEQ ID NO: 6 deleted, substituted, inserted and / or A poly comprising the added amino acid sequence and comprising the amino acid sequence of SEQ ID NO: 6 A polynucleotide comprising a polynucleotide encoding a polypeptide having substantially the same activity or function as a peptide; or (i) an amino acid sequence having 80% or more identity to SEQ ID NO: 6; And a polynucleotide containing a polynucleotide encoding a polypeptide having substantially the same activity or function as the polypeptide consisting of the amino acid sequence of SEQ ID NO: 6.

  Here, “a polynucleotide that hybridizes under stringent conditions (for example, DNA)” is a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 5. Alternatively, a colony hybridization method, a plaque hybridization method, a southern hybridization method, or the like is used by using all or part of the polynucleotide encoding the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 6 as a probe. The polynucleotide obtained by this. Specifically, hybridization was performed at 65 ° C. in the presence of 0.7 to 1.0 mol / L NaCl using a filter on which colony or plaque-derived DNA was immobilized, and then 0.1 to 2 times. By using a SSC (Saline-sodium citrate) solution at a concentration (composition of 1-fold concentration of SSC solution is 150 mmol / L sodium chloride, 15 mmol / L sodium citrate) and washing the filter under 65 ° C. Polynucleotides that can be identified can be mentioned.

  Hybridization is described in Sambrook J. et al., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press (2001) (hereinafter abbreviated as Molecular Cloning 3rd Edition), Ausbel FM et al., Current Protocols. In experimental documents such as in Molecular Biology, Supplement 1-38, John Wiley and Sons (1987-1997), Glover DM and Hames BD, DNA Cloning 1: Core Techniques, A practical Approach, Second Edition, Oxford University Press (1995) It can be carried out according to the method described.

  As used herein, the “stringent conditions” may be any of low stringency conditions, moderate stringency conditions, and high stringency conditions. “Low stringent conditions” are, for example, conditions of 5 × SSC, 5 × Denhardt's solution, 0.5% (w / v) SDS, 50% (v / v) formamide, 32 ° C. The “medium stringent conditions” are, for example, conditions of 5 × SSC, 5 × Denhardt's solution, 0.5% (w / v) SDS, 50% (v / v) formamide, 42 ° C. “High stringent conditions” are, for example, conditions of 5 × SSC, 5 × Denhardt's solution, 0.5 (w / v)% SDS, 50% (v / v) formamide, 50 ° C. The tighter the conditions, the higher the complementarity required for duplex formation. Specifically, for example, under these conditions, it can be expected that DNA having higher homology can be efficiently obtained as the temperature is increased. However, multiple factors such as temperature, probe concentration, probe length, ionic strength, time, and salt concentration can be considered as factors that affect hybridization stringency. Those skilled in the art will select these factors as appropriate. It is possible to achieve similar stringency.

  In addition, when using a commercially available kit for hybridization, Alkphos Direct Labeling Reagents (made by Amersham Pharmacia) can be used, for example. In this case, follow the protocol supplied with the kit, incubate with the labeled probe overnight, and then wash the membrane with a primary wash buffer containing 0.1% (w / v) SDS at 55 ° C. , Hybridized DNA can be detected.

  Other hybridizable polynucleotides include a polypeptide or sequence comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 when calculated using default parameters by an analysis program such as FASTA or BLAST. Number: A polynucleotide encoding a protein comprising the amino acid sequence of 6 and about 80% or more, 85% or more, 88% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, 99% As mentioned above, a polynucleotide containing a polynucleotide having the identity of 99.3% or more, 99.5% or more, 99.7% or more, 99.8% or more, 99.9% or more can be mentioned. The identity of amino acid sequences and base sequences can be determined using the method described above.

A polynucleotide encoding a polypeptide having an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted and / or added to a certain amino acid sequence can be synthesized by site-directed mutagenesis (eg, Gotoh, T et al., Gene 152, 271-275 (1995), Zoller, MJ, and Smith, M., Methods Enzymol. 100, 468-500 (1983), Kramer, W. et al., Nucleic Acids Res. 12 , 9441-9456 (1984), Kramer W, and Fritz HJ, Methods.Enzymol. 154, 350-367 (1987), Kunkel, TA, Proc. Natl. Acad. Sci. USA. 82, 488-492 (1985) , Kunkel, Methods Enzymol. 85, 2763-2766 (1988), etc.), methods utilizing amber mutation (eg, Gapped duplex method, Nucleic Acids Res. 12, 9441-9456 (1984), etc.) It can be obtained by using.
PCR (for example, Ho SN et al., Gene 77, 51 (for example, Ho SN et al., Gene 77, 51 ( 1989), etc.) can also introduce mutations into polynucleotides.

  A polynucleotide encoding a partial fragment of a polypeptide that is a kind of deletion mutant is a sequence that matches the 5'-end base sequence of the region encoding the partial fragment to be prepared in the polynucleotide encoding the polypeptide. And an oligonucleotide having a sequence complementary to the nucleotide sequence at the 3 ′ end as a primer and performing PCR using a polynucleotide encoding the polypeptide as a template.

Specific examples of the polynucleotide of the present invention include, for example, a polynucleotide containing a polynucleotide encoding a polypeptide containing the amino acid sequence of SEQ ID NO: 1, and a polypeptide containing the amino acid sequence of SEQ ID NO: 3. A polynucleotide containing a polynucleotide, a polynucleotide containing a polynucleotide encoding a polypeptide containing the amino acid sequence of SEQ ID NO: 13, and a polynucleotide encoding a polypeptide containing the amino acid sequence of SEQ ID NO: 17. A polynucleotide containing a polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3; a polynucleotide containing a polynucleotide encoding the polypeptide consisting of the amino acid sequence of SEQ ID NO: 3 And a polynucleotide containing a polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 13, a polynucleotide containing a polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 17, and the like. .
Specific examples of the polynucleotide of the present invention include, for example, a polynucleotide containing a polynucleotide encoding a polypeptide containing the amino acid sequence of SEQ ID NO: 6, and a polypeptide containing the amino acid sequence of SEQ ID NO: 8. A polynucleotide comprising a polynucleotide comprising: a polynucleotide comprising a polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 6; a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 8; Examples thereof include polynucleotides.

  Examples of the polynucleotide encoding a polypeptide containing the amino acid sequence of SEQ ID NO: 1 include a polynucleotide containing a polynucleotide consisting of the base sequence shown in SEQ ID NO: 2. Examples of the polynucleotide encoding the polypeptide containing the amino acid sequence shown in SEQ ID NO: 3 include a polynucleotide containing a polynucleotide consisting of the base sequence shown in SEQ ID NO: 4. Examples of the polynucleotide encoding the polypeptide containing the amino acid sequence shown in SEQ ID NO: 13 include a polynucleotide containing a polynucleotide consisting of the base sequence shown in SEQ ID NO: 14. Examples of the polynucleotide encoding the polypeptide containing the amino acid sequence shown in SEQ ID NO: 17 include a polynucleotide containing a polynucleotide consisting of the base sequence shown in SEQ ID NO: 18.

