JP5540501B2 - Method for producing photoprotein - Google Patents

Description

  The present invention relates to a photoprotein, a method for producing the same, and the like.

One of the proteins involved in bioluminescence is a calcium-binding photoprotein. This photoprotein specifically binds to Ca ²⁺ and emits light instantaneously. A calcium-binding photoprotein is useful as a reporter protein because it has a weak luminescence background and a high S / N ratio. In addition, since the calcium-binding photoprotein specifically binds to Ca ²⁺ and emits light, it can be used for quantification of calcium concentration. Further, it is desired that the calcium-binding photoprotein is used in a bioluminescence resonance energy transfer (BRET) method. This BRET is a method in which energy generated by a luminescence reaction is transferred to a fluorescent organic compound or a fluorescent protein to cause the fluorescent material to shine.

  The calcium-binding photoprotein is a complex containing a protein having an oxygenation function and a peroxide of luciferin, which is a luminescent substrate. In the calcium-binding photoprotein, a protein having an oxygenation function is called apoprotein, and the luciferin peroxide is coelenterazine peroxide (2-hydroperoxycoelenterazine). Specific examples of such calcium-binding proteins include aequorin, clytin-I, clytin-II, mitrocomin, and obelin. The thing derived from coelenterate such as is known. The maximum emission wavelength of these calcium-binding photoproteins is about 460 nm to about 480 nm, and the full width at half maximum (FWHM) is about 80 nm to 90 nm. Among aequorin, clytin, and mitrocomin, the photoprotein having the best sensitivity to calcium concentration is aequorin, and it has been reported that the sensitivity to calcium concentration decreases in the order of mitrocomin and clytin (Non-patent Document). 1).

Maitrocomin (also referred to as Halistaurin) was isolated in 1963 from Shimomura et al. (Mitrocoma cellularia (formally Halistaura microcoma)) (Non-patent Document 2). Apomitrocomin, the apoprotein of Mytrocomin, contains 6 cysteine residues, an EF-hand domain that binds Ca ²⁺ , and a C-terminal tyrosine residue. GenBank Accession No L31623 is known as an apomytrocomin cDNA (base sequence is shown in SEQ ID NO: 7 and amino acid sequence is shown in SEQ ID NO: 8). The amino acid sequence of apomaitrocomin is highly identical to the amino acid sequence of the apoprotein of other calcium-binding photoproteins such as apoaequorin or apocritin (Non-patent Document 3). Further, it has been reported that the emission spectrum of mitrocomin prepared from apomytrocomin expressed in E. coli is the same as the emission spectrum of aequorin, and the maximum emission wavelength is 470 nm ( Non-patent document 3).

  Here, among the calcium-binding photoproteins, aequorin, oberin, and clytin are used in the host E. coli using a genetic recombination technique to produce recombinant apoaequorin, recombinant apooverin, or recombinant apocrytin. And a method for purifying the recombinant apoprotein has been reported. However, there has been no report on a method for purifying recombinant apomytrocomin and a method for preparing purified recombinant mitrocomin.

O. Shimomura, A. Shimomura, Halistaurin, phialidin and modified forms of aequorin as Ca2 + indicator in biological systems, Biochem. J. 228 (1985) 745-749 O. Shimomura, F.H. Johnson, Y. Saiga, Extraction and properties of Halistaurin, a bioluminescent protein from the Hydromedusan Halistaura, J. Cell. Comp. Physiol. 59 (1963) 223-240 T.F. Fagan, Y. Ohmiya, J.R.Blinks, S. Inouye, F.I.Tsuji, Cloning, expression and sequence analysis of cDNA for the Ca2 + -binding photoprotein, mitrocomin, FEBS Lett. 333 (1993) 301-305

  In the above situation, there has been a demand for a method for preparing a calcium-binding photoprotein having a different emission spectrum from a known photoprotein, a recombinant mitrocomin, and the like.

As a result of intensive studies in order to solve the above problems, the present inventors have made various studies using an expression system in which specific sequences are combined, and as a result, the present invention has been completed.
That is, this invention provides the manufacturing method of a photoprotein shown below, a photoprotein, etc.

