JP5736764B2 - Fusion protein with luminescent activity - Google Patents

Description

  The present invention relates to a fusion protein having a luminescence catalytic activity using luciferase as a substrate.

  A luminescent enzyme (luciferase) using coelenterazine as a luminescent substrate (luciferin) is scattered in marine luminescent organisms, and the enzyme is called coelenterazine-based luciferase. Up to now, luciferase genes derived from luminescent organisms such as Renilla luciferase, Gaussia luciferase, Pre-Urman luciferase, Oproforus luciferase, and Metriciluciferase have been reported (Table 1). The molecular weight of luciferase shows various molecular weight distributions from 19 kDa to 35 kDa. Furthermore, there is no homology between the amino acid sequences of the primary structures of these luciferases, and the luminescence mechanism by coelenterazine luciferase is unclear.

  Since the luminescence reaction of coelenterazine is the simplest luminescence system performed only by coelenterazine, luciferase and oxygen, luciferase is widely used as a reporter enzyme (gene) in various assay systems.

  On the other hand, as a luminescence reaction of coelenterazine by a protein other than a luciferase derived from a luminescent organism, a luminescence reaction by a protein such as bovine serum albumin having a molecular weight of 69 kDa is known (Non-Patent Document 5: Zhao, H., Doyle, TC, Wong, RJ, Cao, Y., Stevenson, DK, Piwnica-Worms D., Contag, CH (2004) Mol. Imaging. 3, 43-54.). However, with respect to the luminescence reaction involving bovine serum albumin, there is no detailed report on the luminescence mechanism, and furthermore, proteins that catalyze the luminescence reaction with proteins other than luciferases derived from luminescent organisms have not been found.

WO 99/49019

Lorenz, W.W., McCann, R.O., Longiaru, M., Cormier, M.J. (1991) Proc. Natl. Acad. Sci. USA. 88, 4438-4442. Inouye, S., Watanabe, K., Nakamura, H., Shimomura, O. (2000) FEBS Lett. 481, 19-25. Markova, S. V., Golz S., Frank, L.A., Kalthof, B., Vysotski, E. S (2004) J. Biol. Chem. 279, 3212-3217 Takenaka, Y., Masuda, H., Yamaguchi, A., Nishikawa, S., Shigeri, Y .. Yoshida, Y., Mizuno, H. (2008) Gene 425, 28-35. Zhao, H., Doyle, T.C., Wong, R.J., Cao, Y., Stevenson, D.K., Piwnica-Worms D., Contag, C.H. (2004) Mol. Imaging. 3,43-54.

  In the above situation, there has been a demand for a protein other than a luciferase derived from a luminescent organism and having a luminescent catalytic activity using luciferin as a luminescent substrate.

  The present inventors screened proteins having luminescent catalytic activity using coelenterazine as a luminescent substrate for various proteins. As a result, it was found that ZZ domain-gCys has a luminescent catalytic activity. ZZ domain-gCys is a fusion protein in which a flexible linker (gCys) having a modifiable cysteine at the C-terminus is inserted into the C-terminus of the ZZ domain. ZZ domain-The ZZ domain of gCys is an artificial domain derived from the IgG binding domain of Protein A, a cell wall component of Staphylococcus aureus (Staphylococcus aureus). As a result of further studies based on these findings, the present invention has been completed.

  That is, the present invention provides the following fusion protein, polynucleotide, recombinant vector, transformant, method for producing a fusion protein, a complex in which another useful compound is bound to the fusion protein, and the like.

[1] (1) Formula (Z) _n
(In the formula, n represents an integer of 1 to 5, and Z represents a polypeptide selected from the group consisting of the following (a) to (d):
(a) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 8,
(b) a polypeptide comprising 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: 8;
(c) a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 8, and
(d) a polypeptide 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 of SEQ ID NO: 7)
A first region containing the amino acid sequence of a polypeptide having a luminescence catalytic activity using luciferin as a substrate when expressed as a fusion protein with a polypeptide that is represented by
(2) a second region containing an amino acid sequence of a modifiable polypeptide having one or more cysteine residues for binding other useful compounds via a thiol group;
[2] The group in which the second region is composed of the following (e) to (h):
(e) a polypeptide comprising the amino acid sequence of SEQ ID NO: 6,
(f) The amino acid sequence of SEQ ID NO: 6 consists of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added, and binds other useful compounds via a thiol group A polypeptide having one or more cysteine residues of
(g) One or more cysteine residues consisting of an amino acid sequence having 70% or more identity to the amino acid sequence of SEQ ID NO: 6 and for binding other useful compounds via a thiol group A polypeptide having, and
(h) consisting of 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 other via a thiol group A polypeptide having one or more cysteine residues for binding useful compounds;
The fusion protein according to Item [1], which is a region containing the amino acid sequence of a polypeptide selected from the group consisting of:
[3] The group in which the second region is composed of the following (e) to (h):
(e) a polypeptide comprising the amino acid sequence of SEQ ID NO: 6,
(f) The amino acid sequence of SEQ ID NO: 6 consists of an amino acid sequence in which 1 to 3 amino acids are deleted, substituted, inserted and / or added, and for binding other useful compounds via a thiol group A polypeptide having one or more cysteine residues of
(g) One or more cysteine residues consisting of an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 6 for binding other useful compounds via a thiol group A polypeptide having, and
(h) It consists of an amino acid sequence encoded by a polynucleotide that hybridizes with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 5 under highly stringent conditions, and other via a thiol group A polypeptide having one or more cysteine residues for binding a useful compound thereof;
The fusion protein according to Item [2], which is a region containing the amino acid sequence of a polypeptide selected from the group consisting of:
[4] The fusion protein according to any one of items [1] to [3], wherein the polypeptide represented by the formula (Z) _n is a polypeptide represented by (Z) ₂ .
[5] The polypeptide represented by the formula (Z) ₂ is:
(a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 10,
(b) When expressed as a fusion protein consisting of 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: 10, and a modifiable polypeptide A polypeptide having a luminescent catalytic activity using luciferin as a substrate,
(c) An amino acid sequence comprising 90% or more identity to the amino acid sequence of SEQ ID NO: 10 and expressed as a fusion protein with a modifiable polypeptide, using luciferin as a substrate A polypeptide having photocatalytic activity, and
(d) a polypeptide 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 of SEQ ID NO: 9, and a modifiable polypeptide Item 4. The fusion protein according to item [4], which is a polypeptide selected from the group consisting of polypeptides having luminescence catalytic activity using luciferin as a substrate when expressed as a fusion protein.
[6] The fusion protein according to any one of items [1] to [5], wherein the polypeptide represented by the formula (Z) n has a function capable of binding to the Fc region of IgG.
[7] (1) The first region is a region consisting of the amino acid sequence of SEQ ID NO: 10;
(2) The fusion protein according to item [1], wherein the second region is a region consisting of the amino acid sequence of SEQ ID NO: 6.
[8] The fusion protein according to any one of items [1] to [7], further comprising an amino acid sequence for promoting translation and / or an amino acid sequence for purification.
[9] A fusion protein consisting of the amino acid sequence of SEQ ID NO: 2.
[10] A polynucleotide containing a polynucleotide encoding the fusion protein according to any one of items [1] to [9].
[11] (1) Formula (Z) _n
(In the formula, n represents an integer of 1 to 5, and Z represents a polypeptide selected from the group consisting of the following (a) to (d):
(a) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 8,
(b) a polypeptide comprising 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: 8;
(c) a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 8, and
(d) a polypeptide 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 of SEQ ID NO: 7)
A first coding sequence comprising a polynucleotide that encodes a polypeptide having a luminescence catalytic activity when expressed as a fusion protein with a polypeptide that is represented by
(2) a second coding sequence comprising a polynucleotide that encodes a modifiable polypeptide having one or more cysteine residues for attachment of other useful compounds via a thiol group; Polynucleotide.
[12] The group consisting of the following (e) to (h):
(e) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 5,
(f) Residue of cysteine for hybridizing under stringent conditions with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 5 and binding other useful compounds via a thiol group A polynucleotide encoding a polypeptide having one or more groups,
(g) a polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 6, and
(h) To bind other useful compounds via a thiol group, comprising 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: 6. A polynucleotide encoding a polypeptide having one or more cysteine residues of
The polynucleotide of paragraph [11], which is a coding sequence containing a polynucleotide selected from.
[13] The group consisting of the second coding sequences (e) to (h):
(e) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 5,
(f) Cysteine that hybridizes with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 5 under highly stringent conditions and binds other useful compounds via a thiol group A polynucleotide encoding a polypeptide having one or more residues,
(g) a polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 6, and
(h) To bind other useful compounds via a thiol group, comprising an amino acid sequence in which 1 to 3 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 6. A polynucleotide encoding a polypeptide having one or more cysteine residues of
The polynucleotide of paragraph [12], which is a coding sequence containing a polynucleotide selected from.
[14] The polynucleotide according to any one of items [11] to [13], wherein the polypeptide represented by the formula (Z) _n is a polypeptide represented by (Z) ₂ .
[15] The polypeptide represented by the formula (Z) ₂ is:
(a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 10,
(b) When expressed as a fusion protein consisting of 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: 10, and a modifiable polypeptide A polypeptide having a luminescent catalytic activity using luciferin as a substrate,
(c) An amino acid sequence comprising 90% or more identity to the amino acid sequence of SEQ ID NO: 10 and expressed as a fusion protein with a modifiable polypeptide, using luciferin as a substrate A polypeptide having photocatalytic activity, and
(d) a polypeptide 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 of SEQ ID NO: 9, and a modifiable polypeptide The polynucleotide according to Item [14], which is a polypeptide selected from the group consisting of polypeptides having a luminescence catalytic activity using luciferin as a substrate when expressed as a fusion protein.
[16] The polynucleotide according to any one of items [11] to [15], wherein the polypeptide represented by the formula (Z) n has a function capable of binding to the Fc region of IgG.
[17] (1) The first coding sequence is a coding sequence consisting of a polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 10;
(2) The polynucleotide according to Item [11], wherein the second coding sequence is a coding sequence consisting of a polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 6.
[18] (1) The first coding sequence is a coding sequence comprising a polynucleotide comprising the base sequence of SEQ ID NO: 9;
(2) The polynucleotide according to Item [11], wherein the second coding sequence is a coding sequence comprising a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 5.
[19] Any one of Items [11] to [18], further comprising a polypeptide comprising an amino acid sequence for promoting translation and / or a polynucleotide encoding a polypeptide comprising an amino acid sequence for purification. The polynucleotide according to Item.
[20] A polynucleotide comprising a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1.
[21] A recombinant vector containing the polynucleotide according to any one of items [10] to [20].
[22] A transformant into which the recombinant vector according to item [21] has been introduced.
[23] Any of items [1] to [9], comprising culturing the transformant according to item [22] and generating the fusion protein according to any one of items [1] to [9]. A method for producing the fusion protein according to claim 1.
[24] A complex in which another useful compound is bound to the fusion protein according to any one of items [1] to [9] via the thiol group of the cysteine residue in the second region of the fusion protein body.
[25] The complex according to item [24], wherein the other useful compound is a fluorescent substance and / or a ligand specific to the substance to be detected.
[26] A kit comprising the fusion protein according to any one of items [1] to [9].
[27] A kit comprising the polynucleotide according to any one of items [10] to [20], the recombinant vector according to item [21], or the transformant according to item [22].
[28] A kit comprising the complex according to item [24] or [25].
[29] The kit according to any one of items [26] to [28], further comprising luciferin.
[30] The kit according to item [29], wherein the luciferin is coelenterazines.
[31] The kit according to item [30], wherein the coelenterazine is coelenterazine.
[32] A method for performing a luminescence reaction, comprising bringing the fusion protein according to any one of [1] to [9] or the complex according to [24] or [25] into contact with luciferin.
[33] A bioluminescence resonance energy transfer (BRET) method is performed using the fusion protein according to item [1] to [9] or the complex according to item [24] or [25] as a donor protein. To analyze physiological function or measure enzyme activity.
[34] A method for measuring a substance specific to a ligand, using the complex according to item [25].
[35] An immunoassay method using the fusion protein according to any one of items [1] to [9].