  The polynucleotide of the present invention may comprise a polynucleotide encoding a peptide sequence for purification and / or a polynucleotide encoding a secretory signal peptide. As a polynucleotide encoding a peptide sequence for purification, a polynucleotide containing a base sequence encoding a peptide sequence for purification used in the art can be used. Examples of the peptide sequence for purification include those described above. As a polynucleotide encoding a secretory signal peptide, a polynucleotide containing a nucleic acid sequence encoding a secretory signal peptide known in the art can be used. Examples of the secretory signal peptide include those described above.

3. Recombinant vector and transformant of the present invention Furthermore, the present invention provides a recombinant vector and a transformant containing the above-described polynucleotide of the present invention.
(1) Preparation of recombinant vector The recombinant vector of the present invention can be obtained by ligating (inserting) the polynucleotide (for example, DNA) of the present invention into an appropriate vector. More specifically, it can be obtained by cleaving a purified polynucleotide (DNA) with a suitable restriction enzyme, inserting it into a restriction enzyme site or a multiple cloning site of a suitable vector, and ligating the vector. The vector for inserting the polynucleotide of the present invention is not particularly limited as long as it can replicate in the host, and examples thereof include plasmids, bacteriophages, animal viruses and the like. Examples of plasmids include plasmids derived from E. coli (eg, pBR322, pBR325, pUC118, pUC119, etc.), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, etc.), and plasmids derived from yeast (eg, YEp13, YEp24, YCp50, etc.). can give. Examples of the bacteriophage include λ phage. Examples of animal viruses include retroviruses, vaccinia viruses, insect viruses (eg, baculoviruses) and the like.

  The polynucleotide of the present invention is usually operably linked downstream of a promoter in an appropriate vector. The promoter used is preferably an SV40-derived promoter, a retrovirus promoter, a metallothionein promoter, a heat shock promoter, a cytomegalovirus promoter, an SRα promoter or the like when the host to be transformed is an animal cell. When the host is Escherichia, a Trp promoter, T7 promoter, lac promoter, recA promoter, λPL promoter, lpp promoter and the like are preferable. When the host is Bacillus, SPO1 promoter, SPO2 promoter, penP promoter and the like are preferable. When the host is yeast, PHO5 promoter, PGK promoter, GAP promoter, ADH1 promoter, GAL promoter, etc. are preferable. When the host is an insect cell, polyhedrin promoter, P10 promoter and the like are preferable.

  In addition to the above, the recombinant vector of the present invention may include those containing an enhancer, a splicing signal, a poly A addition signal, a ribosome binding sequence (SD sequence), a selection marker, and the like as desired. Examples of the selection marker include a dihydrofolate reductase gene, an ampicillin resistance gene, a neomycin resistance gene, and the like.

(2) Preparation of transformant A recombinant vector containing the polynucleotide of the present invention (that is, the polynucleotide encoding the polypeptide of the present invention or the protein used in the present invention) thus obtained, A transformant can be prepared by introducing it into a suitable host. The host is not particularly limited as long as it can express the polynucleotide (DNA) of the present invention. For example, Escherichia, Bacillus, Pseudomonas, Rhizobium, yeast, animal cells or Insect cells. Examples of the genus Escherichia include Escherichia coli. Examples of the genus Bacillus include Bacillus subtilis. Examples of Pseudomonas spp. Include Pseudomonas putida. Examples of the genus Rhizobium include Rhizobium meliloti. Examples of the yeast include Saccharomyces cerevisiae and Schizosaccharomyces pombe. Examples of animal cells include COS cells and CHO cells. Examples of insect cells include Sf9 and Sf21.

  The introduction method of the recombinant vector into the host and the transformation method thereby can be performed by various general methods. As a method for introducing a recombinant vector into a host cell, for example, calcium phosphate method (Virology, 52, 456-457 (1973)), lipofection method (Proc. Natl. Acad. Sci. USA, 84, 7413 (1987) ), Electroporation method (EMBO J., 1, 841-845 (1982)). Examples of the transformation method of the genus Escherichia include the methods described in Proc. Natl. Acad. Sci. USA, 69, 2110 (1972), Gene, 17, 107 (1982) and the like. Examples of the method for transforming Bacillus include the method described in Molecular & General Genetics, 168, 111 (1979). Examples of yeast transformation methods include the method described in Proc. Natl. Acad. Sci. USA, 75, 1929 (1978). Examples of methods for transforming animal cells include the method described in Virology, 52, 456 (1973). Examples of the method for transforming insect cells include the method described in Bio / Technology, 6, 47-55 (1988). In this manner, a transformant transformed with a recombinant vector containing a polynucleotide encoding the polypeptide of the present invention or the protein used in the present invention (polynucleotide of the present invention) can be obtained.

4). Production of the polypeptide of the present invention or the protein used in the present invention The present invention also includes a step of culturing the transformant to produce the polypeptide of the present invention or the protein used in the present invention. A method for producing a polypeptide or a protein used in the present invention is provided. The polypeptide of the present invention or the protein used in the present invention is obtained by culturing the transformant under conditions capable of expressing the polypeptide of the present invention or a polynucleotide (DNA) encoding the protein used in the present invention. The polypeptide of the invention or the protein used in the present invention can be produced / accumulated, separated and purified.

(1) Culture of transformant The culture of the transformant of the present invention can be performed according to a usual method used for culturing a host. By the culture, the polypeptide of the present invention or the protein used in the present invention is produced by the transformant, and the polypeptide of the present invention or the protein used in the present invention is accumulated in the transformant or in the culture solution.

  As a medium for cultivating transformants whose hosts are Escherichia or Bacillus genus, it contains a carbon source, nitrogen source, inorganic salts, etc. necessary for the growth of the transformant, so that transformants can be cultured efficiently. Any natural or synthetic medium may be used as long as it can be performed automatically. As the carbon source, carbohydrates such as glucose, fructose, sucrose and starch, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol are used. As the nitrogen source, ammonia, ammonium salts of inorganic or organic acids such as ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, or other nitrogen-containing compounds, peptone, meat extract, corn steep liquor, and the like are used. Examples of inorganic salts include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate. During culture, an antibiotic such as ampicillin or tetracycline may be added to the medium as necessary. When cultivating a transformant transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, when cultivating a transformant transformed with an expression vector using the Lac promoter, transformation by transforming isopropyl-β-D-thiogalactopyranoside (IPTG) with an expression vector using the trp promoter When culturing the body, indoleacrylic acid (IAA) or the like may be added to the medium.