(1) (1) a promoter sequence capable of inducing expression;
(2) 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 comprising the amino acid sequence of SEQ ID NO: 2,
(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: 2.
(C) a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 2, and (d) a polynucleotide comprising a base sequence complementary to the base sequence of SEQ ID NO: 1. A polypeptide comprising an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions)
And a polyprotein having a function that allows the fusion protein to be expressed as a soluble protein when expressed as a fusion protein of a linker peptide and a polypeptide selected from the group consisting of (i) to (l). A first coding sequence containing a polynucleotide encoding a peptide;
(3) A polypeptide having an amino acid sequence of Gly-Pro-Glu-Phe at the N-terminus of a polypeptide selected from the group consisting of (i) to (l) when cleaved. A coding sequence comprising a polynucleotide that encodes a linker peptide that adds; and (4) a second code comprising a polynucleotide that encodes a polypeptide selected from the group consisting of: Sequence;
(I) a polypeptide comprising the ninth to 198th amino acid sequence of SEQ ID NO: 8,
(J) consisting of an amino acid sequence in which one to a plurality of amino acids are deleted, substituted, inserted and / or added in the ninth to 198th amino acid sequences of SEQ ID NO: 8, and the calcium-binding photoprotein A polypeptide having apoprotein activity,
(K) a polypeptide comprising an amino acid sequence having 90% or more identity to the 9th to 198th amino acid sequences of SEQ ID NO: 8, and having an apoprotein activity of a calcium-binding photoprotein, And (l) an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a base sequence complementary to the 42nd to 614th base sequences of SEQ ID NO: 7, And a polypeptide having an apoprotein activity of a calcium-binding photoprotein,
Expressing the fusion protein as a soluble protein using an expression vector containing
Cleaving the fusion protein at the cleavage site of the linker peptide, and obtaining a protein comprising a polypeptide selected from the group consisting of (i) to (l), wherein Gly-Pro-Glu-Phe is added to the N-terminus. A method for producing a protein, comprising:
(2) The method according to (1) above, wherein the polypeptide represented by the formula (Z) _n is a polypeptide represented by (Z) ₂ .
(3) The polypeptide represented by the formula (Z) ₂ is
(E) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 4,
(F) consisting of 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: 4, and from the group consisting of a linker peptide and (i) to (l) A polypeptide having a function of allowing the fusion protein to be expressed as a soluble protein when expressed as a fusion protein with the selected polypeptide;
(G) Fusion of a linker peptide and a polypeptide selected from the group consisting of (i) to (l), comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 4. A polypeptide having a function capable of expressing the fusion protein as a soluble protein when expressed as a protein, and (h) a polynucleotide comprising a base sequence complementary to the base sequence of SEQ ID NO: 3 and stringent conditions A fusion protein comprising an amino acid sequence encoded by a polynucleotide that hybridizes underneath and expressed as a fusion protein of a linker peptide and a polypeptide selected from the group consisting of (i) to (l) A polypeptide selected from the group consisting of polypeptides having the function of allowing a protein to be expressed as a soluble protein That, the above method (2) described.
(4) The method according to any one of (1) to (3), wherein the promoter sequence capable of inducing expression is a promoter sequence capable of inducing expression at low temperature.
(5) The method according to (4) above, wherein the promoter sequence capable of inducing expression at a low temperature is a cold shock gene promoter sequence.
(6) The method according to (5) above, wherein the cold shock gene promoter sequence is an E. coli cold shock gene promoter sequence.
(7) The method according to (6) above, wherein the promoter sequence of the E. coli cold shock gene is a promoter sequence of the E. coli cold shock gene cspA, cspB, cspG, cspI or csdA.
(8) In any one of the above (1) to (7), further comprising a coding sequence containing a polynucleotide encoding an amino acid sequence for purification on the 5 ′ side of the first coding sequence. The method described.
(9) The method according to (8) above, wherein the amino acid sequence for purification is a histidine tag sequence.
(10) Calcium binding, comprising contacting the protein produced by the method according to any one of (1) to (9) above with coelenterazine or a coelenterazine analog to obtain a calcium-binding photoprotein. Type photoprotein production method.
(11) The coelenterazine analog is selected from the group consisting of n-coelenterazine, cp-coelenterazine, hcp-coelenterazine, f-coelenterazine, fcp-coelenterazine, h-coelenterazine, Bis-coelenterazine, e-coelenterazine, and methoxycoelenterazine. The method according to (10) above.
(12) The coelenterazine analog is selected from the group consisting of n-coelenterazine, cp-coelenterazine, hcp-coelenterazine, f-coelenterazine, fcp-coelenterazine, h-coelenterazine, and e-coelenterazine, according to (11) above Method.
(13) A protein selected from the group consisting of the following (m) to (p):
(M) a protein consisting of the amino acid sequence of SEQ ID NO: 10;
(N) the amino acid sequence of SEQ ID NO: 10, consisting of an amino acid sequence in which one or more amino acids other than the first to fourth amino acids are deleted, substituted, inserted and / or added, and calcium binding A protein having an apoprotein activity of a type photoprotein;
(O) 90% or more identity to the amino acid sequence of SEQ ID NO: 10, consisting of an amino acid sequence in which the first to fourth amino acids are Gly-Pro-Glu-Phe, and calcium A protein having an apoprotein activity of a binding photoprotein; and (p) 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: 9; A protein comprising an amino acid sequence in which the first to fourth amino acids are Gly-Pro-Glu-Phe and having apoprotein activity of a calcium-binding photoprotein.
(14) A calcium-binding photoprotein comprising the protein according to (13) above and a peroxide of coelenterazine or a coelenterazine analog.
(15) The coelenterazine analog is selected from the group consisting of n-coelenterazine, cp-coelenterazine, hcp-coelenterazine, f-coelenterazine, fcp-coelenterazine, h-coelenterazine, Bis-coelenterazine, e-coelenterazine, and methoxycoelenterazine. The protein according to (14) above.
(16) The coelenterazine analog is selected from the group consisting of n-coelenterazine, cp-coelenterazine, hcp-coelenterazine, f-coelenterazine, fcp-coelenterazine, h-coelenterazine, and e-coelenterazine, according to (15) above protein.
(17) A method for detecting or quantifying calcium ions, comprising using the calcium-binding photoprotein produced by the method according to any one of (10) to (12) above.
(18) A method for detecting or quantifying calcium ions, comprising using the calcium-binding photoprotein according to any one of (13) to (15) above.
(19) The method according to (17) or (18) above, wherein the calcium ions to be detected or quantified are in the concentration range of 10 ⁻⁶ M to 10 ⁻⁴ M.
(20) A bioluminescence resonance energy transfer method is performed using the calcium-binding photoprotein produced by the method according to any one of (10) to (12) as a donor protein. Active substance analysis method.
(21) Physiological function characterized by performing bioluminescence resonance energy transfer (BRET) method using the calcium-binding photoprotein of any one of (13) to (15) as a donor protein Analysis method.
(22) Enzymatic activity characterized by performing a bioluminescence resonance energy transfer (BRET) method using the calcium-binding photoprotein of any one of (13) to (15) as a donor protein Measuring method.