  The fusion protein of the present invention has a luminescent catalytic activity using luciferin as a substrate. The fusion protein of the present invention can be modified by a chemical modification method and is highly versatile. Luminescence in the luminescence reaction catalyzed by the fusion protein of a preferred embodiment of the present invention is greater than the luminescence intensity in the luminescence reaction catalyzed by bovine serum albumin.

FIG. 1 (a) is a schematic diagram showing the vector pCold-ZZ-gCys used in the examples. pCold-ZZ-gCys expresses a fusion protein (ZZ domain-gCys) having a histidine sequence at the amino terminus and having a peptide linker and a cysteine residue downstream of the ZZ domain. FIG. 1 (b) is a schematic diagram of the amino acid sequence (SEQ ID NO: 2) of ZZ domain-gCys. FIG. 2 is a diagram showing the results of SDS-PAGE analysis during the purification process of ZZ domain-gCys. SDS-PAGE analysis was performed under the following conditions: 12% separation gel, reduced state, heat treatment at 95 ° C. for 3 minutes. Samples in each lane are as follows. Lane 1: β-galactosidase (116,000 Da), phospholipase B (97,400 Da), bovine serum albumin (69,000 Da), glutamate dehydrogenase (55,000 Da), lactate dehydrogenase (36,500 Da), carbonic acid dehydration as protein molecular weight markers (Tefco) Elementary enzyme (29,000 Da), trypsin inhibitor (20,100 Da), lane 2: supernatant obtained by centrifuging an ultrasonic disruption of E. coli transformant expressing recombinant ZZ domain-gCys at 12,000 g (protein 22.0 μg), lane 3: elution fraction when the soluble fraction was subjected to an IgG sepharose column (protein 15.3 μg), lane 4: elution fraction when the IgG sepharose column was washed with TBST solution (protein 3.3 μg) Lane 5: Elution fraction (protein 1.2 μg) when IgG sepharose column was washed with 5 mM ammonium acetate solution (pH 5.0), Lane 6: Elution fraction 3 from IgG sepharose column (protein 25.5 μg) , Lane 7: Elution fraction 4 from IgG sepharose column (protein 15.3 μg), Lane 8: Elution fraction 5 from IgG sepharose column (protein 1.5 μg). FIG. 3 shows the results of SDS-PAGE analysis after 72 hours of dialysis of the eluted ZZ domain-gCys fraction from an IgG sepharose column. SDS-PAGE analysis was performed under the following conditions: 12% separation gel, non-reduced state, heat treated at 95 ° C. for 3 minutes. Samples in each lane are as follows. Lane 1: β-galactosidase (116,000 Da), phospholipase B (97,400 Da), bovine serum albumin (69,000 Da), glutamate dehydrogenase (55,000 Da), lactate dehydrogenase (36,500 Da), carbonic acid dehydration as protein molecular weight markers (Tefco) Elementary enzyme (29,000 Da), trypsin inhibitor (20,100 Da), lane 2: ZZ domain-gCys after dialysis of the eluted fraction from the IgG sepharose column (protein 10.0 μg). FIG. 4 is a diagram showing a light emission pattern of ZZ domain-gCys when coelenterazine is used as a substrate. FIG. 5 is a diagram showing an emission spectrum of ZZ domain-gCys when coelenterazine is used as a substrate. FIG. 6 is a diagram showing the optimum pH in the luminescence reaction of ZZ domain-gCys when coelenterazine is used as a substrate. FIG. 7 shows activation of the ZZ domain-gCys luminescence activity at various concentrations of sodium chloride. FIG. 8 is a diagram showing activation of the luminescence activity of ZZ domain-gCys at each concentration of Tween 20.

Hereinafter, the present invention will be described in detail.
1. Fusion protein of the present invention The fusion protein of the present invention comprises (1) a polypeptide represented by the formula (Z) n and having substantially the same activity or function as the polypeptide consisting of the amino acid sequence of SEQ ID NO: 10. A first region containing the amino acid sequence of (2), and (2) a second region consisting of the amino acid sequence of a polypeptide having one or more cysteine residues for binding other useful compounds via a thiol group; Is a fusion protein containing

  The fusion protein of the present invention may be one in which the first region and the second region are arranged in the order of “N-terminal-first region-second region-C-terminal” or “N It may be arranged in the order of "terminal-second region-first region-C-terminal". In a preferred embodiment of the fusion protein of the present invention, the first region and the second region are arranged in the order of “N-terminal-first region-second region-C-terminal”.

  The first region is a region containing the amino acid sequence of a polypeptide represented by the formula (Z) n and having substantially the same activity or function as the polypeptide consisting of the amino acid sequence of SEQ ID NO: 10.

  “Substantially the same activity or function as the polypeptide consisting of the amino acid sequence of SEQ ID NO: 10” means, for example, a luminescent catalyst using luciferin as a substrate when expressed as a fusion protein with a modifiable polypeptide. Activity (sometimes referred to herein simply as “luminescence activity”), that is, to catalyze a reaction in which luciferin (eg, coelenterazines) is oxidized with oxygen molecules to produce oxyluciferin in an excited state. Means activity. Note that oxyluciferin generated in an excited state emits visible light and becomes a ground state.

  Such activity or function can be measured, for example, by the method described in the literature Inouye, S. & Sahara, Y. (2008) Biochem. Biophys. Res. Commun. 365, 96-101. Specifically, the luminescence reaction can be started by mixing the fusion protein of the present invention with luciferin, and the luminescence catalytic activity can be measured using a luminescence measuring device. As the luminescence measuring device, a commercially available device such as Luminescencer-PSN AB2200 (manufactured by Atto Corporation), Centro 960 luminometer (manufactured by Bertoor Corporation) or the like can be used.

  The luciferin used in the present invention may be luciferin that serves as a substrate for the fusion protein of 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, 3iso-coelenterazine, 3meo-coelenterazine, cf3-coelenterazine, i-coelenterazine, et-coelenterazine, me-coelenterazine, 3me-coelenterazine, αmeh-coelenterazine 8- (1-naphthyl) -coelenterazine, 8- (2-naphthyl) -coelenterazine , 8- (2-thienyl) -coelenterazine, 6,8-di (2-thienyl) -coelenterazine, 8- (4-hydroxyphenyl) -coelenterazine, 8- (2-benzothienyl) -coelenterazine, 8- (b-styryl ) -Coelenterazine, 8-phenyl-coelenterazine, 6-deoxy-coelenterazine, 8- (3-thienyl) -coelenterazine and 8- (3-benzo [b] th ienyl) -coelenterazine and the like. Among these coelenterazines, coelenterazine is particularly preferable in the present invention. These coelenterazines may be synthesized by a known method, or commercially available products can be obtained.

As a method for synthesizing coelenterazines, for example, Shimomura et al. (1988) Biochem. J. 251 , 405-410, Shimomura et al. (1989) Biochem. J. 261 , 913-920, Shimomura et al. 1990) Biochem. J. 270 , 309-312, WO2010 / 090319 publication, Inouye et al. (2010) Anal. Biochem. 407 , 247-252, etc.

  Regarding the coelenterazine commercial products, for example, coelenterazine and h-coelenterazine manufactured by Chisso Corporation; hcp-coelenterazine, cp-coelenterazine, f-coelenterazine, fcp-coelenterazine and n-coelenterazine manufactured by Promega Corporation Examples include coelenterazine, Bis-coelenterazine, hcp-coelenterazine, and h-coelenterazine.

  In some embodiments of the invention, the first region further has an activity or function capable of binding to IgG, eg, an activity or function capable of binding to the FC region of IgG. Preferably, the fusion protein of the present invention can bind to IgG without substantially inhibiting the luminescent catalytic activity using luciferin as a substrate. The activity or function capable of binding to IgG can be measured, for example, according to the Western blotting method, the method described in the examples described later, and the like.

Z represents a polypeptide selected from the group consisting of the following (a) to (d):
(a) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 8,
(b) a polypeptide comprising 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: 8;
(c) a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 8, and
(d) A polypeptide comprising an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence of SEQ ID NO: 7.

n represents an integer of 1 to 5, preferably 2 or 3, and particularly preferably 2.
In the polypeptide represented by the formula (Z) _n , each Z may be the same or different.
As the polypeptide represented by the formula (Z) _n , a polypeptide represented by the formula (Z) ₂ is particularly preferable.

The polypeptide represented by the formula (Z) ₂ has, for example, substantially the same activity or function as the polypeptide consisting of the amino acid sequence of SEQ ID NO: 10 or the polypeptide consisting of the amino acid sequence of SEQ ID NO: 10. A polypeptide. In the present specification, a polypeptide consisting of the amino acid sequence of SEQ ID NO: 10 or a polypeptide having substantially the same activity or function as the polypeptide consisting of the amino acid sequence of SEQ ID NO: 10 is referred to as a “ZZ domain”. is there.

As a polypeptide represented by the formula (Z) ₂ , more specifically, for example,
(a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 10,
(b) When expressed as a fusion protein consisting of 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: 10, and a modifiable polypeptide A polypeptide having a luminescent catalytic activity using luciferin as a substrate,
(c) An amino acid sequence comprising 90% or more identity to the amino acid sequence of SEQ ID NO: 10 and expressed as a fusion protein with a modifiable polypeptide, using luciferin as a substrate A polypeptide having photocatalytic activity, and
(d) a polypeptide 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 of SEQ ID NO: 9, and a modifiable polypeptide Examples thereof include a polypeptide selected from the group consisting of polypeptides having luminescence catalytic activity using luciferin as a substrate when expressed as a fusion protein.

  In the first region, “one or more amino acids are deleted, substituted, inserted and / or added” means one or more amino acids at any position in one or more amino acid sequences in the same sequence It means that there is a deletion, substitution, insertion and / or addition of a residue.

  In the first region, the range of “1 to plural” in “amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added” is, for example, 1 to 20, 1 to 15 , 1-10, 1-9, 1-8, 1-7, 1-6 (1 to several), 1-5, 1-4, 1-3, 1-2 One piece. 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 regions are described in “Sambrook J. et al., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press (2001)”, “Ausbel FM et al., Current Protocols in Molecular Biology, Supplement 1- 38, John Wiley and Sons (1987-1997) ”,“ 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) ”, etc. It can be obtained using a mutagenesis method.