  When the host is an Escherichia bacterium, the culture is usually carried out at about 15 to 43 ° C. for about 3 to 24 hours, and if necessary, aeration or agitation is added. When the host is a genus Bacillus, the culture is usually performed at about 30 to 40 ° C. for about 6 to 24 hours, and if necessary, aeration or agitation is added.

  As a medium for cultivating a transformant whose host is yeast, for example, a Burkholder minimum medium (Proc. Natl. Acad. Sci. USA, 77, 4505 (1980)) or 0.5% (w / v ) SD medium containing casamino acid (Proc. Natl. Acad. Sci. USA, 81, 5330 (1984)). The pH of the medium is preferably adjusted to about 5-8. Culture is usually performed at about 20 ° C. to 35 ° C. for about 24 to 72 hours, and aeration and agitation are added as necessary.

  As a medium for culturing a transformant whose host is an animal cell, for example, a MEM medium (Science, 122, 501 (1952)) containing about 5 to 20% (v / v) fetal calf serum, a DMEM medium (Virology) , 8, 396 (1959)). The pH is preferably about 6-8. The culture is usually performed at about 30 ° C. to 40 ° C. for about 15 to 60 hours, and aeration and agitation are added as necessary.

  As a medium for cultivating transformants whose host is an insect cell, an appropriate addition of an additive such as 10% (v / v) bovine serum inactivated in Grace's Insect Medium (Nature, 195, 788 (1962)) Is used. The pH of the medium is preferably adjusted to about 6.2 to 6.4. The culture is usually performed at about 27 ° C. for about 3 to 5 days, and aeration and agitation are added as necessary.

(2) Separation / Purification of Polypeptide of the Present Invention or Protein Used in the Present Invention By separating / purifying the polypeptide of the present invention or the protein used in the present invention from the culture, the polypeptide of the present invention or The protein used in the present invention can be obtained. Here, the culture means any of a culture solution, cultured cells or cultured cells, or disrupted cultured cells or cultured cells. Separation / purification of the polypeptide of the present invention or the protein used in the present invention can be performed according to a usual method.

  Specifically, when the polypeptide of the present invention or the protein used in the present invention is accumulated in cultured cells or cultured cells, after culturing, a usual method (for example, ultrasound, lysozyme, freeze-thawing, etc.) ), And then the crude extract of the polypeptide of the present invention or the protein used in the present invention can be obtained by an ordinary method (for example, centrifugation, filtration, etc.). When the polypeptide of the present invention or the protein used in the present invention accumulates in the periplasmic space, the polypeptide of the present invention or the present invention is used by the usual method (for example, osmotic shock method etc.) after completion of the culture. An extract containing the obtained protein can be obtained. When the polypeptide of the present invention or the protein used in the present invention is accumulated in the culture solution, the cells or cells and the culture supernatant are collected by a conventional method (for example, centrifugation, filtration, etc.) after completion of the culture. A culture supernatant containing the polypeptide of the present invention or the protein used in the present invention can be obtained.

  Purification of the polypeptide of the present invention or the protein used in the present invention contained in the extract or culture supernatant thus obtained can be performed according to a conventional separation / purification method. Separation / purification methods include, for example, ammonium sulfate precipitation, gel filtration chromatography, ion exchange chromatography, affinity chromatography, reverse phase high performance liquid chromatography, dialysis, ultrafiltration, etc., alone or in appropriate combination. be able to. When the polypeptide of the present invention or the protein used in the present invention contains the above-described peptide sequence for purification, it is preferably purified using this. Specifically, when the polypeptide of the present invention or the protein used in the present invention contains a histidine tag sequence, nickel chelate affinity chromatography, and when it contains a binding domain to glutathione of S-transferase, glutathione binding. In the case of containing an amino acid sequence of protein A or gel A, antibody affinity chromatography can be used.

5). Use of the polypeptide of the present invention or the protein used in the present invention (use as a reporter protein)
The polypeptide of the present invention or the protein used in the present invention can also be used as a reporter protein for measurement of transcriptional activity of a promoter or the like. Construction of a vector in which a polynucleotide encoding the polypeptide of the present invention or the protein used in the present invention (ie, the polynucleotide of the present invention) is fused to a target promoter or other expression control sequence (for example, an enhancer). . Introducing the vector into a host cell and measuring the activity of the target promoter or other expression control sequence by detecting luminescence derived from the polypeptide of the present invention or the protein used in the present invention in the presence of luciferin. Can do.
The polynucleotide of the present invention can be used as a reporter gene as described above.

(Use as a detection marker by luminescence)
The polypeptide of the present invention or the protein used in the present invention can be used as a detection marker by luminescence in the presence of luciferin. The detection marker of the present invention can be used for detecting a target substance in, for example, an immunoassay or a hybridization assay. The polypeptide of the present invention or the protein used in the present invention can be used by binding to the target protein or target nucleic acid by a commonly used method such as a chemical modification method. The detection method using such a detection marker can be performed by a normal method. In addition, the detection marker of the present invention can be used for measuring the distribution of the target protein by expressing it as a fusion protein with the target protein and introducing it into the cell by a technique such as microinjection. it can. Such measurement of the distribution of the target protein or the like can also be performed using a detection method such as luminescence imaging. In addition, the polypeptide of the present invention or the protein used in the present invention can be used by being expressed in a cell in addition to being introduced into the cell by a technique such as a microinjection method.

(Materials for amusement supplies)
The polypeptide of the present invention or the protein used in the present invention has an activity of catalyzing a reaction in which luciferin is oxidized with oxygen molecules and oxyluciferin is generated in an excited state. Excited oxyluciferin emits visible light to the ground state. Therefore, the polypeptide of the present invention or the protein used in the present invention can be suitably used as a light-emitting base material for amusement goods. Examples of the amusement goods include a light emitting soap bubble, a light emitting ice, a light emitting bowl, and a light emitting paint. The amusement product of the present invention can be manufactured by a usual method.

6). Kit of the present invention The present invention also includes a kit comprising any one selected from the polypeptide of the present invention or the protein used in the present invention, the polynucleotide of the present invention, the recombinant vector of the present invention and the transformant of the present invention. provide. The kit of the present invention may further contain luciferin (for example, coelenterazine or a derivative thereof). The kit of the present invention can be produced by commonly used materials and methods. The kits of the invention may include, for example, sample tubes, plates, instructions for kit users, solutions, buffers, reagents, samples suitable for standardization or control samples. The kit of the present invention may further contain a salt containing a halide ion.

  The kit of the present invention can be used for measurement using the above-described reporter protein or reporter gene, a fluorescent marker, and the like.