  According to one embodiment of the present invention, a new calcium-binding photoprotein apoprotein is provided. The calcium-binding photoprotein prepared from the apoprotein according to a preferred embodiment of the present invention exhibits an emission spectrum different from that of aequorin, mitrocomine, or clytin. The calcium-binding photoprotein prepared from the apoprotein according to a further preferred embodiment of the present invention is less sensitive to calcium concentration than aequorin, mitrocomine, or clitin, and can be used for quantification of high concentrations of calcium.

  Hereinafter, the present invention will be described in detail.

1. Protein of the Present Invention The protein of the present invention means a protein consisting of the amino acid sequence of SEQ ID NO: 10 and a protein having substantially the same activity or function as the protein consisting of the amino acid sequence of SEQ ID NO: 10. In the present specification, a protein consisting of the amino acid sequence of SEQ ID NO: 10 and a protein having substantially the same activity or function as the protein consisting of the amino acid sequence of SEQ ID NO: 10 are referred to as “recombinant apomitrocomin”. There is.

  Substantially the same activity or function means, for example, (i) a function in which the protein can be combined with a coelenterazine peroxide or a coelenterazine analog peroxide to form a photoprotein, and (ii) the protein is coelenterazine. A function capable of forming a photoprotein that emits light by the action of calcium ions by binding to peroxides or peroxides of coelenterazine analogs, and (iii) the maximum emission intensity of light emitted by binding of the photoprotein to calcium ions (Imax ) Is ¼ or more, preferably １ ／ or more, more preferably ½ or more, more preferably 1/1. Or more of the maximum luminescence intensity (Imax) of the protein consisting of the amino acid sequence set forth in SEQ ID NO: 10. (Iv) the photoprotein binds to calcium ions. The half-life (T1 / 2, sec) of the luminescence generated by the above is 4 times or less, preferably 3 times or less than the half-life (T1 / 2, sec) of the protein comprising the amino acid sequence set forth in SEQ ID NO: 10. Preferably, it means 2 times or less, more preferably 1.5 times or less, or the like. The above-described measurement of luminescence activity and luminescence pattern is performed, for example, by Shimomura 0. et al (1988) Biochem. J. 251,405-410 and Shimomura 0. et al. Biochem. J. (1989) 261, 913-920. It can be measured by the method described in 1. Specifically, for example, a luminescence reaction can be started by adding a calcium solution to the photoprotein, and the luminescence activity or luminescence pattern can be measured using a luminescence measuring device. As the luminescence measuring device, a commercially available device such as TD-4000 (manufactured by Labo Science), Centro LB 960 (manufactured by Bertolud) or the like can be used.

  In the present specification, “apoprotein activity or function of calcium-binding photoprotein” means, for example, activity or function of apoprotein binding to peroxide of coelenterazine or coelenterazine analog to form calcium-binding photoprotein. Means. Specifically, “a protein binds to a peroxide of coelenterazine or a coelenterazine analogue to form a calcium-binding photoprotein” specifically, (1) the protein binds to a peroxide of coelenterazine or a peroxide of coelenterazine analogue. (2) A photoprotein containing a protein and a coelenterazine peroxide or a coelenterazine analog peroxide by contacting the coelenterazine or a derivative thereof in the presence of oxygen. It also means forming a (complex).

  In the present specification, the coelenterazine analog means a compound capable of binding to the protein of the present invention and forming a photoprotein capable of emitting light by the action of calcium ions.

The protein of one embodiment of the present invention is a protein selected from the group consisting of the following (m) to (p).
(M) a protein consisting of the amino acid sequence of SEQ ID NO: 10;
(N) the amino acid sequence of SEQ ID NO: 10, consisting of an amino acid sequence in which one or more amino acids other than the first to fourth amino acids are deleted, substituted, inserted and / or added, and calcium binding A protein having an apoprotein activity or function of a type photoprotein;
(O) 90% or more identity to the amino acid sequence of SEQ ID NO: 10, consisting of an amino acid sequence in which the first to fourth amino acids are Gly-Pro-Glu-Phe, and calcium A protein having an apoprotein activity or function of a binding photoprotein; and (p) 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: 9 A protein having an amino acid sequence in which the first to fourth amino acids are Gly-Pro-Glu-Phe and having an apoprotein activity or function of a calcium-binding photoprotein.

  The range of “1 to plural” in the above “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 to 10 1-9, 1-8, 1-7, 1-6 (1 to several), 1-5, 1-4, 1-3, 1-2, 1 is there. Generally, 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 “Molecular Cloning 3rd Edition”, “Current Protocols in Molecular Biology”, “Nuc. Acids. Res., 10, 6487 (1982)”, “Proc. Natl. Acad. Sci. USA, 79, 6409 (1982) ”,“ Gene, 34, 315 (1985) ”,“ Nuc. Acids. Res., 13, 4431 (1985) ”,“ Proc. Natl. Acad. Sci. USA , 82, 488 (1985) ”and the like.

  In addition, the range of “90% or more” in the “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 one embodiment of the present invention, the base sequence identity value is determined using BLAST.

  The above-mentioned “polynucleotide hybridizing under stringent conditions” refers to a polynucleotide comprising a base sequence complementary to the base sequence of SEQ ID NO: 9 or a polynucleotide encoding the amino acid sequence of SEQ ID NO: 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 the probe 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 polynucleotide derived from colony or plaque was immobilized, and then 0.1 to 2 Wash the filter at 65 ° C using double-concentration SSC (Saline-sodium citrate) solution (composition of single-concentration SSC solution is 150 mmol / L sodium chloride, 15 mmol / L sodium citrate) Polynucleotides that can be identified by:

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

  The “stringent conditions” referred to herein may be low stringent conditions, medium stringent conditions, or high stringent 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, Alkphos Direct Labeling Reagents (made by Amersham Pharmacia) can be used, for example. 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 hybridizable polynucleotides include a polynucleotide encoding the amino acid sequence of SEQ ID NO: 10 and about 60% or more and 65% when calculated using the default parameters by an analysis program such as BLAST. 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, 98% or more, 99% or more, 99.3% or more, Examples include DNA having 99.5% or more, 99.7% or more, 99.8% or more, and 99.9% or more identity. The identity of amino acid sequences and base sequences can be determined using the method described above. In one embodiment of the present invention, the base sequence identity value is determined using BLAST.