Examples of amino acid residues that can be substituted with each other are shown below. Amino acid residues contained in the same group can be substituted for each other.
Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, o-methylserine, t-butylglycine, t-butylalanine, cyclohexylalanine;
Group B: aspartic acid, glutamic acid, isoaspartic acid, isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid;
Group C: asparagine, glutamine;
Group D: lysine, arginine, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid;
Group E: proline, 3-hydroxyproline, 4-hydroxyproline;
Group F: serine, threonine, homoserine;
Group G: phenylalanine, tyrosine.

  In the first region, the range of “90% or more” in the “polypeptide consisting of an amino acid sequence having 90% or more identity” is, for example, 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, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99. 5% or more, 99.6% or more, 99.7% or more, 99.8% or more, 99.9% or more. In general, the larger the numerical value of identity, the better. The identity of amino acid sequences and base sequences can be determined using an analysis program such as BLAST (see, for example, Altzshul S. F. et al., J. Mol. Biol. 215, 403 (1990)). When using BLAST, the default parameters of each program are used.

  In the first region, “a polynucleotide that hybridizes under stringent conditions” means a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 7 or 9, or the amino acid of SEQ ID NO: 8 or 10 A polynucleotide (for example, DNA) obtained by using a colony hybridization method, a plaque hybridization method, a Southern hybridization method, or the like using all or part of a polynucleotide encoding a sequence as a probe. Specifically, hybridization was performed at 65 ° C. in the presence of 0.7 to 1.0 mol / L NaCl using a filter on which a colony or plaque-derived polynucleotide was immobilized, and then 0.1 to 2 times the concentration of SSC ( Saline-sodium citrate) solution (composition of 1-fold concentration of SSC solution consists of 150 mmol / L sodium chloride and 15 mmol / L sodium citrate), and a polynucleotide that can be identified by washing the filter under 65 ° C conditions Can give.

  Hybridization is described in Sambrook J. et al., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press (2001), Ausbel FM et al., Current Protocols 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), etc. .

  “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, it can be expected that a polynucleotide (eg, DNA) having high homology as the temperature is increased can be efficiently obtained under these conditions. 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, for example, Alkphos Direct Labeling Reagents (manufactured by Amersham Pharmacia) can be used. In this case, follow the protocol attached to 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 polynucleotides that can be hybridized include polynucleotides consisting of the nucleotide sequence of SEQ ID NO: 7 or 9 or SEQ ID NO: 8 or 10 when calculated using the default parameters by an analysis program such as BLAST. About 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 88% or more, 90% or more, 92% or more, 95% or more, Examples include DNA having 97% or more, 98% or more, 99% or more, 99.3% or more, 99.5% or more, 99.7% or more, 99.8% or more, 99.9% or more. The identity of the base sequence or amino acid sequence can be determined using the method described above.

  More preferably, the first region is a region consisting of the amino acid sequence of SEQ ID NO: 10.

  The second region is a region containing the amino acid sequence of a polypeptide having one or more cysteine residues for binding other useful compounds via a thiol group. The “one or more” in the above “having one or more cysteine residues for binding other useful compounds via a thiol group” is, for example, 1, 2, or 3, preferably Is one or two, and particularly preferably one. It is possible to introduce other useful compounds into the fusion protein of the present invention by chemical modification via a thiol group derived from a cysteine residue contained in the second region. The fusion protein according to a preferred embodiment of the present invention has another useful compound by chemical modification via a thiol group derived from a cysteine residue contained in the second region, while hardly affecting the photocatalytic activity of the fusion protein. It is possible to introduce.

  The length of the polypeptide in the second region is, for example, 2 to 40 amino acids, preferably 5 to 35 amino acids, more preferably 7 to 30 amino acids, and still more preferably 10 to 20 amino acids. And most preferably 15 amino acids.

  Other useful compounds for binding to the second region include fluorescent substances, ligands specific to the substance to be detected (e.g. biotin, biotin-binding protein, enzyme, substrate, antibody, antigen, nucleic acid, polysaccharide). , A receptor, or a compound capable of binding to these).

  Examples of the “fluorescent substance” and the “ligand” include those described later.

In some embodiments of the present invention, the second region is a group consisting of the following (e) to (h):
(e) a polypeptide comprising the amino acid sequence of SEQ ID NO: 6,
(f) The amino acid sequence of SEQ ID NO: 6 contains an amino acid sequence in which one to a plurality of amino acids are deleted, substituted, inserted and / or added, and another useful compound is bonded via a thiol group. A polypeptide having one or more cysteine residues for
(g) one amino acid sequence having 70% or more identity to the amino acid sequence of SEQ ID NO: 6 and one cysteine residue for binding other useful compounds via a thiol group A polypeptide having, and
(h) containing 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: 5, and others via a thiol group A polypeptide having one or more cysteine residues for binding a useful compound thereof;
A region containing the amino acid sequence of a polypeptide selected from.

  In the second region, “one or more amino acids are deleted, substituted, inserted and / or added” means one or more amino acids at any position in one or more amino acid sequences in the same sequence It means that there is a deletion, substitution, insertion and / or addition of a residue.

  In the second region, the range of “1 to plural” in the “amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added” is, for example, 1 to 5, 1 to 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 regions are described in “Sambrook J. et al., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press (2001)”, “Ausbel FM et al., Current Protocols in Molecular Biology, Supplement 1- 38, John Wiley and Sons (1987-1997) ”,“ 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) ”, etc. It can be obtained using a mutagenesis method.

Examples of amino acid residues that can be substituted with each other are shown below. Amino acid residues contained in the same group can be substituted for each other.
Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, o-methylserine, t-butylglycine, t-butylalanine, cyclohexylalanine;
Group B: aspartic acid, glutamic acid, isoaspartic acid, isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid;
Group C: asparagine, glutamine;
Group D: lysine, arginine, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid;
Group E: proline, 3-hydroxyproline, 4-hydroxyproline;
Group F: serine, threonine, homoserine;
Group G: phenylalanine, tyrosine.

  In the second region, the range of “70% or more” in the “amino acid sequence having 70% or more identity” is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 88% 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% or more, 99.1% or more, 99.2% or more, 99.3% Above, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, 99.9% or more. In general, the larger the numerical value of identity, the better. The identity of the base sequence or amino acid sequence can be determined using an analysis program such as BLAST (see, for example, Altzshul S. F. et al., J. Mol. Biol. 215, 403 (1990)). When using BLAST, the default parameters of each program are used.

  In the second region, “a polynucleotide that hybridizes under stringent conditions” encodes a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 5 or the amino acid sequence of SEQ ID NO: 6. A polynucleotide (for example, DNA) obtained by using a colony hybridization method, a plaque hybridization method, a Southern hybridization method, or the like using all or part of the polynucleotide as a probe. Specifically, hybridization was performed at 65 ° C. in the presence of 0.7 to 1.0 mol / L NaCl using a filter on which a colony or plaque-derived polynucleotide was immobilized, and then 0.1 to 2 times the concentration of SSC ( Saline-sodium citrate) solution (composition of 1-fold concentration of SSC solution consists of 150 mmol / L sodium chloride and 15 mmol / L sodium citrate), and a polynucleotide that can be identified by washing the filter under 65 ° C conditions Can give.

  Hybridization is described in Sambrook J. et al., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press (2001), Ausbel FM et al., Current Protocols 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), etc. .

  “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, it can be expected that a polynucleotide (eg, DNA) having high homology as the temperature is increased can be efficiently obtained under these conditions. 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, for example, Alkphos Direct Labeling Reagents (manufactured by Amersham Pharmacia) can be used. In this case, follow the protocol attached to 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.

  As other hybridizable polynucleotides, the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 5 or the amino acid sequence of SEQ ID NO: 6 when calculated using the default parameters by an analysis program such as BLAST About 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 88% or more, 90% or more, 92% or more, 95% or more, 97% or more Examples of the DNA include 98% or more, 99% or more, 99.3% or more, 99.5% or more, 99.7% or more, 99.8% or more, 99.9% or more. The identity of the base sequence or amino acid sequence can be determined using the method described above.

In a preferred embodiment of the present invention, the second region is a group consisting of the following (e) to (h):
(e) a polypeptide comprising the amino acid sequence of SEQ ID NO: 6,
(f) contains an amino acid sequence in which 1 to 3 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 6, and binds other useful compounds via a thiol group A polypeptide having one or more cysteine residues for
(g) one amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 6, and one cysteine residue for binding other useful compounds via a thiol group A polypeptide having, and
(h) containing an amino acid sequence encoded by a polynucleotide that hybridizes with a polynucleotide consisting of a base sequence complementary to the base sequence of SEQ ID NO: 5 under highly stringent conditions, and via a thiol group A polypeptide having one or more cysteine residues for binding to other useful compounds;
A region containing the amino acid sequence of a polypeptide selected from.

  In a further preferred embodiment of the present invention, the second region is a region consisting of the amino acid sequence of SEQ ID NO: 6.

The fusion protein of a preferred embodiment of the present invention is
(1) A fusion protein comprising a first region consisting of the amino acid sequence of SEQ ID NO: 10 and (2) a second region consisting of the amino acid sequence of SEQ ID NO: 6.
As a further preferred embodiment of the fusion protein of the present invention, for example, a fusion protein comprising the amino acid sequence of SEQ ID NO: 2 can be mentioned.

  The fusion protein of the present invention may further contain other peptide sequences at the N-terminus and / or C-terminus, preferably at the N-terminus, like the amino acid sequence of SEQ ID NO: 2. Other peptide sequences include, for example, a peptide sequence for promoting translation, a peptide sequence for purification, a secretory signal peptide sequence, a peptide sequence for expressing the fusion protein of the present invention as a soluble protein, and an antibody-recognizable epitope. Mention may be made of at least one peptide sequence selected from the group consisting of sequences and the like. The other peptide sequence is preferably a peptide sequence for purification and / or a secretory signal peptide sequence. In another preferred embodiment of the present invention, the other peptide sequence is at least selected from the group consisting of a peptide sequence for purification, a secretory signal peptide sequence, and a sequence for expressing the fusion protein of the present invention as a soluble protein. An array.

  The fusion protein of the present invention may further contain a linker sequence of a restriction enzyme site, like the amino acid sequence of SEQ ID NO: 2.

  As a peptide sequence for promoting translation, a peptide sequence used in this technical field can be used. Examples of peptide sequences for promoting translation include TEE sequences.

  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 of glutathione S-transferase to glutathione, Examples include protein A amino acid sequence and avidin tag sequence.

  The secretory signal peptide means a peptide region that plays a role of allowing a protein 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 that encode 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, the secretion signal peptide is, for example, 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 fungal cholera toxin.

  The peptide sequence used in this technical field can be used as the linker sequence for the restriction enzyme site.

  In the fusion protein of the present invention, when the first region and the second region are arranged in the order of "N-terminal-first region-second region-C-terminal", the first region The length of the portion excluding the first region from to the C-terminal is, for example, 4 to 50 amino acids, preferably 7 to 45 amino acids, more preferably 14 to 43 amino acids, still more preferably 17-40 amino acids, most preferably 21-36 amino acids.

  In the fusion protein of the present invention, when the first region and the second region are arranged in the order of “N-terminal-second region-first region-C-terminal”, The length of the portion excluding the first region between up to 1 region is, for example, 4 to 50 amino acids, preferably 7 to 45 amino acids, more preferably 14 to 43 amino acids, still more preferably 17-40 amino acids, most preferably 21-36 amino acids.