7). Luminescence Reaction Method and Method for Activating Luminescence Activity The present invention uses a halide ion to produce a luminescence activity using the luciferin (eg, coelenterazines) of the polypeptide of the present invention or the protein used in the present invention as a substrate ( Also provided is a method of activating the luminescent catalyst activity.
(Luminescence activity)
The polypeptide of the present invention or the protein used in the present invention has an activity of catalyzing a reaction in which luciferin (for example, coelenterazines) is oxidized with oxygen molecules to produce excited oxyluciferin. Excited oxyluciferin emits visible light when it reaches the ground state. That is, the polypeptide of the present invention or the protein used in the present invention has an activity of catalyzing a luminescence reaction using luciferin (for example, coelenterazines) as a substrate to generate luminescence. This activity is sometimes referred to herein as “luminescence activity”.
(Luminescence reaction)
The luminescence reaction using the polypeptide of the present invention or the protein used in the present invention and luciferin (for example, coelenterazines) as a substrate is brought into contact with the polypeptide of the present invention or the protein luciferin used in the present invention. It can be carried out. As the reaction conditions, it can be carried out under conditions usually used for luminescence reaction using Gaussia luciferase or conditions based thereon (for example, WO99 / 49019, J. Biol. CHem. 279, 3212-3217 (2004), And their references).
Specifically, as the reaction solvent, for example, a buffer such as a Tris-HCl buffer or a sodium phosphate buffer, water, or the like is used.
The reaction temperature is generally about 10 ° C to about 40 ° C, preferably about 20 ° C to about 40 ° C.
The pH of the reaction solution is usually about 5 to about 10, preferably about 6 to about 9, more preferably about 7 to about 8, and particularly preferably about 7.5.
As luciferin, coelenterazines are preferable, and coelenterazine is particularly preferable.
Luciferin may be added to the reaction system as a polar solvent such as dimethylformamide or dimethylsulfoxide, or an alcohol solution such as methanol, ethanol or butanol.
(Activation of luminous activity)
Luminescent activity (luminescent catalytic activity) of the polypeptide of the present invention or the protein used in the present invention using luciferin (for example, coelenterazines) as a substrate is activated by halide ions.
Examples of the halide ion include fluorine ion, chlorine ion, bromine ion and iodine ion, and chlorine ion, bromine ion and iodine ion are preferable.
The concentration of halide ions is usually about 10 μM to about 100 mM, preferably about 100 μM to about 50 mM, particularly preferably about 1 mM to about 20 mM.
Examples of the method of adding halide ions to the reaction system include a method of adding as a salt. Examples of the salt used include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salt, magnesium salt and barium salt. More specifically, NaF, NaCl, NaBr, NaI , KF, KCl, KBr, KI, CaF 2, CaCl 2, CaBr 2, CaI 2, MgF 2, MgCl 2, MgBr 2, MgI 2, and the like .

  J. Sambrook, EF Fritsch & T. Maniatis (Ed.), Molecular cloning, a laboratory manual (3rd edition), Cold Spring Harbor, unless otherwise stated in the best mode for carrying out the invention and the examples. Press, Cold Spring Harbor, New York (2001); FM Ausubel, R. Brent, RE Kingston, DD Moore, JG Seidman, JA Smith, K. Struhl (Ed.), Current Protocols in Molecular Biology, John Wiley & Sons Ltd A method described in a standard protocol collection such as. Or a modified or modified method thereof is used. In addition, when using commercially available reagent kits and measuring devices, unless otherwise explained, protocols attached to them are used.

  The objects, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description of the present specification, and those skilled in the art can easily implement the present invention from the description of the present specification. it can. The best mode for carrying out the invention and specific examples show preferred embodiments of the present invention and are shown for illustration or description, and the present invention is not limited thereto. Not what you want. It will be apparent to those skilled in the art that various modifications can be made based on the description of the present specification within the spirit and scope of the present invention disclosed herein.

The sequence numbers in the sequence listing in the present specification indicate the following sequences.
[SEQ ID NO: 1] This shows the amino acid sequence of carboxyterminal domain-deleted Gaussia luciferase (polypeptide I of the present invention). This amino acid sequence corresponds to the 27th to 97th amino acid sequence of the amino acid sequence of SEQ ID NO: 6.
[SEQ ID NO: 2] This represents the nucleotide sequence of a polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1.
[SEQ ID NO: 3] This shows the amino acid sequence of amino terminal domain-deleted Gaussia luciferase (polypeptide II of the present invention). This amino acid sequence corresponds to the 98th to 168th amino acid sequence of the amino acid sequence of SEQ ID NO: 6.
[SEQ ID NO: 4] This shows the base sequence of a polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3.
[SEQ ID NO: 5] This represents the nucleotide sequence of a polynucleotide excluding the region encoding the secretory signal sequence from the Gaussia luciferase gene (hGL gene).
[SEQ ID NO: 6] This represents the amino acid sequence of a protein encoded by a polynucleotide consisting of the base sequence of SEQ ID NO: 5, ie, a protein obtained by removing a secretory signal sequence from Gaussia luciferase.
[SEQ ID NO: 7] This shows the base sequence of the polynucleotide encoding the recombinant Gaussia luciferase inserted into the Gaussia luciferase expression vector pCold-hGL prepared in Example 1.
[SEQ ID NO: 8] This shows the amino acid sequence of the recombinant protein encoded by the polynucleotide consisting of the base sequence of SEQ ID NO: 7.
[SEQ ID NO: 9] This shows the base sequence of the primer used in Example 1.
[SEQ ID NO: 10] This represents the base sequence of the primer used in Example 1.
[SEQ ID NO: 11] This shows the base sequence of the primer used in EXAMPLE 2.
[SEQ ID NO: 12] This shows the base sequence of the primer used in EXAMPLE 2.
[SEQ ID NO: 13] This represents the amino acid sequence of the carboxyterminal domain-deleted Gaussia luciferase recombinant protein (polypeptide) prepared in Example 2.
[SEQ ID NO: 14] Carboxy terminal domain inserted into the expression vector pCold-hGL-27 / 97 of a recombinant protein (polypeptide) from which the carboxy terminal domain of Gaussia gaucus luciferase prepared in Example 2 has been deleted It represents the nucleotide sequence of a polynucleotide encoding a deleted Gaussia luciferase recombinant protein (polypeptide).
[SEQ ID NO: 15] This shows the base sequence of the primer used in EXAMPLE 2.
[SEQ ID NO: 16] This shows the base sequence of the primer used in EXAMPLE 2.
[SEQ ID NO: 17] This shows the amino acid sequence of amino terminal domain-deleted Gaussia luciferase recombinant protein (polypeptide) prepared in Example 2.
[SEQ ID NO: 18] Amino-terminal domain inserted into an expression vector pCold-hGL-98 / 168 of a recombinant protein (polypeptide) from which the amino-terminal domain of Gaussia gaucus luciferase prepared in Example 2 has been deleted It represents the nucleotide sequence of a polynucleotide encoding a deleted Gaussia luciferase recombinant protein (polypeptide).

  EXAMPLES The present invention will be described below with reference to examples, but the examples are not intended to limit the present invention.