2. Calcium-binding Photoprotein of the Present Invention The calcium-binding photoprotein of the present invention comprises the protein of the present invention and coelenterazine peroxide or coelenterazine analog peroxide. According to one aspect of the present invention, the coelenterazine analog comprises n-coelenterazine, cp-coelenterazine, hcp-coelenterazine, f-coelenterazine, fcp-coelenterazine, h-coelenterazine, bis-coelenterazine, e-coelenterazine, and methoxycoelenterazine Selected from the group consisting of According to a preferred embodiment of the present invention, the coelenterazine analog is selected from the group consisting of n-coelenterazine, cp-coelenterazine, hcp-coelenterazine, f-coelenterazine, fcp-coelenterazine, h-coelenterazine, and e-coelenterazine. According to another aspect of the invention, the coelenterazine analog is n-coelenterazine. According to yet another aspect of the invention, the coelenterazine analog is cp-coelenterazine. According to yet another aspect of the invention, the coelenterazine analog is hcp-coelenterazine. According to yet another aspect of the invention, the coelenterazine analog is f-coelenterazine. According to yet another aspect of the invention, the coelenterazine analog is fcp-coelenterazine. According to yet another aspect of the invention, the coelenterazine analog is h-coelenterazine. According to yet another aspect of the invention, the coelenterazine analog is e-coelenterazine.
The calcium-binding photoprotein of a preferred embodiment of the present invention exhibits an emission spectrum different from that of aequorin, mitrocomin, or clytin. The calcium-binding photoprotein of a further preferred embodiment of the present invention is less sensitive to calcium concentration than aequorin, mitrocomine, or clitin, and can be used for quantification of high-concentration calcium.
The calcium-binding photoprotein of a preferred embodiment of the present invention containing coelenterazine is recombinant mitrocomine. The calcium-binding photoprotein of a preferred embodiment of the present invention containing a coelenterazine analog is semisynthetic recombinant mitrocomin.

3. Method for Producing the Protein of the Present Invention According to one aspect of the present invention, the following protein production method is provided:
(1) a promoter sequence capable of inducing expression;
(2) is represented by the formula (Z) _n, and, when expressed as a fusion protein with a polypeptide selected from the group consisting of a linker peptide (i) ~ (l), the fusion protein is soluble proteins A first coding sequence comprising a polynucleotide encoding a polypeptide having a function or activity that can be expressed as:
(3) A polypeptide having an amino acid sequence of Gly-Pro-Glu-Phe at the N-terminus of a polypeptide selected from the group consisting of (i) to (l) when cleaved. A coding sequence comprising a polynucleotide that encodes a linker peptide that adds; and (4) a second coding sequence comprising a polynucleotide encoding a polypeptide selected from the group consisting of (i)-(l);
Expressing the fusion protein as a soluble protein using an expression vector containing
Cleaving the fusion protein at the cleavage site of the linker peptide, and obtaining a protein comprising a polypeptide selected from the group consisting of (i) to (l), wherein Gly-Pro-Glu-Phe is added to the N-terminus. A method for producing a protein, comprising:
According to this production method, a new apoprotein of calcium-binding protein can be provided.

3.1. Fusion protein Hereinafter, the fusion protein will be described in more detail. The fusion protein includes the following peptides:
(1) is represented by the formula (Z) _n, and, when expressed as a fusion protein with a polypeptide selected from the group consisting of a linker peptide (i) ~ (l), the fusion protein is soluble proteins A polypeptide having a function that can be expressed as;
(2) A polypeptide having an amino acid sequence of Gly-Pro-Glu-Phe at the N-terminus of a polypeptide having a cleavage site and selected from the group consisting of (i) to (l) when cleaved And (3) a polypeptide selected from the group consisting of (i) to (l).

(1) Polypeptide represented by formula (Z) _{n In the} fusion protein, the polypeptide represented by formula (Z) _n is a polypeptide selected from the group consisting of a linker peptide and (i) to (l) When expressed as a fusion protein, the fusion protein has an activity or function capable of being expressed as a soluble protein.
Z represents a polypeptide selected from the group consisting of the following (a) to (d):
(A) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2,
(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: 2.
(C) a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 2, and (d) a polynucleotide comprising a base sequence complementary to the base sequence of SEQ ID NO: 1. A polypeptide comprising an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions.

  The range of “1 to plural” in the above “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 to 10 1-9, 1-8, 1-7, 1-6 (1 to several), 1-5, 1-4, 1-3, 1-2, 1 is there. Generally, 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 “Molecular Cloning 3rd Edition”, “Current Protocols in Molecular Biology”, “Nuc. Acids. Res., 10, 6487 (1982)”, “Proc. Natl. Acad. Sci. USA, 79, 6409 (1982) ”,“ Gene, 34, 315 (1985) ”,“ Nuc. Acids. Res., 13, 4431 (1985) ”,“ Proc. Natl. Acad. Sci. USA , 82, 488 (1985) ”and the like.

  In addition, the range of “90% or more” in the “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 one embodiment of the present invention, the numerical value of amino acid sequence or base sequence identity is determined using BLAST.