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

2. Polynucleotide of the Present Invention The present invention also provides a polynucleotide encoding the above-described fusion protein of the present invention. The polynucleotide of the present invention may be any polynucleotide as long as it contains the base sequence encoding the fusion protein of 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. Further, 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.

As the polynucleotide of the present invention, specifically,
(1) A polynucleotide encoding a polypeptide having a luminescent catalytic activity using luciferin as a substrate when expressed as a fusion protein with a polypeptide that is represented by the formula (Z) _n and can be modified A first coding sequence; and
(2) a second coding sequence comprising a polynucleotide that encodes a modifiable polypeptide having one or more cysteine residues for binding other useful compounds via a thiol group; Nucleotides are included.

Here, the formula (Z) _n represents the same meaning as described above.

Among the polynucleotides of the present invention, among others,
(1) A polynucleotide encoding a polypeptide having a luminescent catalytic activity using luciferin as a substrate when expressed as a fusion protein with a polypeptide represented by formula (Z) ₂ and a modifiable polypeptide. A first coding sequence; and
(2) a second coding sequence comprising a polynucleotide that encodes a modifiable polypeptide having one or more cysteine residues for binding other useful compounds via a thiol group; Nucleotides are preferred.

Here, the formula (Z) ₂ represents the same meaning as described above.

As the polynucleotide encoding the polypeptide represented by the formula (Z) ₂ , more specifically, for example,
(a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 10,
(b) When expressed as a fusion protein consisting of 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: 10, and a modifiable polypeptide A polypeptide having a luminescent catalytic activity using luciferin as a substrate,
(c) An amino acid sequence comprising 90% or more identity to the amino acid sequence of SEQ ID NO: 10 and expressed as a fusion protein with a modifiable polypeptide, using luciferin as a substrate A polypeptide having photocatalytic activity, and
(d) a polypeptide 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 of SEQ ID NO: 9, and a modifiable polypeptide Examples thereof include a polynucleotide encoding a polypeptide selected from the group consisting of polypeptides having a luminescence catalytic activity using luciferin as a substrate when expressed as a fusion protein.

The polynucleotide encoding the polypeptide represented by the formula (Z) ₂ is preferably a polynucleotide selected from the group consisting of the following (a) to (d):
(a) a polynucleotide containing a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 9,
(b) When hybridized with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 9 under stringent conditions and expressed as a fusion protein with a modifiable polypeptide, luciferin A polynucleotide containing a polynucleotide that encodes a polypeptide having a luminescent catalytic activity, wherein
(c) a polynucleotide containing a polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 10, and
(d) When the amino acid sequence of SEQ ID NO: 10 comprises an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added, and is expressed as a fusion protein with a modifiable polypeptide. And a polynucleotide containing a polynucleotide encoding a polypeptide having a luminescence catalytic activity using luciferin as a substrate.

The second coding sequence is preferably a group consisting of the following (e) to (h):
(e) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 5,
(f) Residue of cysteine for hybridizing under stringent conditions with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 5 and binding other useful compounds via a thiol group A polynucleotide encoding a polypeptide having one or more groups,
(g) a polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 6, and
(h) To bind other useful compounds via a thiol group, comprising 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: 6. A polynucleotide encoding a polypeptide having one or more cysteine residues of
A coding sequence containing a polynucleotide selected from

  Here, the “polynucleotide hybridizing under stringent conditions” in the first and second coding sequences refers to a polynucleotide comprising a base sequence complementary to the base sequence of SEQ ID NO: 5, 7, or 9 or A polynucleotide (for example, a polynucleotide obtained by using a colony hybridization method, a plaque hybridization method, a Southern hybridization method, or the like using, as a probe, all or part of a polynucleotide encoding the amino acid sequence of SEQ ID NO: 6, 8, or 10. , DNA). Specifically, hybridization was performed at 65 ° C. in the presence of 0.7 to 1.0 mol / L NaCl using a filter on which a colony or plaque-derived polynucleotide was immobilized, and then 0.1 to 2 times the concentration of SSC ( Saline-sodium citrate) solution (composition of 1-fold concentration of SSC solution consists of 150 mmol / L sodium chloride and 15 mmol / L sodium citrate), and a polynucleotide that can be identified by washing the filter under 65 ° C conditions Can give.

  Hybridization is described in Sambrook J. et al., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press (2001), Ausbel FM et al., Current Protocols 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), etc. .

  “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, it can be expected that a polynucleotide (eg, DNA) having high homology as the temperature is increased can be efficiently obtained under these conditions. 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, for example, Alkphos Direct Labeling Reagents (manufactured by Amersham Pharmacia) can be used. In this case, follow the protocol attached to 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 polynucleotides that can be hybridized include polynucleotides consisting of the nucleotide sequence of SEQ ID NO: 5, 7, or 9 or SEQ ID NO: 6 when calculated using the default parameters by an analysis program such as BLAST. About 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 88% or more, 90% or more, 92% or more, and a polynucleotide encoding an amino acid sequence of 8 or 10 Examples include DNA having 95% or more, 97% or more, 98% or more, 99% or more, 99.3% or more, 99.5% or more, 99.7% or more, 99.8% or more, 99.9% or more. The identity of amino acid sequences and base sequences can be determined using the method described above.

  In the first and second coding sequences, “an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added” is as described in the first and second regions, respectively. is there.

  A polynucleotide encoding a region 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 obtained by site-directed mutagenesis (see, for example, 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.), a method utilizing amber mutation (for example, see Gapped duplex method, Nucleic Acids Res. 12, 9441-9456 (1984), etc.) Can be obtained.

  PCR (for example, Ho SN et al., Gene 77, 51 (for example, Ho SN et al., Gene 77, 51 ( 1989) and the like) can also introduce a mutation into a polynucleotide.

  In addition, a polynucleotide encoding a partial fragment of a protein that is a kind of deletion mutant has a sequence that matches the base sequence of the 5 'end of the region encoding the partial fragment to be prepared in the polynucleotide encoding the protein. It can be obtained by performing PCR using an oligonucleotide and an oligonucleotide having a sequence complementary to the base sequence at the 3 ′ end as a primer and using a polynucleotide encoding the protein as a template.

In a preferred embodiment of the polynucleotide of the present invention, the first coding sequence consists of the following groups (a) to (d):
(a) a polynucleotide comprising the base sequence of SEQ ID NO: 9;
(b) When hybridized with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 9 under highly stringent conditions and expressed as a fusion protein with a modifiable polypeptide, A polynucleotide encoding a polynucleotide having luminescence catalytic activity using luciferin as a substrate;
(c) a polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 10, and
(d) When expressed as a fusion protein consisting of an amino acid sequence in which 1 to 3 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 10, and a modifiable polypeptide And a polynucleotide encoding a polypeptide having a luminescence catalytic activity using luciferin as a substrate;
A coding sequence containing a polynucleotide selected from

In a preferred embodiment of the polynucleotide of the present invention, the second coding sequence comprises the following group (e) to (h):
(e) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 5,
(f) Cysteine that hybridizes with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 5 under highly stringent conditions and binds other useful compounds via a thiol group A polynucleotide having one or more residues,
(g) a polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 6, and
(h) To bind other useful compounds via a thiol group, comprising an amino acid sequence in which 1 to 3 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 6. A polynucleotide encoding a polypeptide having one or more cysteine residues of
A coding sequence containing a polynucleotide selected from

The polynucleotide of a further preferred embodiment of the present invention is
(1) a polynucleotide comprising a first coding sequence comprising a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 9 and (2) a second coding sequence comprising a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 5 It is.

  As a particularly preferred embodiment of the polynucleotide of the present invention, for example, a polynucleotide containing a polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 can be mentioned. Examples of the polynucleotide containing a polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 include a polynucleotide containing a polynucleotide consisting of the base sequence of SEQ ID NO: 1.

  The polynucleotide of the present invention may include a polynucleotide containing a polynucleotide encoding another peptide sequence, such as a polynucleotide containing a polynucleotide consisting of the base sequence of SEQ ID NO: 1. Other peptide sequences include, for example, a peptide sequence for promoting translation, a peptide sequence for purification, a secretory signal peptide sequence, a peptide sequence for expressing the fusion protein of the present invention as a soluble protein, and an antibody-recognizable epitope. Mention may be made of at least one peptide sequence selected from the group consisting of sequences and the like.

  The polynucleotide of the present invention may further include a polynucleotide encoding a linker sequence of a restriction enzyme site, such as a polynucleotide containing a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1.

  As the polynucleotide containing a polynucleotide encoding a peptide sequence for promoting translation, a polynucleotide containing a polynucleotide encoding a peptide sequence for promoting translation used in the art may be used. it can. Examples of peptide sequences for promoting translation include those described above.

  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.

Examples of the polynucleotide encoding a peptide sequence for expressing the protein of the present invention as a soluble protein include a polypeptide represented by the formula (Z) _n . Examples of the amino acid sequence of the polypeptide represented by the formula (Z) _n and the nucleic acid sequence encoding it include those described above.

  As the polynucleotide encoding the linker sequence of the restriction enzyme site, a polynucleotide containing a base sequence encoding a peptide sequence for purification used in the art can be used.

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.

Production of Recombinant Vector The recombinant vector of the present invention can be obtained by ligating (inserting) the polynucleotide (DNA) of the present invention into an appropriate vector. More specifically, it can be obtained by cleaving a purified polynucleotide (DNA) with an appropriate restriction enzyme, inserting it into a restriction enzyme site or a multiple cloning site of an appropriate vector, and ligating it to 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 the plasmid include Escherichia coli-derived plasmids (e.g., pBR322, pBR325, pUC118, pUC119), Bacillus subtilis-derived plasmids (e.g., pUB110, pTP5), and yeast-derived plasmids (e.g., YEp13, YEp24, YCp50). can give. Examples of the bacteriophage include λ phage. Examples of animal viruses include retroviruses, vaccinia viruses, insect viruses (eg, baculoviruses) and the like. Further, a pCold I vector, a pCold II vector, a pCold III vector, a pCold IV vector (above, manufactured by Takara Bio Inc.), a PICZa vector (manufactured by Invitrogen), and the like can be suitably used.

  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 an Escherichia bacterium, 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, the PHO5 promoter, PGK promoter, GAP promoter, ADH1 promoter, GAL promoter and the like are preferable. When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable.

  A promoter capable of inducing expression at a low temperature can also be suitably used. Examples of the promoter capable of inducing expression at a low temperature include a promoter sequence of a cold shock gene. Cold shock genes include, for example, E. coli cold shock genes (e.g., cspA, cspB, cspG, cspI, csdA, etc.), Bacillus caldolyticus cold shock genes (e.g., Bc-Csp, etc.), Salmonella enterica cold shock genes (e.g., cspE Erwinia carotovora cold shock gene (for example, cspG etc.) and the like. Among them, for example, a cspA promoter, a cspB promoter, a cspG promoter, a cspI promoter, a csdA promoter and the like can be preferably used as a promoter capable of inducing expression at a low temperature.

  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.