Example 1 Construction of Gaussia luciferase expression vector pCold-hGL A region encoding a secretory signal sequence from the Gaussia luciferase gene (hGL gene) in order to express Gaussia luciferase derived from Gausia princess, a deep-sea copepoda, in Escherichia coli. An expression vector, pCold-hGL, was constructed by inserting the polynucleotide (SEQ ID NO: 5) from which the DNA was removed into the expression vector pCold II (Takara Bio Inc.) (FIG. 1). The amino acid sequence of the protein encoded by the polynucleotide consisting of the base sequence represented by SEQ ID NO: 5 is shown in SEQ ID NO: 6. The nucleotide sequence of DNA encoding recombinant Gaussia luciferase inserted into pCold-hGL is shown in SEQ ID NO: 7. The amino acid sequence of the protein encoded by the polynucleotide consisting of the base sequence represented by SEQ ID NO: 7 is shown in SEQ ID NO: 8.
Specifically, the Gaussia luciferase expression vector pCold-hGL was constructed according to the following method. Two types of PCR primers using pcDNA3-hGL (Prolmi) with Gaussia luciferase gene as a template:
GL5-N / SacI (5 'gcc- GAG-CTC- AAG-CCC-ACC-GAG-AAC-AAC-GAA 3') (SEQ ID NO: 9; SacI restriction enzyme site underlined) and GL2-C / EcoRI (5 'gcc- GAA-TTC- TTA-GTC-ACC-ACC-GGC-CCC-CTT 3') (SEQ ID NO: 10; EcoRI restriction enzyme site is underlined), PCR kit (manufactured by Takara Bio Inc.) ), PCR (cycle conditions 25 cycles; 1 minute / 94 ° C., 1 minute / 50 ° C., 1 minute / 72 ° C.) was performed to amplify the desired DNA region. The resulting fragment was purified with a PCR purification kit (Qiagen), digested with restriction enzyme SacI / EcoRI by a conventional method, and then ligated to the restriction enzyme SacI / EcoRI site of pColdII (Takara Bio). An expression vector pCold-hGL was constructed (FIG. 2).

Example 2 Construction of Domain Deletion Recombinant Protein Expression Vector In order to express a protein lacking the amino terminal and carboxy terminal domains of Gaussia luciferase (hGL), the full length of Gaussia luciferase gene (hGL gene) was expressed. The domain-deleted hGL gene is obtained by PCR from the phGL (Prorumi), and the expression vector, pCold-hGL-27 / 97 and pCold- are inserted into the expression vector, pCold II (Takara Bio). hGL-98 / 168, was constructed (FIG. 1).
Specifically, the carboxy-terminal of Gaussia luciferase having 27th to 97th amino acid sequence of the polypeptide (SEQ ID NO: 6) excluding the 17-residue secretion signal sequence at the amino terminus from Gaussia luciferase. The expression vector for the recombinant protein lacking the domain consists of two PCR primers using phGL as a template: GL13-N-27 / EcoRI (5'ggc- GAA-TTC- CGC-GGG-AAG-TTG-CCC-GGC -AAG 3 ') (SEQ ID NO: 11; EcoRI restriction enzyme site is underlined) and GL8-C-71 / XbaI (5' cgg- TCT-AGA- TTA-GTC-GAC-GAT-CGC-CTC-GCC 3 '(SEQ ID NO: 12; XbaI restriction enzyme site is underlined) using PCR kit (manufactured by Takara Bio Inc.) for PCR (cycle conditions 25 cycles; 1 minute / 94 ° C, 1 minute / 50 ° C, 1 minute) The desired DNA region was amplified by purifying with a PCR purification kit (Qiagen). After digesting with restriction enzymes EcoRI / XbaI, pCold-hGL, a recombinant protein expression vector lacking the carboxy-terminal domain by ligation to the restriction enzyme EcoRI / XbaI site of pColdII (Takara Bio) -27/97 was constructed (Fig. 3) The recombinant protein (SEQ ID NO: 13) lacking the domain at the carboxy terminus added a 22 amino acid residue sequence containing 6 histidine sequences to the amino terminus. It consists of 93 amino acids.
Construction of an expression vector for a recombinant protein lacking the amino terminal domain having amino acids 98 to 168 of the amino acid sequence of the polypeptide (SEQ ID NO: 6) excluding the secretory signal sequence from Gaussia luciferase is as follows. Went according to. Two PCR primers using phGL as a template: GL15-N-97 / EcoRI (5 'ggc- GAA-TTC -ATT-CCT-GAG-ATT-CCC-GGG-TTC 3' (SEQ ID NO: 15; EcoRI restriction enzyme) Site is underlined) and GL7-C / XbaI (5 'gcc- TCT-AGA -TTA-GTC-ACC-ACC-GGC-CCC-CTT 3' (SEQ ID NO: 16; XbaI restriction enzyme site is underlined) Using PCR kit (manufactured by Takara Bio Inc.), PCR (cycle conditions 25 cycles; 1 min / 94 ° C., 1 min / 50 ° C., 1 min / 72 ° C.) was performed to amplify the desired DNA region The resulting fragment is purified with a PCR purification kit (Qiagen), digested with restriction enzymes EcoRI / XbaI by a conventional method, and then ligated to the restriction enzyme EcoRI / XbaI sites of pColdII (Takara Bio). To construct a recombinant protein expression vector pCold-hGL-98 / 168 lacking the amino-terminal domain (FIG. 4). 17) is composed of 93 amino acids with a sequence consisting of 22 amino acid residues including 6 histidine sequences added to the amino terminal side of the polynucleotide encoding the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 17. The sequence is shown in SEQ ID NO: 18.
This expression vector is inducible at low temperatures, and the expressed recombinant protein lacking the carboxy-terminal or amino-terminal domain of Gaussia luciferase has a histidine tag at the amino terminus.
In the present specification, a protein in which the carboxy-terminal or amino-terminal domain of Gaussia luciferase is deleted may be referred to as “domain-deleted Gaussia luciferase”. A protein in which the carboxy-terminal domain of Gaussia luciferase is deleted may be referred to as “carboxy-terminal domain-deleted Gaussia luciferase”. A protein in which the amino terminal domain of Gaussia luciferase has been deleted may be referred to as “amino terminal domain-deleted Gaussia luciferase”. A polypeptide comprising the amino acid sequence of SEQ ID NO: 1 may be referred to as “carboxy terminal domain deleted Gaussia luciferase”. The polypeptide comprising the amino acid sequence of SEQ ID NO: 3 may be referred to as “amino terminal domain-deleted Gaussia luciferase”. A recombinant protein of a protein lacking the carboxy-terminal or amino-terminal domain of Gaussia luciferase may be referred to as a “domain-deleted recombinant protein”.