  The above-mentioned “polynucleotide hybridizing under stringent conditions” refers to a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 or a polynucleotide encoding the amino acid sequence of SEQ ID NO: 2. 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 probe 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 polynucleotide derived from colony or plaque was immobilized, and then 0.1 to 2 Wash the filter at 65 ° C using double-concentration SSC (Saline-sodium citrate) solution (composition of single-concentration SSC solution is 150 mmol / L sodium chloride, 15 mmol / L sodium citrate) Polynucleotides that can be identified by:

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

  The “stringent conditions” referred to herein may be low stringent conditions, medium stringent conditions, or high stringent 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, Alkphos Direct Labeling Reagents (made by Amersham Pharmacia) can be used, for example. 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 polynucleotides that can be hybridized, the polynucleotide encoding the amino acid sequence of SEQ ID NO: 2 is approximately 60% or more and 65% when calculated using the default parameters by an analysis program such as BLAST. 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, 98% or more, 99% or more, 99.3% or more, Examples include DNA having 99.5% or more, 99.7% or more, 99.8% or more, and 99.9% or more identity. The identity of amino acid sequences and base sequences can be determined using the method described above. In one embodiment of the present invention, the base sequence identity value is determined using BLAST.

n represents an integer of 1 to 5, preferably 2 or 3, and particularly preferably 2.
Formula (Z) In the polypeptide represented by _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: 4 or the polypeptide consisting of the amino acid sequence of SEQ ID NO: 4. A polypeptide. In the present specification, a polypeptide consisting of the amino acid sequence of SEQ ID NO: 4 or a polypeptide having substantially the same activity or function as the polypeptide consisting of the amino acid sequence of SEQ ID NO: 4 is referred to as a “ZZ domain”. is there.
The “substantially the same activity or function” means, for example, when the fusion protein is expressed as a fusion protein of a linker peptide and a polypeptide selected from the group consisting of (i) to (l). It means an activity or function that can be expressed as a soluble protein. Such activity or function, for example, the IgG binding ability of the ZZ domain can be measured by an IgG binding assay.

More specifically, examples of the polypeptide represented by the formula (Z) ₂ include:
(E) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 4,
(F) consisting of 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: 4, and from the group consisting of a linker peptide and (i) to (l) A polypeptide having an activity or function capable of being expressed as a soluble protein when expressed as a fusion protein with a selected polypeptide;
(G) Fusion of a linker peptide and a polypeptide selected from the group consisting of (i) to (l), comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 4. A polypeptide having an activity or function that allows the fusion protein to be expressed as a soluble protein when expressed as a protein, and (h) a polynucleotide comprising a base sequence complementary to the base sequence of SEQ ID NO: 3 and a stringent When expressed as a fusion protein consisting of an amino acid sequence encoded by a polynucleotide that hybridizes under various conditions and a linker peptide and a polypeptide selected from the group consisting of (i) to (l), The fusion protein is selected from the group consisting of polypeptides having activity or function that can be expressed as soluble proteins. Such polypeptides-option and the like.

(2) A linker peptide having a cleavage site The fusion protein is selected from the group consisting of a first amino acid sequence containing the amino acid sequence of the polypeptide represented by formula (Z) _n and (i) to (l) An amino acid sequence containing the amino acid sequence of the linker peptide having a cleavage site between the second amino acid sequence containing the amino acid sequence of the polypeptide. In one embodiment of the present invention, when the linker peptide is cleaved, the linker peptide removes the polypeptide represented by the formula (Z) _n , and N of the polypeptide selected from the group consisting of (i) to (l) A protein containing a polypeptide to which Gly-Pro-Glu-Phe is added at the end is generated.
The linker peptide having a cleavage site means a linker peptide having a cleavage site that can be cleaved by an enzymatic or chemical cleavage substance. Many peptides are known that are cleaved by enzymes (proteases) or chemicals (eg, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1988); Walsh, Proteins Biochemistry and Biotechnology, John Wiley & Sons, LTD., West Sussex, England (2002)). A cleaving substance is a chemical substance or enzyme that recognizes a cleaving site in a peptide and divides the peptide into two peptides by cleaving the bond in the peptide. Examples of the cleaving substance include chemical substances and proteases.
The linker peptide having a cleavage site is preferably a linker peptide having a protease cleavage site. Examples of protease cleavage sites include thrombin cleavage sites, human rhinovirus 3C protease cleavage sites, and factor Xa cleavage sites.
The linker peptide having a cleavage site is preferably a linker peptide consisting of the amino acid sequence of SEQ ID NO: 6. The linker peptide consisting of the amino acid sequence of SEQ ID NO: 6 has a human rhinovirus 3C protease cleavage site.

(3) A polypeptide selected from the group consisting of (i) to (l) The fusion protein includes a polypeptide selected from the group consisting of the following (i) to (l):
(I) a polypeptide comprising the ninth to 198th amino acid sequence of SEQ ID NO: 8,
(J) consisting of an amino acid sequence in which one to a plurality of amino acids are deleted, substituted, inserted and / or added in the ninth to 198th amino acid sequences of SEQ ID NO: 8, and the calcium-binding photoprotein A polynucleotide encoding a polypeptide having apoprotein activity or function,
(K) a polymorphic amino acid sequence having 90% or more identity to the 9th to 198th amino acid sequences of SEQ ID NO: 8, and having an apoprotein activity or function of a calcium-binding photoprotein Encoded by a polynucleotide encoding a peptide, and (1) a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a base sequence complementary to the 42nd to 614th base sequences of SEQ ID NO: 7 And a polypeptide having an apoprotein activity or function of a calcium-binding photoprotein.

  “Amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added” and “Amino acid sequence having 90% or more identity” and “Polynucleotide hybridizing under stringent conditions” The meaning of is the same as described above.

  Examples of the polypeptide selected from the group consisting of (i) to (l) include, for example, a polypeptide comprising the ninth to 198th amino acid sequence of SEQ ID NO: 8, or the ninth to And a polypeptide having substantially the same activity or function as the polypeptide having the 198th amino acid sequence.