Production of a transformant By introducing a recombinant vector containing the polynucleotide of the present invention (that is, the polynucleotide encoding the fusion protein of the present invention) thus obtained into a suitable host, Transformants can be created. 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. Examples of methods for introducing a recombinant vector into a host cell include the calcium phosphate method (Virology, 52, 456-457 (1973)) and the 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). Thus, a transformant transformed with a recombinant vector containing a polynucleotide encoding the protein of the present invention (polynucleotide of the present invention) can be obtained.

As an expression vector and a transformant expression vector containing a promoter sequence capable of inducing expression at a low temperature, an expression vector containing a promoter sequence capable of inducing expression at a low temperature is particularly preferable.
The expression vector containing a promoter sequence capable of inducing expression at a low temperature specifically means an expression vector containing the following promoter sequence and coding sequence:
(1) a promoter sequence capable of inducing expression at low temperature; and
(2) A coding sequence containing the polynucleotide of the present invention.
The promoter sequence capable of inducing expression at a low temperature means a promoter sequence capable of inducing the expression of the fusion protein by lowering the temperature from the culture conditions for growing the host cells. Examples of the promoter capable of inducing expression at a low temperature include a promoter of a gene encoding a cold shock protein (cold shock gene). Examples of the cold shock gene promoter include those described above.

  The temperature at which the promoter capable of inducing expression at a low temperature used in the present invention is usually 30 ° C. or lower, preferably 25 ° C. or lower, more preferably 20 ° C. or lower, and particularly preferably 15 ° C. or lower. However, in order to induce the expression more efficiently, the expression is usually induced at 5 ° C. or higher, preferably 10 ° C. or higher, particularly preferably about 15 ° C.

  When preparing an expression vector containing a promoter sequence capable of inducing expression at low temperature of the present invention, vectors for inserting the polynucleotide of the present invention include pCold I vector, pCold II vector, pCold III vector, pCold IV vector (Above, manufactured by Takara Bio Inc.) can be preferably used. When these vectors are used to express a prokaryotic cell as a host, the fusion protein can be produced as a soluble protein in the cytoplasm of the host cell.

  As a host into which an expression vector containing a promoter sequence capable of inducing expression at a low temperature is introduced, prokaryotic cells are preferable, E. coli is more preferable, particularly BL21 strain and JM109 strain are preferable, and BL21 strain is particularly preferable.

  The culture temperature for cell growth of a transformant introduced with an expression vector containing a promoter sequence capable of inducing expression at low temperature is usually 25 to 40 ° C, preferably 30 to 37 ° C. The temperature for inducing expression is usually 4 to 25 ° C, preferably 10 to 20 ° C, more preferably 12 to 18 ° C, and particularly preferably 15 ° C.

4. Production of fusion protein of the present invention The present invention also provides a method for producing the fusion protein of the present invention, comprising the steps of culturing the transformant to produce the fusion protein of the present invention. The fusion protein of the present invention can be obtained, for example, by culturing the transformant under conditions that allow expression of the polynucleotide (DNA) encoding the fusion protein of the present invention, generating and accumulating the fusion protein of the present invention, It can be manufactured by purification.

Culturing of the transformant The transformant of the present invention can be cultured according to a usual method used for culturing a host. By the cultivation, the fusion protein of the present invention is produced by the transformant, and the fusion protein of 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 Lac promoter, transformation with isopropyl-β-D-thiogalactopyranoside (IPTG) or the like transformed with an expression vector using trp promoter When culturing the body, indoleacrylic acid (IAA) or the like may be added to the medium.

  When the host is Escherichia, the culture is usually performed at about 15 to 43 ° C. for about 3 to 24 hours, and if necessary, aeration or agitation is added. When the host is Bacillus, the culture is usually carried out 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, Burkholder minimum medium (Proc. Natl. Acad. Sci. USA, 77, 4505 (1980)) or 0.5% (w / v) casamino An SD medium containing acid (Proc. Natl. Acad. Sci. USA, 81, 5330 (1984)) can be mentioned. The pH of the medium is preferably adjusted to about 5-8. The culture is usually carried out 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 bovine serum, a DMEM medium (Virology , 8, 396 (1959)). The pH is preferably about 6-8. 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 additives such as 10% (v / v) bovine serum deactivated in Grace's Insect Medium (Nature, 195,788 (1962)), etc. Is used. The pH of the medium is preferably adjusted to about 6.2 to 6.4. The culture is usually carried out at about 27 ° C. for about 3 to 5 days, and aeration and agitation are added as necessary.

  The culture temperature for cell growth and the temperature for inducing expression of a transformant introduced with an expression vector containing a promoter sequence capable of inducing expression at low temperatures are as described above.

Separation and purification of the fusion protein of the present invention The fusion protein of the present invention can be obtained by separating and purifying the fusion protein of the present invention from the above culture. Here, the culture means any of a culture solution, cultured cells or cultured cells, or disrupted cultured cells or cultured cells. Separation and purification of the fusion protein of the present invention can be carried out according to ordinary methods.

  Specifically, when the fusion protein of the present invention is accumulated in cultured cells or cultured cells, after culturing, the cells or cells are removed by a usual method (for example, ultrasonic wave, lysozyme, freeze-thawing, etc.). After crushing, a crude extract of the fusion protein of the present invention can be obtained by ordinary methods (eg, centrifugation, filtration, etc.). When the fusion protein of the present invention is accumulated in the periplasmic space, an extract containing the fusion protein of the present invention can be obtained by the usual method (for example, osmotic shock method) after completion of the culture. When the fusion protein of the present invention is accumulated in the culture solution, the cells or cells and the culture supernatant are separated from each other by a conventional method (for example, centrifugation, filtration, etc.) after completion of the culture. A culture supernatant containing the fusion protein of the invention can be obtained.

  Purification of the fusion protein of 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 fusion protein of the present invention contains the above-described peptide sequence for purification, it is preferably purified using this. Specifically, when the fusion protein of the present invention contains a histidine tag sequence, nickel chelate affinity chromatography, and when it contains a binding domain to glutathione of S-transferase, affinity chromatography using glutathione-binding gel, protein When the amino acid sequence of A is contained, antibody affinity chromatography can be used.

5. Complex of the Present Invention The fusion protein of the present invention (hereinafter sometimes referred to as “the luciferase of the present invention”) is another useful compound (eg, fluorescent substance, ligand specific to the substance to be detected). Etc.) to form a complex.

  The complex of the present invention is obtained by linking other useful compounds via the thiol group of the cysteine residue in the second region of the luciferase of the present invention. In some embodiments of the present invention, the complex is a combination of the luciferase of the present invention and a fluorescent substance or a ligand specific for the substance to be detected. In the complex of some embodiments of the present invention, the binding ratio between the thiol group of luciferase and the fluorescent substance or ligand is a ratio of 1: 1 or a ratio close thereto.

  Among other useful compounds, fluorescent materials include organic compounds such as Hoechist3342, Indo-1, and DAP1; fluorescent proteins such as green fluorescent protein (GFP), blue fluorescent protein (BFP), mutant GFP fluorescent protein, and phycobilin Can be mentioned.

  The ligand specific to the substance to be detected may be either a substance that directly binds to the substance to be detected or a substance that indirectly binds to the substance to be detected. Examples of the ligand include biotin, biotin-binding protein, enzyme, substrate, antibody, antigen, nucleic acid, polysaccharide, receptor, or a compound capable of binding to these.

  Among these, examples of the biotin-binding protein include avidin, streptavidin, mutant avidin (neutravidin) and the like. These biotin-binding proteins can be obtained commercially. A commercially available biotin-binding protein can also be prepared by a conventional method so that it can be modified.

  As antigens and antibodies, antigens of various substances (e.g., in vivo trace components of humans and animals and plants such as tumor markers and hormones, trace contaminants in the environment, etc.), and antibodies against these antigens have been commercially available. Thus, an antibody having the analyte to be measured as an antigen as appropriate can be obtained and used as necessary.

  Among them, tumor markers that increase in serum and urine with tumor growth include fetal antigen, CA19-9, serial Lex-i antigen, serial Tn antigen, thymidine kinase activity, tissue polypeptide antigen, basic fetoprotein , Immunosuppressive acidic protein, CA72-4, CA125, DUPAN-2, SPan-1, elastase 1, BCA-225, CA15-3, SCC antigen, cytokeratin 19 fragment, prostate specific antigen, gamma-seminoprotein, prostate Acid phosphatase, α-fetoprotein, AFP lectin fraction, PIVKA-II, nerve specific enolase, NCC-ST-439, CA130, type I collagen-C-telopeptide, etc. Increasing markers are known so far. These antigens are commercially available, and can be appropriately used as standard substances for measuring the marker in serum or urine. Furthermore, antibodies of various classes or subclasses against these antigens are commercially available, and these can be used as appropriate.

  Examples of the nucleic acid include any complementary DNA and RNA, and examples thereof include DNA and RNA having a base sequence that can be used for detection of pathogenic genes, genetic diagnosis, and the like. These nucleic acids can be appropriately chemically synthesized by conventional methods.

  Depending on the physicochemical properties, etc., in consideration of the molecular size of the luciferase of the present invention and steric hindrance with other useful compounds, other useful compounds are directly or via a linker or spacer. To luciferase.

  The linker or spacer used in the present invention is not particularly limited as long as it can specifically react with the —SH group, but preferably has a length of 20 angstroms or more. Various —SH group-modifying reagents that can be used as linkers or spacers are commercially available, and these can be used as appropriate.

  The cross-linking reagent having a functional group that reacts with the thiol group of cysteine (also referred to as “sulfhydryl group”) is not particularly limited, but specifically, N- (4- [p-azidosalicylamido ] Butyl) -3′-2′pyridyldithio) propionamide (APDP), 1,4-di- (3 ′-[2′pyridyldithio] propionamide) butane, 1,6-hexane-bis-vinylsulfone ( HBVS), succinimidyl 3- (bromoacetamide) propionate (SBAP), N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP), N- (α-maleimidoacetoxy) -succinimide ester (AMAS), succinimidyl 4- (N -Maleimidomethyl) cyclohexane (SMCC), sulfonated derivative of SMCC (sulfo-SMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), sulfonated derivative of MBS (sulfo-MBS), succini Gills 4- (p-maleimidophenyl) butyrate (SMPB), sulfonated derivatives of SMPB (sulfo-SMPB), succinimidyl-6- (N-maleimide-n-hexanoate), succinimidyl (4-iodoacetyl) aminobenzoate (SIAB) ), N-hydroxysuccinimidyl bromoacetate, bis- (maleimido) methyl ester, bis-maleimidohexane (BMH), and the like. In the present invention, among them, a crosslinking reagent having a maleimide group as a functional group that reacts with a sulfhydryl group is preferable.

Reactions for conjugating other useful compounds to the luciferase of the present invention are described in Hermanson GT. "Bioconjugate Techniques, 2" nd Edition (2008) Elsevier Inc. can be carried out by a method known in the art, such as the method described in Elsevier Inc. More specifically, in the case of a ligand specific to the substance to be detected, the reaction is desirably performed at 30 ° C. or lower, preferably 25 ° C. or lower, pH 6-8, preferably pH 7-7.5. In the case of a fluorescent substance, the reaction is desirably carried out at 25 ° C. or lower, preferably 4 ° C. or lower, pH 6-8, preferably pH 7-7.5.