Example 3 Protein Purification Method 1) Expression of Protein in E. coli Expression vectors pCold-hGL, pCold-hGL-27 / 97 and pCold-hGL-98 / 168 were introduced into E. coli BL21 strain by a conventional method. The transformed strain is inoculated into 10 ml of LB liquid medium (10 g of bactotryptone, 5 g of yeast extract, 5 g of sodium chloride per liter of water) containing ampicillin (50 μg / ml), and cultured at 37 ° C. for 18 hours. went. Then, the cultured cell fluid was added to 400 ml of a new LB liquid medium, cultured at 37 ° C. for 4.5 hours, cooled on ice water, and isopropyl-β-D (−)-thiogalactopyranoside (IPTG (Manufactured by Wako Pure Chemical Industries, Ltd.) was added to the culture solution to a final concentration of 0.2 mM, and cultured at 15 ° C for 17 hours. The cultured cells were collected by centrifugation (5,000 rpm, 5 minutes) and used as a starting material for protein extraction.
2) Extraction of protein from cultured cells Suspended collected cells in 80 ml of 50 mM Tris-HCl (pH 7.6) and subjected to ultrasonic disruption treatment (Branson, Sonifier model cycle 250) under ice-cooling. 3 times for 2 minutes. The cell disruption solution was centrifuged at 10,000 rpm (12,000 × g) for 20 minutes to obtain a soluble fraction and an insoluble precipitate fraction. The soluble fraction was directly used as a starting material for protein purification. On the other hand, the insoluble precipitate fraction was suspended and dissolved in 50 mM Tris-HCl (pH 7.6) containing 50 ml of 6M urea, and centrifuged at 10,000 rpm (12,000 × g) for 20 minutes. The obtained 6M urea solubilized fraction was used as a starting material for protein purification from the insoluble precipitate fraction.
3) Purification of soluble fraction of protein by nickel chelate column chromatography method Nickel chelate column (Amersham Biosciences, Inc.) equilibrated with the soluble fraction (80 ml) with 50 mM Tris-HCl (pH 7.6). Column size: diameter 2.5 × 6.5 cm) was added and adsorbed. The protein adsorbed on the column was eluted with 50 mM Tris-HCl (pH 7.6) containing 0.1 M imidazole (manufactured by Wako Pure Chemical Industries, Ltd.) and then with 50 mM Tris-HCl (pH 7.6) containing 0.3 M imidazole. In the fraction containing 0.3 M imidazole, luminescence activity and protein elution were confirmed by 12% SDS-polyacrylamide electrophoresis.
4) Purification of insoluble fraction of protein by nickel chelate column chromatography The nickel chelate column (Amersham) was prepared by equilibrating the insoluble fraction (50 ml) with 50 mM Tris-HCl (pH 7.6) containing 6 M urea. Bioscience, column size: diameter 2.5 × 6.5 cm) and adsorbed. The protein adsorbed on the column was eluted with 6M Urea (pH 8.0) containing 0.1 M imidazole (manufactured by Wako Pure Chemical Industries, Ltd.) and then with 6M urea (pH 8.0) containing 0.3 M imidazole. Luminescent activity and protein elution were confirmed by 12% SDS-polyacrylamide electrophoresis in a 6 M urea (pH 8.0) fraction containing 0.3 M imidazole.
The purity of the protein by 16% SDS-polyacrylamide electrophoresis was 95% or more. (FIG. 5).
Protein concentration was measured by Bradford's dye binding method (Anal. Biochem. 72, 248-254 (1976)) using a commercially available kit (Biorad) and bovine serum albumin (Pierce) as a standard substance. . SDS-PAGE analysis was performed according to the Remli method (Nature 227, 680-658 (1970)) using a 12% separation gel (manufactured by Tefco) under reducing conditions.
As for Gaussia luciferase protein, 15.0 mg of purified protein was obtained from the soluble fraction of the 2 L culture solution and 217.7 mg of purified protein from the insoluble fraction.
As for the recombinant protein of Gaussia luciferase lacking the carboxy terminal domain, 2.0 mg of purified protein was obtained from the soluble fraction and 15.4 mg of purified protein from the insoluble fraction of 800 ml of the culture solution. As for the recombinant protein of Gaussia luciferase lacking the amino terminal domain, 9.2 mg of purified protein was obtained from the soluble fraction and 36.8 mg of purified protein from the insoluble fraction. Table 2 shows the specific activity (activity per protein) of the purified protein. As a result, it was revealed that only the amino-terminal and carboxy-terminal domains of Gaussia luciferase, ie, only 71 amino acid sequence, showed activity.

Example 4 Method for confirming molecular weight by mass spectrometry
According to analysis by SDS-polyacrylamide electrophoresis (FIG. 5), the estimated molecular weight of amino terminal domain-deleted Gaussia luciferase is 6.5 KDa. More accurate molecular weight analysis using matrix-assisted laser ionization time-of-flight mass spectrometry (MALDI-TOF-MS) on auto FLEX (Bruker Daltonics) and sinapinic acid (manufactured by Invitrogen) as a matrix, Anal. Biochem 316, 216-222 (2003). Data were acquired in positive reflector mode. Time-mass conversion was performed by external calibration using apomyoglobin (horse, m / z = 16952.6) as a standard. The calculated molecular weight was obtained using the program ExPASy PeptideMass (http://www.expasy.ch/cgi-bin/peptide-mass.pl).
In order to confirm that the purified Gaussia luciferase recombinant protein or domain-deleted Gaussia luciferase recombinant protein has a histidine hexamer, mass spectrometry was performed using MALDI-TOF-MS.
For Gaussia luciferase recombinant protein, a mass value of m / z 20019.1 for [M + H] + was observed. This value was in good agreement with the calculated average mass of 19977.1 for Gaussia luciferase recombinant protein (SEQ ID NO: 8) with histidine hexamer. For the Gaussia luciferase recombinant protein lacking the carboxy-terminal domain, a mass value of m / z 10742.8 relative to [M + H] + was observed. This value was in good agreement with the calculated average mass of 10696.5 for the carboxy terminal deleted Gaussia luciferase recombinant protein (SEQ ID NO: 13) with histidine hexamer. In the Gaussia luciferase recombinant protein lacking the amino terminal domain, a mass value of m / z 10354.5 relative to [M + H] + was observed. This value was in good agreement with the calculated average mass of 10309.9 for the amino terminal deleted Gaussia luciferase recombinant protein with histidine hexamer (SEQ ID NO: 17).

Example 5 Method for Measuring Luminescent Activity Gaelian luciferase or domain-deleted Gaussia luciferase (1 μl) was suspended in 100 μl of 50 mM Tris-HCl (pH 7.6) containing 10 mM EDTA, and the substrate coelenterazine (1 μg / μl) dissolved in ethanol. ) To start the luminescence reaction, and the luminescence activity was measured for 60 seconds with a luminescence measuring device Luminescencer-PSN AB2200 (manufactured by ATTO). Luminescent activity was shown as a maximum value (Imax).