  Substantially the same activity or function means, for example, (i) a function that allows the polypeptide to bind with a coelenterazine peroxide or a coelenterazine analog peroxide to form a photoprotein, and (ii) Function capable of forming a photoprotein that emits light by the action of calcium ions by binding with peroxide of coelenterazine or coelenterazine analogs, (iii) Maximum emission intensity of light emission generated by binding of the photoprotein to calcium ions (Imax) is ¼ or more, preferably ３ or more, more preferably ½ of the maximum luminescence intensity (Imax) of the polypeptide consisting of the ninth to 198th amino acid sequences of SEQ ID NO: 8. Or more, preferably 1 / 1.5 or more, and (iv) the luminescent protein. The half-life of light emission (T1 / 2, sec) generated by binding of white matter to calcium ions is the half-life of the polypeptide consisting of the 9th to 198th amino acid sequences of SEQ ID NO: 8 (T1 / 2, Or less), preferably 3 times or less, more preferably 2 times or less, and even more preferably 1.5 times or less. The above-described measurement of luminescence activity and luminescence pattern is performed, for example, by Shimomura 0. et al (1988) Biochem. J. 251,405-410 and Shimomura 0. et al. Biochem. J. (1989) 261, 913-920. It can be measured by the method described in 1. Specifically, for example, a luminescence reaction can be started by adding a calcium solution to the photoprotein, and the luminescence activity or luminescence pattern can be measured using a luminescence measuring device. As the luminescence measuring device, a commercially available device such as TD-4000 (manufactured by Labo Science), Centro LB 960 (manufactured by Bertolud) or the like can be used.

  The fusion protein may further contain an amino acid sequence for promoting translation and / or an amino acid sequence for purification. As an amino acid sequence for promoting translation, an amino acid sequence used in this technical field can be used. Examples of amino acid sequences for promoting translation include TEE sequences. As the amino acid sequence for purification, an amino acid sequence used in this technical field can be used. Examples of the amino acid sequence for purification include a histidine tag sequence having an amino acid sequence of 4 or more, preferably 6 or more histidine residues, and an amino acid sequence of a binding domain to glutathione of glutathione S-transferase Etc. The amino acid sequence for purification is preferably present on the amino terminal side of the first amino acid sequence.

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

Specifically, the polynucleotide is a polynucleotide containing the following sequences (1) to (3):
(1) Formula (Z) represented by _n ,
(In the formula, n and Z have the same meaning as described above.)
And a polynucleotide encoding a polypeptide having a function capable of expressing the fusion protein as a soluble protein when expressed as a fusion protein with a polypeptide selected from the group consisting of (i) to (l). A first coding sequence containing;
(2) A polypeptide having an amino acid sequence of Gly-Pro-Glu-Phe at the N-terminus of a polypeptide having a cleavage site and selected from the group consisting of (i) to (l) when cleaved A coding sequence comprising a polynucleotide that encodes a linker peptide that adds; and (3) a second coding sequence comprising a polynucleotide encoding a polypeptide selected from the group consisting of (i) to (l).

Examples of the polynucleotide encoding the polypeptide represented by the formula (Z) ₂ include a polynucleotide selected from the group consisting of the following (e) to (h):
(E) a polynucleotide comprising a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3,
(F) When hybridized with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 3 under stringent conditions and expressed as a fusion protein with a target protein, the fusion protein is soluble A polynucleotide comprising a polynucleotide encoding a polypeptide having an activity or function capable of being expressed as a protein;
(G) a polynucleotide containing a polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 4, and (h) one to a plurality of amino acids in the amino acid sequence of SEQ ID NO: 4 are deleted, substituted, or inserted And / or the fusion protein is expressed as a soluble protein when expressed as a fusion protein comprising a linker peptide and a polypeptide selected from the group consisting of (i) to (l). A polynucleotide comprising a polynucleotide encoding a polypeptide having a possible activity or function.

Such preferred polynucleotides include, for example, polynucleotides containing the following coding sequence:
(1) a first coding sequence selected from the group consisting of the following (e) to (h);
(E) a polynucleotide comprising a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3,
(F) a polynucleotide selected from the group consisting of a linker peptide and (i) to (l), which hybridizes with a polynucleotide consisting of a base sequence complementary to the base sequence of SEQ ID NO: 3 under stringent conditions. A polynucleotide comprising a polynucleotide encoding a polypeptide having an activity or function capable of being expressed as a soluble protein when expressed as a fusion protein with a peptide;
(G) a polynucleotide containing a polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 4, and (h) one to a plurality of amino acids in the amino acid sequence of SEQ ID NO: 4 are deleted, substituted, or inserted And / or the fusion protein is expressed as a soluble protein when expressed as a fusion protein comprising a linker peptide and a polypeptide selected from the group consisting of (i) to (l). A polynucleotide comprising a polynucleotide encoding a polypeptide having a possible activity or function;
(2) A polypeptide having an amino acid sequence of Gly-Pro-Glu-Phe at the N-terminus of a polypeptide having a cleavage site and selected from the group consisting of (i) to (l) when cleaved A coding sequence comprising a polynucleotide that encodes a linker peptide that adds; and (3) a second coding sequence comprising a polynucleotide encoding a polypeptide selected from the group consisting of (i) to (l).