  In particular, in the case of a biotin-binding protein, the reaction is desirably performed at 25 ° C. or lower, preferably 4 ° C. or lower, pH 6-8, preferably pH 7-7.5. In the case of antibodies, the reaction is desirably carried out at 25 ° C. or lower, preferably 4 ° C. or lower, pH 6-8, preferably pH 7-7.5. In the case of nucleic acids, the reaction is desirably carried out at 25 ° C. or lower, preferably 4 ° C. or lower, pH 6-8, preferably pH 7-7.5.

6. Use of the fusion protein and complex of the present invention
Use as a detection marker by luminescence The fusion protein of the present invention or the complex of 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 example, for detection of a target substance in an immunoassay, a hybridization assay, or the like. When the complex of the present invention is used as a detection marker, other useful compounds in the complex of the present invention are ligands specific to the substance to be detected.

  The fusion protein of a preferred embodiment of the present invention can bind to IgG without substantially inhibiting the luminescent catalytic activity using luciferin as a substrate. Such a fusion protein can be used for detection of IgG in an immunoassay. Such detection of IgG can be performed by a usual method. Specifically, IgG can be detected by binding the fusion protein of the present invention to IgG and emitting light with luciferin.

  When the complex of the present invention is used in an immunoassay, the ligand of the complex of the present invention is, for example, a primary antibody that specifically recognizes a target substance. Since the primary antibody in the complex of the present invention specifically binds to a substance to be detected (antigen) present in the sample, by measuring the luminescence of the fusion protein in the complex of the present invention, The part and amount of the substance to be detected can be detected.

  In order to increase the sensitivity of detection, a method using a secondary antibody that specifically recognizes the primary antibody is also well known. In this case, the ligand in the complex of the present invention is, for example, a secondary antibody.

  Alternatively, biotin can be bound to the secondary antibody, and this biotinylated secondary antibody can be reacted with avidin or streptavidin bound to the fusion protein of the present invention. In this case, the ligand in the complex of the present invention is avidin or streptavidin.

  Furthermore, the property that one molecule of avidin or one molecule of streptavidin binds to four molecules of biotin can be used. That is, a biotinylated secondary antibody is reacted with avidin or streptavidin, and further reacted with biotin bound to the fusion protein of the present invention. In this case, the ligand in the complex of the present invention is biotin.

  When detecting a receptor using the complex of the present invention, a signal peptide (hormone such as insulin, cytokine, TNF, Fas ligand, etc.) that binds to the receptor is used as a ligand in the complex of the present invention. Can do. On the other hand, when detecting a signal peptide, a protein constituting the receptor can be used as a ligand in the complex of the present invention. That is, when detecting a receptor to which a certain drug binds, the drug can be used as a ligand in the complex of the present invention. When detecting a drug that binds to a certain receptor, the receptor is defined as that of the present invention. It can be a ligand in a complex.

  When an enzyme is detected using the complex of the present invention, the enzyme substrate can be used as a ligand in the complex of the present invention. On the other hand, when detecting a substrate of the enzyme, the enzyme can be used as a ligand in the complex of the present invention.

  When the complex of the present invention is used in a hybridization assay, a nucleic acid complementary to the nucleic acid to be detected is detected in the complex of the present invention in order to detect other nucleic acids that specifically bind to one nucleic acid. It can be a ligand.

  When other substances that specifically bind to polysaccharides are detected using the complex of the present invention, the polysaccharide can be used as a ligand in the complex of the present invention.

  In addition, a DNA-binding protein such as a lectin or a transcription factor that can specifically bind to a blood coagulation factor can be used as a ligand in the complex of the present invention.

  Since the complex of the present invention can be directly or indirectly bound to a target substance via a ligand as described above, it can be used as a detection marker by luminescence in the presence of luciferin. Detection of a target substance using such a detection marker can be performed by a usual method.

  In addition, the fusion protein of the present invention can be used for measuring the distribution of the target protein, for example, 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 fusion protein of the present invention can be expressed in a cell and used in addition to being introduced into a cell by a technique such as a microinjection method.

Use as a reporter protein The fusion protein of the present invention can also be used as a reporter protein to measure transcriptional activity of a promoter or the like. A vector is constructed in which a polynucleotide encoding the fusion protein of 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). By introducing the vector into a host cell and detecting luminescence derived from the fusion protein of the present invention in the presence of luciferin, the activity of the target promoter or other expression control sequence can be measured.

  The polynucleotide of the present invention can be used as a reporter gene as described above.

Materials for Amusement Products The fusion protein of 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 fusion protein of the present invention can be suitably used as a luminescent base material for amusement materials. 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.

Bioluminescence Resonance Energy Transfer (BRET) Method The fusion protein of the present invention or the complex of the present invention is analyzed for physiological functions and measurement of enzyme activity using the principle of intermolecular interaction by the bioluminescence resonance energy transfer (BRET) method. It can be used for analysis methods such as.

  For example, in the fusion protein of the present invention or the complex of the present invention, the fusion protein of the present invention or the complex of the present invention (sometimes referred to as “the luciferase of the present invention”) is used as a donor, and a fluorescent substance (eg, The interaction between the donor and the acceptor can be detected by causing bioluminescence resonance energy transfer (BRET) between them using an organic compound, a fluorescent protein, etc.) as an acceptor.

  In one embodiment of the present invention, the organic compound used as an acceptor is Hoechist3342, Indo-1, DAP1, or the like. In another embodiment of the present invention, the fluorescent protein used as an acceptor is green fluorescent protein (GFP), blue fluorescent protein (BFP), mutant GFP fluorescent protein, phycobilin, and the like.

  In a preferred embodiment of the present invention, the physiological function to be analyzed is orphan receptor (especially G protein-coupled receptor), apoptosis, or transcriptional regulation by gene expression. In a preferred embodiment of the present invention, the enzyme to be analyzed is a protease, esterase or phosphorylase.

  Analysis of physiological functions by the BRET method can be performed by a known method, for example, the method described in Biochem. J. 2005, 385, 625-637 or Expert Opin. Ther Tarets, 2007 11: 541-556, etc. It can be performed according to. The enzyme activity can also be measured by a known method, for example, according to the method described in Nature Methods 2006, 3: 165-174, Biotechnol. J. 2008, 3: 311-324, or the like. be able to.

  The complex of some embodiments of the present invention is obtained by binding a fluorescent substance as another useful compound to the fusion protein of the present invention. Examples of the fluorescent material include those described above. BRET can be caused between the fusion protein of the present invention in this complex as a donor and the fluorescent substance in the same complex as an acceptor.

7. Kit of the Present Invention The present invention is a kit comprising any one selected from the fusion protein of the present invention, the polynucleotide of the present invention, the recombinant vector of the present invention, the transformant of the present invention and the complex of the present invention. Also provide. The kit of the present invention may further contain luciferin (for example, coelenterazines). 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, detection marker by luminescence, analysis of physiological function by BRET method, measurement of enzyme activity, and the like.

8. Luminescent reaction method
Luminescent Catalytic Activity The fusion protein of the present invention or the complex of the present invention (hereinafter sometimes referred to as “the fusion protein of the present invention”) is an excited state by oxidizing luciferin (for example, coelenterazines) with oxygen molecules. It has an activity to catalyze a reaction for producing oxyluciferin. Excited oxyluciferin emits visible light when it reaches the ground state. That is, the fusion protein of the present invention has an activity of catalyzing a luminescence reaction using luciferin (for example, coelenterazines) as a substrate and generating luminescence. This activity is sometimes referred to herein as “luminescence catalytic activity”.

Luminescent reaction A luminescent reaction using a luciferin (for example, coelenterazines) as a substrate using the fusion protein of the present invention can be carried out by bringing the fusion protein of the present invention into contact with luciferin. 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 4 ° C to about 40 ° C, preferably about 4 ° C to about 25 ° 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 Luminescent Activity Luminescent activity (luminescent catalytic activity) using luciferin (for example, coelenterazines) as a substrate, such as the fusion protein of the present invention, is activated by halide ions, nonionic surfactants, and the like.

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 can be mentioned.

Nonionic surfactants include Tween 20 (polyoxyethylene sorbitan monolaurate), Tween 80 (polyoxyethylene sorbitan monooleate), Triton X-100 (polyethylene glycol-p-isooctyl phenyl ether), Briji-58 ( Polyoxyethylene (20) cetyl ether), Nonidet P-40 (ethylphenol poly (ethylene glycol ether) n) and the like, and Tween 20, Triton X-100 and the like are preferable.
The concentration of the nonionic surfactant is usually about 0.0002% (w / v) to about 0.2% (w / v), preferably about 0.001% (w / v) to about 0.1% (w / v), especially Preferably, it is about 0.05% (w / v) to about 0.02% (w / v).

  It should be noted that all documents and publications described in this specification are incorporated herein by reference in their entirety regardless of their purposes.

  Unless otherwise stated in the embodiments and examples, J. Sambrook, EF Fritsch & T. Maniatis (Ed.), Molecular cloning, a laboratory manual (3rd edition), Cold Spring Harbor Press, Cold Spring Harbor, New York (2001); FM Ausubel, R. Brent, RE Kingston, DD Moore, JG Seidman, JA Smith, K. Struhl (Ed.), Standard Protocols in Molecular Biology, John Wiley & Sons Ltd. The method described in the protocol collection, 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 reproduce the present invention from the description of the present specification. it can.

  The embodiments and specific examples of the invention show preferred embodiments of the present invention, and are shown for illustration or explanation, and the present invention is not limited thereto. . 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.

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 a new basic vector pCold-ZZ-gCys for expressing a protein having a histidine sequence at the amino terminus and a ZZ domain that is an IgG binding domain A new expression vector pCold-ZZ-gCys was prepared by the following procedure. Built with. pCold-ZZ-gCys has a histidine sequence that enables purification by nickel chelate gel chromatography, a ZZ domain that is an IgG-binding domain of protein A, and a peptide linker and cysteine residue on the C-terminal side of the ZZ domain. A fusion protein (recombinant ZZ domain-gCys) is expressed.

  In the same manner as described in Example 1 of JP-A-2008-99669, peptide linker and cysteine residue (Gly-Gly-Gly -Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Cys) (SEQ ID NO: 6) was inserted to construct pCold-ZZ-gCys. .

GGGGS Linker-F (5 'AG CTT GGT GGT GGT GGT TCT GGT GGT GGT GGT TCT GGT GGT GGT GGT TGC TAA GCT AGC CTG CA 3') (SEQ ID NO: 3); and
GGGGS Linker-R (5 'G GCT AGC TTA GCA ACC ACC ACC ACC AGA ACC ACC ACC ACC AGA ACC ACC ACC ACC A 3') (SEQ ID NO: 4).

  The insert DNA was confirmed by determining the nucleotide sequence with a DNA sequencer (manufactured by ABI). The nucleotide sequence of the polynucleotide encoding the recombinant ZZ domain-gCys is shown in SEQ ID NO: 1, and the amino acid sequence of the recombinant ZZ domain-gCys is shown in SEQ ID NO: 2.

  The new expression vector pCold-ZZ-gCys has E. coli cold shock protein A promoter as the promoter and lactose operator as the operator, and has 6 histidine sequences that can be purified by chelating gel and IgG binding ability. And a sequence encoding a flexible peptide linker and a cysteine residue downstream thereof (FIG. 1).