Example 6 Examination of optimum pH Using the above-mentioned method for measuring luminescence activity, optimum pH of purified Gaussia luciferase (hGL) or domain-deleted Gaussia luciferase (hGL-27 / 97, hGL-98 / 168) It was investigated. Specifically, purified Gaussia luciferase or domain-deleted Gaussia luciferase is mixed with sodium acetate buffer (pH 5.0, pH 5.5), sodium phosphate buffer (pH 6.0, pH 6.5, pH 7.0, pH 7.5, pH 8.0). ), Glycine-sodium hydroxide buffer (pH 9.0, pH 9.5, pH 10.0, pH 10.5) suspended in 100 μl and mixed with ethanol coelenterazine (1 μg / μl) to start the luminescence reaction and measure luminescence Luminescence activity was measured for 60 seconds with an apparatus Luminescencer-PSN AB2200 (manufactured by Ato). The luminescence intensity of each buffer was shown as relative activity (%) (FIG. 6). The amount of protein used was 1.0 μg for hGL, 0.45 μg for hGL-27 / 97, and 0.3 μg for hGL-98 / 168. This result indicates that Gaussia luciferase lacking the amino-terminal and carboxy-terminal domains has the same optimum pH as Gaussia luciferase.

Example 7 Measurement of emission spectrum The emission spectrum of purified Gaussia luciferase or domain-deleted Gaussia luciferase was confirmed. Specifically, in 30 mM Tris-HCl (pH 7.6) containing 10 mM EDTA, Jusco (Tokyo) FP-560 fluorescence emission meter (bandpass 20 nm; response 0.5 nm, Medium; scan speed 2000 nm / min), Measurements were made at 22-25 ° C. using a quartz cuvette (10 mm optical path). The protein concentration of Gaussia luciferase (hGL) used was 140 μg / ml. The protein amounts of hGL-27 / 97 and hGL-98 / 168 of domain-deleted Gaussia luciferase were 80 μg / ml and 210 μg / ml, respectively. The luminescence intensity was shown as relative intensity (%) (FIG. 7).
From this result, it was revealed that the amino-terminal and carboxy-terminal domain-deleted Gaussia luciferase has the same emission spectrum as Gaussia luciferase. This indicates that the excited molecular species in the luminescence reaction are the same.

Example 8 Inhibition by copper ions and activation by chloride ions
A substrate coelenterazine (1 μg / μl) dissolved in ethanol and a copper chloride solution or a calcium chloride solution were suspended in 200 μl of 100 mM sodium phosphate buffer (pH 7.5). This suspension, purified Gaussia luciferase (hGL) or domain-deleted Gaussia luciferase (hGL-27 / 97, hGL-98 / 168) (2 μl) are mixed to start the luminescence reaction, and the luminescence measuring device Luminescencer -Luminous activity was measured for 10 seconds with PSN AB2200 (manufactured by Atto Corporation). The amount of protein used was 0.4 μg for hGL, 0.08 μg for hGL-27 / 97, and 0.1 μg for hGL-98 / 168. The final concentration of the copper chloride solution or calcium chloride solution was 0.001 μM, 0.01 μM, 0.1 μM, 1 μM, 10 μM, 100 μM, and 1000 μM. Luminescent activity showed the maximum value (Imax) as relative activity (%) (FIG. 8). From this result, it was revealed that Gaussia luciferase lacking the amino-terminal or carboxy-terminal domain is subject to inhibition by copper ions and activation by chloride ions, like Gaussia luciferase.

Example 9 Activation with halide ions
Substrate coelenterazine (1 μg / μl) dissolved in ethanol and a halide ion solution (final concentration 5 mM) were suspended in 200 μl of 100 mM sodium phosphate buffer (pH 7.5). The suspension was mixed with purified Gaussia luciferase (hGL) or domain-deleted Gaussia luciferase (hGL-27 / 97, hGL-98 / 168) (2 μl) to initiate the luminescence reaction, and the luminescence measurement device Luminescence-PSN AB2200 (manufactured by ATTO) was used to measure luminescence activity for 10 seconds. The amount of protein used was 0.32 μg for hGL, 0.08 μg for hGL-27 / 97, and 0.04 μg for hGL-98 / 168. Luminescent activity showed the maximum value (Imax) as relative activity (%) (Table 3). From this result, it is clear that Gaussia luciferase and Gaussia luciferase lacking the amino-terminal or carboxy-terminal domain are similarly activated by chloride ion, bromine ion and iodine ion (up to about 17 times). It became.

Example 10 Substrate specificity
A substrate coelenterazine derivative (1 μg / μl) dissolved in ethanol was suspended in 100 μl of 50 mM Tris-HCl (pH 7.6) containing 10 mM EDTA. The suspension was mixed with purified Gaussia luciferase (hGL) or domain-deleted Gaussia luciferase (hGL-27 / 97, hGL-98 / 168) (2 μl) to initiate the luminescence reaction, and the luminescence measurement device Luminescence-PSN AB2200 (manufactured by ATTO) was used to measure luminescence activity for 10 seconds. The amount of protein used was 0.32 μg for hGL, 0.08 μg for hGL-27 / 97, and 0.10 μg for hGL-98 / 168. Luminescent activity showed the maximum value (Imax) as relative activity (%) (Table 4). Coelenterazine was the best substrate for Gaussia luciferase lacking the amino-terminal domain, Gaussia luciferase and Gaussia luciferase lacking the carboxy-terminal domain. From these results, it was revealed that Gaussia luciferase lacking the amino-terminal domain, Gaussia luciferase lacking the carboxy-terminal domain, and Gaussia luciferase all show the same substrate specificity.

The polypeptide of the present invention can be used as a reporter protein for measuring transcriptional activity of a promoter or the like. Furthermore, the polypeptide of the present invention can also be used as a detection marker, a material for an amusement product, and the like.
Since the polynucleotide of the present invention encodes the polypeptide of the present invention described above, it can be used as a reporter gene.
Moreover, the polynucleotide of the present invention, the vector of the present invention, the transformant of the present invention and the like can be used for the production of the polypeptide of the present invention.
According to the method for activating the luminescence activity of the present invention, the luminescence efficiency of the polypeptide of the present invention or the protein used in the present invention can be improved. Therefore, detection sensitivity of a detection marker using the polypeptide of the present invention or the protein used in the present invention can be improved. In addition, the luminescence intensity of an amusement product using the polypeptide of the present invention or the protein used in the present invention can be improved.