  Here, “a polynucleotide that hybridizes under stringent conditions (for example, DNA)” means a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 3 or the amino acid sequence of SEQ ID NO: 4. 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 encoded 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 polynucleotide derived from colony or plaque was immobilized, and then 0.1 to 2 Wash the filter at 65 ° C using double-concentration SSC (Saline-sodium citrate) solution (composition of single-concentration SSC solution is 150 mmol / L sodium chloride, 15 mmol / L sodium citrate) Polynucleotides that can be identified by:

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

  As used herein, the “stringent conditions” may be any of low stringency conditions, moderate stringency conditions, and high stringency conditions. “Low stringent conditions” are, for example, conditions of 5 × SSC, 5 × Denhardt's solution, 0.5% (w / v) SDS, 50% (v / v) formamide, 32 ° C. The “medium stringent conditions” are, for example, conditions of 5 × SSC, 5 × Denhardt's solution, 0.5% (w / v) SDS, 50% (v / v) formamide, 42 ° C. “High stringent conditions” are, for example, conditions of 5 × SSC, 5 × Denhardt's solution, 0.5 (w / v)% SDS, 50% (v / v) formamide, 50 ° C. The tighter the conditions, the higher the complementarity required for duplex formation. Specifically, for example, 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, Alkphos Direct Labeling Reagents (made by Amersham Pharmacia) can be used, for example. 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 polynucleotides that can be hybridized, a polynucleotide encoding the amino acid sequence of SEQ ID NO: 4 and about 60% or more and 65% when calculated using the default parameters by an analysis program such as BLAST. 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, 98% or more, 99% or more, 99.3% or more, Examples include DNA having 99.5% or more, 99.7% or more, 99.8% or more, and 99.9% or more identity. The identity of amino acid sequences and base sequences can be determined using the method described above. In one embodiment of the present invention, the base sequence identity value is determined using BLAST.

  A polynucleotide encoding a region having an amino acid sequence in which one or a plurality of amino acids have been deleted, substituted, inserted and / or added to a certain amino acid sequence can be obtained by site-directed mutagenesis (for example, Gotoh, T. et al. 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 using 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 ( (See, for example, 1989)).

  A polynucleotide encoding a partial fragment of a protein that is a kind of deletion mutant has a sequence that matches the 5'-end base sequence of the region encoding the partial fragment to be produced 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.

  Specific examples of the polynucleotide include a polynucleotide containing a polynucleotide encoding a fusion protein consisting of the amino acid sequence of SEQ ID NO: 12. As a polynucleotide containing the polynucleotide which codes the fusion protein which consists of an amino acid sequence of sequence number: 12, the polynucleotide containing the polynucleotide which consists of a base sequence of sequence number: 11, etc. are mentioned, for example. These base sequences and amino acid sequences are shown in FIG.

  The polynucleotide of the present invention comprises a polynucleotide (coding sequence) containing a polynucleotide encoding an amino acid sequence for promoting translation and / or a polynucleotide (coding sequence) containing a polynucleotide encoding an amino acid sequence for purification. ) May be included. As a polynucleotide containing a polynucleotide encoding an amino acid sequence for promoting translation, a polynucleotide containing a polynucleotide encoding an amino acid sequence for promoting translation used in the art may be used. it can. Examples of the amino acid sequence for promoting translation include those described above. As a polynucleotide encoding an amino acid sequence for purification, a polynucleotide containing a polynucleotide encoding an amino acid sequence for purification used in the art can be used. Examples of the amino acid sequence for purification include those described above. The polynucleotide (coding sequence) containing the polynucleotide encoding the amino acid sequence for purification is preferably present on the 5 'side of the first coding sequence.

3.3. Expression vector and transformant Furthermore, this invention provides the expression vector and transformant containing the polynucleotide mentioned above.
(1) Expression vector An expression vector can be obtained by ligating (inserting) the aforementioned polynucleotide (DNA) 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.
Specific examples of the expression vector include an expression vector containing the following promoter sequence and coding sequence:
(1) a promoter sequence capable of inducing expression;
(2) Formula (Z) _n
(In the formula, n and Z have the same meaning as described above.)
And a polyprotein having a function that allows the fusion protein to be expressed as a soluble protein when expressed as a fusion protein of a linker peptide and a polypeptide selected from the group consisting of (i) to (l). A first coding sequence containing a polynucleotide encoding a peptide;
(3) A polypeptide having an amino acid sequence of Gly-Pro-Glu-Phe at the N-terminus of a polypeptide selected from the group consisting of (i) to (l) when cleaved. And (4) a second coding sequence containing a polynucleotide encoding a polypeptide selected from the group consisting of (i) to (l).

  The vector for inserting the polynucleotide is not particularly limited as long as it can replicate in the host, and examples thereof include plasmids, bacteriophages, animal viruses and the like. Examples of plasmids include plasmids derived from E. coli (eg, pBR322, pBR325, pUC118, pUC119, etc.), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, etc.), plasmids derived from yeast (eg, YEp13, YEp24, YCp50, etc.), and the like. can give. Examples of the bacteriophage include λ phage. Examples of animal viruses include retroviruses, vaccinia viruses, insect viruses (eg, baculoviruses) and the like. Moreover, a pCold I vector, a pCold II vector, a pCold III vector, a pCold IV vector (manufactured by Takara Bio Inc.) and the like can also be suitably used.

  The polynucleotide is usually ligated so that it can be expressed downstream of a promoter (promoter capable of inducing expression) in an appropriate vector. The promoter used is preferably an SV40-derived promoter, a retrovirus promoter, a metallothionein promoter, a heat shock promoter, a cytomegalovirus promoter, an SRα promoter or the like when the host to be transformed is an animal cell. When the host is Escherichia, a Trp promoter, T7 promoter, lac promoter, recA promoter, λPL promoter, lpp promoter and the like are preferable. When the host is Bacillus, SPO1 promoter, SPO2 promoter, penP promoter and the like are preferable. When the host is yeast, PHO5 promoter, PGK promoter, GAP promoter, ADH1 promoter, GAL promoter, etc. are preferable. When the host is an insect cell, polyhedrin promoter, P10 promoter and the like are preferable. 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. Examples of cold shock genes include E. coli cold shock genes (eg, cspA, cspB, cspG, cspI, csdA, etc.), Bacillus caldolyticus cold shock genes (eg, Bc-Csp, etc.), Salmonella enterica cold shock genes (eg, cspE, etc.). 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 suitably used as a promoter capable of inducing expression at a low temperature.