Example 2 Purification Method of Recombinant ZZ Domain-gCys Recombinant ZZ domain-gCys was obtained as shown below. That is, using the expression vector pCold-ZZ-gCys, recombinant ZZ domain-gCys was expressed in E. coli, and purified by IgG sepharose column chromatography to obtain purified ZZ domain-gCys.

1) Expression of recombinant ZZ domain-gCys in E. coli In order to express recombinant ZZ domain-gCys in E. coli, an expression vector pCold-ZZ-gCys including the ZZ domain-gCys gene was used. The expression vector pCold-ZZ-gCys was introduced into Escherichia coli BL21 by a conventional method, and the obtained transformant was treated with 10 ml of LB liquid medium containing ampicillin (50 μg / ml) (10 g of bactotryptone per liter of water). Inoculated in yeast extract 5 g, sodium chloride 5 g, pH 7.2) and cultured at 37 ° C. for 18 hours. Then, the culture was added to 400 ml x5 new LB liquid medium (total volume 2 L), incubated at 37 ° C. for 2.5 hours, cooled on ice water, and isopropyl-β-D (−)-thiogalacto Pyranoside (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. After culturing, the cells were collected by centrifugation (5,000 rpm, 5 minutes) and used as a starting material for protein extraction.

2) Extraction of ZZ domain-gCys from cultured cells and IgG Sepharose column chromatography The collected cultured cells are suspended in 200 ml of 50 mM Tris-HCl (pH 7.6) and subjected to ultrasonic crushing under ice-cooling. (Branson, Sonifier model cycle 250) was performed 3 times for 3 minutes, and the cell disruption solution was centrifuged at 10,000 rpm (12,000 × g) at 4 ° C. for 20 minutes. An IgG Sepharose 6 FastFlow column (Amersham Biosciences, column size: diameter 2.5 × 6) in which the obtained soluble fraction was equilibrated with a TBST solution (20 mM Tris-HCl (pH7.4), 150 mM NaCl, 0.5% Tween20). cm), and ZZ domain-gCys was adsorbed on the column. The column was washed with 300 ml of TBST solution, then with 100 ml of ammonium acetate solution (pH 5.0), and then eluted with 10 ml of 0.5 M acetic acid solution (pH 3.4) (Wako Pure Chemical Industries). I did it. As a result of measuring the eluted fraction with an absorption spectrum at 280 nm, the ZZ domain-gCys fraction was confirmed. Further, the elution fraction was analyzed by SDS-PAGE using a 12% polyacrylamide gel in a reduced state, and ZZ domain-gCys was confirmed. The results of SDS-PAGE analysis are shown in FIG.

3) Dialysis of IgG Sepharose column eluate
20 ml of the ZZ domain-gCys elution fraction obtained from the IgG sepharose column was dialyzed against 5 L of 100 mM sodium phosphate buffer (pH 7.3) at 4 ° C for 72 hours, and 22 ml of ZZ domain-gCys solution was dialyzed. I got it. The ZZ domain-gCys solution after dialysis was analyzed by SDS-PAGE using 12% polyacrylamide gel in a non-reducing state. The results of SDS-PAGE analysis are shown in FIG. Dimer formation by a newly introduced cysteine residue of ZZ domain-gCys generated during 72 hours of dialysis was confirmed.

  The protein concentration was determined using a commercially available kit (Bio-Rad) based on the Bradford method and using bovine serum albumin (Pierce) as a standard substance.

  The results of SDS-PAGE analysis in each purification process are shown in FIG. As shown in FIG. 2, a single band corresponding to a molecular weight of 18 kDa protein was detected in the final purified fraction, and it was revealed that the purity was 95% or more. As shown in Table 2, 94.6 mg of ZZ domain-gCys was obtained from 2 L of cultured cells.

Example 3 Identification of ZZ Domain-gCys by Mass Spectrometry Mass analysis by Matrix assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS) was performed on the ZZ domain-gCys purified in Example 2. (Bruker Daltonics). Angiotensin I (m / z 1296.69), insulin (m / z 5734.59), apomyoglobin (m / z 16952.60), and apoaequorin (m / z 2163.20) were used as molecular weight standards. Sinapic acid (Invitrogen) was used as a matrix. As a result of measurement, 1792.37 was obtained as m / z of the main peak. This value was in good agreement with the calculated average mass [M + H ⁺ ] = 1804.68 of ZZ domain-gCys. Moreover, the measured value of m / z = 35, 800.38 corresponding to the ZZ domain-gCys dimer ([M + H ⁺ ] = 36,007.7) described in Example 2 was confirmed. The peak intensity conversion calculation revealed that 6.5% ZZ domain-gCys formed dimers in 72 hours of dialysis. Therefore, the ZZ domain-gCys after dialysis was stored by freezing.

Example 4 Detection of Luminescent Activity of ZZ Domain-gCys
The luminescence activity of ZZ domain-gCys was measured as follows. The substrate coelenterazine (1 μg / μl) dissolved in ethanol was mixed with 30 μm Tris-HCl (pH 7.6) solution containing 100 μl of 10 mM EDTA, and then the luminescence reaction was started by adding ZZ domain-gCys (5 μg). . Luminescence activity was measured for 60 seconds with a luminescence measuring device Luminescencer-PSN AB2200 (manufactured by ATTO). Luminescent activity was indicated by the maximum luminescence value (Imax). As a control experiment, luminescence activity was measured in the same manner using 5 μg of Protein A (RepliGen) and bovine serum albumin (Sigma). In order to show that it was an enzymatic reaction, heat treatment was performed at 95 ° C. for 10 minutes, and the luminescence activity was measured in the same manner. The measurement results of the luminescence activity are summarized in Table 3.

  As a result, it became clear that ZZ domain-gCys is a protein (luciferase) that catalyzes luminescence. In addition, it showed an activity about 100 times that of bovine serum albumin, and was found to be sufficiently usable as a luminescent enzyme.

  Furthermore, when the relationship between the protein concentration (1-15 μg) of ZZ domain-gCys and the luminescence activity was examined, as shown in Table 4, the luminescence intensity increased in a concentration-dependent manner, so that ZZ domain-gCys was compared with normal luciferase. It turns out that the same property is shown.

  The light emission pattern of ZZ domain-gCys is shown in FIG. ZZ domain-gCys shows a light emission pattern that decays rapidly after the start of the light emission reaction.

Example 5 Spectroscopic analysis by measurement of emission spectrum Purified ZZ domain--GCys (500 μg) was dissolved in 500 μl of 30 mM Tris-HCl (pH 7.6) solution containing 10 mM EDTA, and then the ZZ domain- Luminescence reaction is started by adding coelenterazine (5μg / 5μl) substrate dissolved in ethanol to gCys solution, and excitation of fluorescence measuring device (manufactured by JASCO Corporation, FP-6500) in quartz cell with optical path length of 10mm The emission spectrum was measured with the light source turned off, and the spectrum was corrected. The measurement conditions are as follows: band pass: 20 nm, response: 0.5 sec, scan speed: 2000 nm / min, temperature: 22-25 ° C. The emission maximum value (λmax, nm) and the half width (nm) were determined from the measured emission spectrum, and the results are summarized in Table 5. The emission spectrum is shown in FIG. This spectral result is very similar to the blue emission of luciferase using coelenterazine as a substrate.

Example 6 Substrate specificity of ZZ domain-gCys
Substrate coelenterazine (CTZ) or its derivative (Bis-CTZ to 8- (3-benzo [b] thienyl) -CTZ) dissolved in ethanol in 30 mM Tris-HCl (pH 7.6) containing 100 μl of 10 mM EDTA (1 μg / μl) was mixed, and 1 μl of ZZ domain-gCys (4.25 μg protein) was added to initiate the luminescence reaction. The substrate coelenterazine or a derivative thereof is commercially available, or prepared by a synthesis method described in WO2010 / 090319 or Inouye et al. (2010) Anal. Biochem. 407 , 247-252 or a method analogous thereto. Using. Specifically, CTZ, Bis-CTZ, hcp-CTZ and h-CTZ were obtained from Promega. n-CTZ, 3iso-CTZ, 3meo-CTZ, cf3-CTZ, i-CTZ, et-CTZ, meo-CTZ, me-CTZ, 3me-CTZ, αmeh-CTZ, 8- (1-naphthyl) -CTZ, 8- (2-naphthyl) -CTZ, 8- (2-thienyl) -CTZ, 8- (4-hydroxyphenyl) -CTZ, 8- (2-benzothienyl) -CTZ, 8- (b-styryl) -CTZ, 8-phenyl-CTZ, 8- (3-thienyl) -CTZ, 8- (3-benzo [b] thienyl) -CTZ, 6,8-di (2-thienyl) -CTZ, 6-deoxy-CTZ A synthesis method described in Inouye et al. (2010) Anal. Biochem. 407 , 247-252 or a method prepared by the same method was used. Luminescence activity was measured for 30 seconds with a luminescence measuring device Luminescencer-PSN AB2200 (manufactured by ATTO). Luminescent activity was expressed as relative activity (%) relative to coelenterazine (CTZ) (Table 6). As a result, it was revealed that the best luminescent substrate for ZZ domain-gCys is coelenterazine, but derivatives of coelenterazine also become luminescent substrates.

Here, the maximum light emission value (Imax) of CTZ is 27,084 rlu (100%), and the light emission integrated value for 30 seconds is 2,052,125 rlu (100%). The relative emission intensity of 1 rlu corresponds to 1.8 x 10 ⁶ photons.

Example 7 Optimal pH of ZZ Domain-gCys
100 μl of 100 mM sodium acetate buffer (pH 5.0), 100 mM sodium phosphate buffer (pH 6.0, pH 6.5, pH 7.0, and pH 7.5), 100 mM Tris-HCl buffer (pH 7. 0, pH 7.5, pH 8.0, pH 8.5, and pH 9.0) or 100 mM glycine-sodium hydroxide buffer (pH 8.0, pH 9.0, and pH 10.0) dissolved in ethanol After mixing the substrate coelenterazine (1 μg / μl), the luminescence reaction was started by adding 1 μl of ZZ domain-gCys (4.25 μg protein) to the mixed solution. Luminescence activity was measured for 60 seconds with a luminescence measuring device Luminescencer-PSN AB2200 (manufactured by ATTO). Luminescent activity showed the maximum luminescence value (Imax) as relative activity (%) (FIG. 6). As a result, it was shown that the optimum buffer and optimum pH of ZZ domain-gCys was 100 mM Tris-HCl (pH 7.5). The activity was detected only with the Tris-HCl buffer solution, not by the effects of sodium ions, acetate ions, and phosphate ions derived from the buffer, but with the trisaminomethane or chloride ions contained in the Tris-HCl buffer solution ( The possibility of the effect by Cl-) was suggested.

Example 8 Activation of Luminescent Activity of ZZ Domain-gCys by Sodium Chloride
The substrate coelenterazine (1 μg / μl) dissolved in ethanol was mixed with various concentrations of sodium phosphate buffer including 100 mM sodium phosphate buffer (pH 7.5) containing 100 μl sodium chloride solution, and then mixed Luminescent reaction was initiated by adding 1 μl of ZZ domain-gCys to the solution. Luminescence activity was measured for 10 seconds with a luminescence measuring device Luminescencer-PSN AB2200 (manufactured by ATTO). Luminescent activity showed the maximum value (Imax) as relative activity (%) (FIG. 7). As a result, the sodium chloride concentration with the highest activation of ZZ domain-gCys was shown to be 100 mM. In other words, it was revealed that activation was caused by the chloride ion (Cl ⁻ ) effect.