FIG. 1 is a diagram showing expression regions of Gaussia luciferase, carboxyterminal domain-deleted Gaussialuciferase, and amino-terminal domain-deleted Gaussialuciferase. FIG. 2 is a diagram showing a Gaussia luciferase expression vector, pCold-hGL. FIG. 3 is a diagram showing an expression vector for p-old-hGL-27 / 97, a Gaussia luciferase lacking a carboxyl terminal domain. FIG. 4 is a diagram showing an expression vector of p-old-hGL-98 / 168, an amino terminal domain-deleted Gaussia luciferase. FIG. 5 is a 16% polyacrylamide electrophoretic analysis of purified Gaussia luciferase recombinant protein and domain-deleted Gaussia luciferase recombinant protein. FIG. 6 is a diagram showing a comparison of optimum pHs between purified Gaussia luciferase and domain-deleted Gaussia luciferase. FIG. 7 is a diagram showing a comparison of emission spectra of purified Gaussia luciferase and domain-deleted Gaussia luciferase. FIG. 8 is a diagram showing inhibition by copper ions and activation by chloride ions of purified Gaussia luciferase and domain-deleted Gaussia luciferase.

Claims (19)

The polypeptide according to any of the following (a) to (d):
(A) a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3;
(B) a luminescent catalytic activity comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 and luciferin as a substrate; A polypeptide having;
(C) a polypeptide having an amino acid sequence having 80% or more identity to SEQ ID NO: 1 or SEQ ID NO: 3 and having a luminescent catalytic activity using luciferin as a substrate; and (d) SEQ ID NO: Luminescent catalytic activity comprising an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence 2 or SEQ ID NO: 4 and using luciferin as a substrate A polypeptide having:
However, the protein containing the amino acid sequence represented by SEQ ID NO: 6 is excluded. The polypeptide according to claim 1, which is any of the following (a) to (d):
(A) a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3;
(B) a luminescence catalytic activity comprising an amino acid sequence in which 1 to 25 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 and luciferin as a substrate; A polypeptide having;
(C) a polypeptide having an amino acid sequence having 90% or more identity to SEQ ID NO: 1 or SEQ ID NO: 3 and having a luminescent catalytic activity using luciferin as a substrate; and (d) SEQ ID NO: A luminescent catalyst comprising an amino acid sequence encoded by a polynucleotide that hybridizes under high stringency conditions with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of 2 or SEQ ID NO: 4, and using luciferin as a substrate A polypeptide having activity.   The polypeptide according to claim 1, which is a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3.   A polynucleotide comprising a polynucleotide encoding the polypeptide according to claim 1. The polynucleotide according to any one of the following (e) to (i):
(E) a polynucleotide containing a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4;
(F) a polypeptide having a luminescent catalytic activity that hybridizes with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4 under stringent conditions and has luciferin as a substrate. A polynucleotide containing the encoding polynucleotide;
(G) a polynucleotide comprising a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3;
(H) a luminescent catalytic activity comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 and luciferin as a substrate; A polynucleotide containing a polynucleotide encoding the polypeptide having; and (i) an amino acid sequence having 80% or more identity to SEQ ID NO: 1 or SEQ ID NO: 3, and using luciferin as a substrate A polynucleotide comprising a polynucleotide encoding a polypeptide having a luminescent catalytic activity. The polynucleotide according to any one of the following (e) to (i):
(E) a polynucleotide containing a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4;
(F) A polypeptide that hybridizes with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 2 or 4 under a highly stringent condition and has a luminescence catalytic activity using luciferin as a substrate. A polynucleotide containing the polynucleotide;
(G) a polynucleotide comprising a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3;
(H) a luminescent catalytic activity comprising an amino acid sequence in which 1 to 25 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 and luciferin as a substrate; A polynucleotide containing a polynucleotide encoding the polypeptide having; and (i) an amino acid sequence having 90% or more identity to SEQ ID NO: 1 or SEQ ID NO: 3, and using luciferin as a substrate A polynucleotide comprising a polynucleotide encoding a polypeptide having a luminescent catalytic activity.   A recombinant vector containing the polynucleotide according to any one of claims 4 to 6.   A transformant into which the recombinant vector according to claim 7 has been introduced.   The method for producing a polypeptide according to any one of claims 1 to 3, comprising a step of culturing the transformant according to claim 8 to produce the polypeptide according to any one of claims 1 to 3.   A kit comprising the polypeptide according to claim 1.   A kit comprising the polynucleotide according to any one of claims 4 to 6, the recombinant vector according to claim 7, or the transformant according to claim 8.   The kit according to claim 10 or 11, further comprising luciferin.   The kit according to claim 12, wherein the luciferin is coelenterazines.   The kit according to claim 13, wherein the coelenterazine is coelenterazine. The polypeptide according to any of the following (a) to (d):
(A) a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3;
(B) a luminescence catalytic activity comprising an amino acid sequence in which 1 to 25 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 and luciferin as a substrate; A polypeptide having;
(C) a polypeptide having an amino acid sequence having 90% or more identity to SEQ ID NO: 1 or SEQ ID NO: 3 and having a luminescent catalytic activity using luciferin as a substrate; and (d) SEQ ID NO: A luminescent catalyst comprising an amino acid sequence encoded by a polynucleotide that hybridizes under high stringency conditions with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of 2 or SEQ ID NO: 4, and using luciferin as a substrate A polypeptide having activity,
And a luminescent reaction comprising contacting luciferin with the luciferin.   The method according to claim 15, wherein the luminescence reaction is carried out in the presence of halide ions.   The method according to claim 16, wherein the halide ion is a fluorine ion, a bromine ion or an iodine ion. The polypeptide according to any one of the following (a) to (d) using a halide ion:
(A) a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3;
(B) a luminescence catalytic activity comprising an amino acid sequence in which 1 to 25 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 and luciferin as a substrate; A polypeptide having;
(C) a polypeptide having an amino acid sequence having 90% or more identity to SEQ ID NO: 1 or SEQ ID NO: 3 and having a luminescent catalytic activity using luciferin as a substrate; and (d) SEQ ID NO: A luminescent catalyst comprising an amino acid sequence encoded by a polynucleotide that hybridizes under high stringency conditions with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of 2 or SEQ ID NO: 4, and using luciferin as a substrate A polypeptide having activity; or a protein according to any one of the following (a) to (d):
(A) a protein containing the amino acid sequence of SEQ ID NO: 6 and having a luminescence catalytic activity using luciferin as a substrate;
(B) a protein having an amino acid sequence in which 1 to 25 amino acids are deleted, substituted, inserted or added in the amino acid sequence of SEQ ID NO: 6 and having a luminescence catalytic activity using luciferin as a substrate;
(C) a protein having an amino acid sequence having about 90% or more identity with the amino acid sequence of SEQ ID NO: 6 and having a luminescence catalytic activity using luciferin as a substrate; and (d) a base sequence of SEQ ID NO: 5. A protein having an amino acid sequence encoded by a polynucleotide that hybridizes under high stringency conditions with a polynucleotide comprising a complementary base sequence, and having a luminescent catalytic activity using luciferin as a substrate,
A method for activating the luminescence activity.   The method according to claim 18, wherein the halide ion is a fluorine ion, a bromine ion or an iodine ion.

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