Example 9 Activation of Luminescent Activity of ZZ Domain-gCys by Halogen Ion In order to investigate the activation effect by other halogen ions, 50 μl of pure phosphine in 50 mM 100 mM sodium phosphate buffer (pH 7.5) was used. After mixing 100 mM halogen compound (MgCl ₂ , NaF, KCl, NaCl, KBr, NaBr, KI, or NaI) dissolved in water (final concentration 50 mM) and substrate coelenterazine (1 μg / μl) dissolved in ethanol The luminescence reaction was initiated by adding 1 μl of ZZ domain-gCys (4.25 μg protein) to the mixed solution. Luminescence activity was measured for 10 seconds with a luminescence measuring device Luminescencer-PSN AB2200 (manufactured by ATTO). Luminescent activity was expressed as the maximum luminescence value (Imax) in relative activity (%) (Table 7). Halogen ions Br ^-, Cl ^-, I ^- addition, the light emission activity significantly activated ZZ domain -gCys as compared with the case of no addition, over 10% as compared with the case of no addition by the addition of NaF in Activation was shown. This revealed that the luminescence activity of ZZ domain-gCys is generally activated by halogen ions.

Example 10 Activation of Luminescence Activity of ZZ Domain-gCys by Tween 20 In order to examine the effect of luminescence activity by addition of a surfactant, various concentrations of nonionic surfactant Tween 20 (manufactured by Sigma) After mixing ZZ domain-gCys 1 μl (4.25 μg protein) with 100 μl PBS (Sigma) containing, the substrate is coelenterazine (1 μg / μl) dissolved in ethanol to start the luminescence reaction. It was. Luminescence activity was measured for 60 seconds with a luminescence measuring device Luminescencer-PSN AB2200 (manufactured by ATTO). Luminescent activity showed the maximum value (Imax) as relative activity (FIG. 8). As a result, it became clear that the addition of the nonionic surfactant Tween 20 activated the luminescent activity of ZZ domain-gCys, and the optimal Tween 20 concentration was 0.01% (w / v). It was.

Example 11 Inhibitory Effect of Luminescent Activity of ZZ Domain-gCys by Human IgG The ZZ domain of ZZ domain-gCys having luminescent catalytic activity has IgG binding ability. In order to confirm whether the IgG binding site and the luminescent catalytic site are identical, human IgG was added to ZZ domain-gCy, and the effect on the luminescent activity was examined.

Specifically, in 100 μl of PBS (manufactured by Sigma), the molar ratio of ZZ domain-gCys 1 μl (18 kDa, 5.0 μg protein) and human IgG (150 kDa, 10 mg / ml, manufactured by Oriental Yeast) Mix at 1: 0, 1: 0.5, 1: 1, 0: 0.5, and 0: 1, let stand at 25 ^° C for 10 minutes, then add substrate coelenterazine (1 μg / μl) dissolved in ethanol This initiated the luminescence reaction. 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). As a result, as shown in Table 8, since the inhibitory effect on the luminescence activity of ZZ domain-gCys by addition of human IgG was not observed, it was revealed that the IgG binding site and the luminescent catalyst site were different.
This indicates that ZZ domain-gCys is a protein having IgG binding and photocatalytic activity.

[SEQ ID NO: 1] This shows the base sequence of DNA encoding ZZ domain-gCys (including ZZ domain, peptide linker and cysteine residue) inserted into expression vector pCold-ZZ-gCys prepared in Example 1 .
[SEQ ID NO: 2] This shows the amino acid sequence of ZZ domain-gCys into which the expression vector pCold-ZZ-gCys prepared in Example 1 has been inserted.
[SEQ ID NO: 3] This shows the base sequence of the peptide linker and cysteine residue used in EXAMPLE 1.
[SEQ ID NO: 4] This shows the base sequence of the peptide linker and cysteine residue used in EXAMPLE 1.
[SEQ ID NO: 5] This shows the base sequence of DNA encoding peptide linker and cysteine residue.
[SEQ ID NO: 6] SEQ ID NO: 5 This shows the amino acid sequence of the peptide linker and cysteine residue.
[SEQ ID NO: 7] This shows the base sequence of DNA encoding the polypeptide represented by formula Z.
[SEQ ID NO: 8] This shows the amino acid sequence of the polypeptide represented by formula Z.
[SEQ ID NO: 9] This shows the base sequence of DNA encoding the polypeptide represented by formula (Z) ₂ .
[SEQ ID NO: 10] This shows the amino acid sequence of the polypeptide represented by formula (Z) ₂ .

Claims (31)

(1) Formula (Z) _n
(Wherein n represents an integer of 1 to 5 and Z represents a polypeptide selected from the group consisting of the following (a) to ( c ):
(a) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 8,
(b) a polypeptide comprising an amino acid sequence in which 1 to 5 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 8; and
(c) a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 8 )
A first region containing the amino acid sequence of a polypeptide having a luminescence catalytic activity using luciferin as a substrate when expressed as a fusion protein with a polypeptide that is represented by
(2) As a modifiable polypeptide having one or more cysteine residues for binding other useful compounds via a thiol group, the group consisting of the following (d) to (f):
(d) a polypeptide comprising the amino acid sequence of SEQ ID NO: 6,
(e) In order to bind another useful compound via a thiol group, comprising an amino acid sequence in which 1 to 3 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 6. A polypeptide having one or more cysteine residues, and
(f) one or more cysteine residues consisting of an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 6 and for binding other useful compounds via a thiol group A polypeptide having,
And a second region containing the amino acid sequence of a polypeptide selected from : a fusion protein. Polypeptide represented by the formula (Z) _n is, (Z) is a polypeptide represented by _2, claim 1 Symbol placement of the fusion protein. The polypeptide represented by the formula (Z) ₂ is:
(a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 10,
(b) When the amino acid sequence of SEQ ID NO: 10 comprises an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted and / or added, and is expressed as a fusion protein with a modifiable polypeptide. And a polypeptide having a luminescence catalytic activity using luciferin as a substrate, and
(c) An amino acid sequence comprising 90% or more identity to the amino acid sequence of SEQ ID NO: 10 and expressed as a fusion protein with a modifiable polypeptide, using luciferin as a substrate A polypeptide having photocatalytic activity,
Or Ranaru a polypeptide selected from the group claim 2 fusion protein according. The fusion protein according to any one of claims 1 to 3 , wherein the polypeptide represented by the formula (Z) n has a function capable of binding to the Fc region of IgG. (1) the first region is a region consisting of the amino acid sequence of SEQ ID NO: 10;
(2) The fusion protein according to claim 1, wherein the second region is a region consisting of the amino acid sequence of SEQ ID NO: 6. The fusion protein according to any one of claims 1 to 5 , further comprising an amino acid sequence for promoting translation and / or an amino acid sequence for purification.   A fusion protein consisting of the amino acid sequence of SEQ ID NO: 2. A polynucleotide comprising a polynucleotide encoding the fusion protein according to any one of claims 1 to 7 . (1) Formula (Z) _n
(Wherein n represents an integer of 1 to 5 and Z represents a polypeptide selected from the group consisting of the following (a) to ( c ):
(a) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 8,
(b) a polypeptide comprising an amino acid sequence in which 1 to 5 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 8; and
(c) a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 8 )
A first coding sequence comprising a polynucleotide that encodes a polypeptide having a luminescence catalytic activity when expressed as a fusion protein with a polypeptide that is represented by
(2) As a polynucleotide encoding a modifiable polypeptide having one or more cysteine residues for binding other useful compounds via a thiol group , the following groups (d) to (f) :
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 5;
(e) a polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 6, and
(f) The amino acid sequence of SEQ ID NO: 6 consists of an amino acid sequence in which 1 to 3 amino acids are deleted, substituted, inserted and / or added, and for binding other useful compounds via a thiol group A polynucleotide encoding a polypeptide having one or more cysteine residues of
And a second coding sequence containing a polynucleotide selected from . Formula (Z) polypeptide represented by _n is a polypeptide represented by (Z) _2, 9. Symbol mounting polynucleotide. The polypeptide represented by the formula (Z) ₂ is:
(a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 10,
(b) When the amino acid sequence of SEQ ID NO: 10 comprises an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted and / or added, and is expressed as a fusion protein with a modifiable polypeptide. And a polypeptide having a luminescence catalytic activity using luciferin as a substrate, and
(c) An amino acid sequence comprising 90% or more identity to the amino acid sequence of SEQ ID NO: 10 and expressed as a fusion protein with a modifiable polypeptide, using luciferin as a substrate A polypeptide having photocatalytic activity,
Or Ranaru a polypeptide selected from the group claim 10, wherein the polynucleotide. The polynucleotide according to any one of claims 9 to 11 , wherein the polypeptide represented by the formula (Z) n has a function capable of binding to an Fc region of IgG. (1) the first coding sequence is a coding sequence consisting of a polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 10,
(2) The polynucleotide according to claim 9 , wherein the second coding sequence is a coding sequence consisting of a polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 6. (1) the first coding sequence is a coding sequence comprising a polynucleotide comprising the base sequence of SEQ ID NO: 9,
(2) The polynucleotide according to claim 9 , wherein the second coding sequence is a coding sequence comprising a polynucleotide comprising the base sequence of SEQ ID NO: 5. The polynucleotide according to any one of claims 9 to 14 , further comprising a polypeptide comprising an amino acid sequence for promoting translation and / or a polynucleotide encoding a polypeptide comprising an amino acid sequence for purification.   A polynucleotide comprising a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1. A recombinant vector containing the polynucleotide according to any one of claims 8 to 16 . A transformant into which the recombinant vector according to claim 17 has been introduced. Culturing the transformant of claim 18, comprising the step of producing a fusion protein of any one of claims 1 to 7, the manufacturing method of the fusion protein of any one of claims 1-7 . A complex obtained by binding another useful compound to the fusion protein according to any one of claims 1 to 7 via a thiol group of a cysteine residue in the second region of the fusion protein. 21. The complex according to claim 20 , wherein the other useful compound is a fluorescent substance and / or a ligand specific for the substance to be detected. A kit comprising the fusion protein according to any one of claims 1 to 7 . A kit comprising the polynucleotide according to any one of claims 8 to 16 , the recombinant vector according to claim 17, or the transformant according to claim 18 . A kit comprising the complex according to claim 20 or 21 . The kit according to any one of claims 22 to 24 , further comprising luciferin. 26. The kit according to claim 25 , wherein the luciferin is coelenterazines. 27. The kit according to claim 26 , wherein the coelenterazine is coelenterazine. A method for performing a luminescent reaction, comprising bringing the fusion protein according to any one of claims 1 to 7 or the complex according to claim 20 or 21 into contact with luciferin. Using the fusion protein according to claim 1 to 7 or the complex according to claim 20 or 21 as a donor protein, bioluminescence resonance energy transfer (BRET) method is performed, analysis of physiological function or enzyme activity Measuring method. A method for measuring a substance specific for a ligand, using the complex according to claim 21 . An immunoassay method using the fusion protein according to any one of claims 1 to 7 .