  In addition to the above, an expression vector containing an enhancer, a splicing signal, a poly A addition signal, a ribosome binding sequence (SD sequence), a selection marker, etc. can be used as desired. Examples of the selection marker include a dihydrofolate reductase gene, an ampicillin resistance gene, and a neomycin resistance gene.

  The expression vector may include a polynucleotide containing a base sequence encoding an amino acid sequence for promoting translation and / or a polynucleotide containing a base sequence encoding an amino acid sequence for purification. As the polynucleotide containing a base sequence encoding an amino acid sequence for promoting translation, a polynucleotide containing a base sequence encoding an amino acid sequence for promoting translation used in the art may be used. it can. Examples of the amino acid sequence for promoting translation include those described above. As a polynucleotide encoding an amino acid sequence for purification, a polynucleotide containing a base sequence encoding an amino acid sequence for purification used in the art can be used. Examples of the amino acid sequence for purification include those described above.

(2) Transformant A transformant is prepared by introducing the thus obtained expression vector containing a polynucleotide (that is, a polynucleotide encoding a fusion protein) into an appropriate host. be able to. The host is not particularly limited as long as it can express a polynucleotide (DNA). For example, Escherichia, Bacillus, Pseudomonas, Rhizobium, yeast, animal cells, insect cells, etc. Can be given. 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.

  A method for introducing an expression vector into a host and a transformation method using the expression vector can be performed by various general methods. Examples of methods for introducing an expression vector into a host cell include, for example, 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). In this way, a transformant transformed with an expression vector containing a polynucleotide encoding the fusion protein can be obtained.

(3) Expression Vector and Transformant Containing a Promoter Sequence Inducible at Low Temperature As an expression vector, an expression vector containing a promoter sequence inducible at 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) Promoter sequence capable of inducing expression at low temperature;
(2) Formula (Z) _n
(In the formula, n and Z have the same meaning as described above.)
And an activity or function that allows the fusion protein to be expressed as a soluble protein when expressed as a fusion protein of a linker peptide and a polypeptide selected from the group consisting of (i) to (l) A first coding sequence containing a polynucleotide encoding a polypeptide having;
(3) A polypeptide having an amino acid sequence of Gly-Pro-Glu-Phe at the N-terminus of a polypeptide selected from the group consisting of (i) to (l) when cleaved. And (4) a second coding sequence containing a polynucleotide encoding a polypeptide selected from the group consisting of (i) to (l).

As an expression vector containing a promoter sequence capable of inducing expression at a low temperature, an expression vector containing the following promoter sequence and coding sequence is particularly preferable:
(1) Promoter sequence capable of inducing expression at low temperature;
(2) Formula (Z) ₂
(In the formula, Z represents the same meaning as described above.)
A first coding sequence comprising a polynucleotide encoding a polypeptide represented by:
(3) A polypeptide having an amino acid sequence of Gly-Pro-Glu-Phe at the N-terminus of a polypeptide selected from the group consisting of (i) to (l) when cleaved. And (4) a second coding sequence containing a polynucleotide encoding a polypeptide selected from the group consisting of (i) to (l).

  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, for example, the cold shock gene promoter of Escherichia coli, the promoter of Bacillus caldolyticus cold shock gene (eg, Bc-Csp), the promoter of Salmonella enterica cold shock gene (eg, cspE), Erwinia carotovora Examples include cold shock gene promoters (eg, cspG). Examples of the E. coli cold shock gene promoter include cspA promoter, cspB promoter, cspG promoter, cspI promoter, csdA promoter and the like, with cspA promoter being preferred. Examples of the Bacillus caldolyticus cold shock gene promoter include Bc-Csp. Examples of the promoter of the Salmonella enterica cold shock gene include cspE. Examples of the Erwinia carotovora cold shock gene promoter include cspG.

  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, 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.) and the like can be suitably 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 a 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.

Examples of expression vectors include the following promoter sequences, coding sequences, and expression vectors containing restriction enzyme sites:
(1) a promoter sequence capable of inducing expression;
(2) Formula (Z) _n
(In the formula, n and Z have the same meaning as described above.)
And an activity or function that allows the fusion protein to be expressed as a soluble protein when expressed as a fusion protein of a linker peptide and a polypeptide selected from the group consisting of (i) to (l) A first coding sequence containing a polynucleotide encoding a polypeptide having;
(3) A polypeptide having an amino acid sequence of Gly-Pro-Glu-Phe at the N-terminus of a polypeptide selected from the group consisting of (i) to (l) when cleaved. A coding sequence comprising a polynucleotide that encodes a linker peptide that adds; and (4) a second coding sequence comprising a polynucleotide that encodes a polypeptide selected from the group consisting of (i)-(l) At least one restriction enzyme site that can be inserted.

  “At least one restriction enzyme site into which a second coding sequence containing a polynucleotide encoding a polypeptide selected from the group consisting of (i) to (l) can be inserted” refers to (i) to A polynucleotide containing a polynucleotide having a restriction enzyme recognition site into which a second coding sequence containing a polynucleotide encoding a polypeptide selected from the group consisting of (l) can be inserted. The restriction enzyme site is not particularly limited as long as it can insert a second coding sequence containing a polynucleotide encoding a polypeptide selected from the group consisting of (i) to (l). A so-called multicloning site is preferred. Restriction enzyme sites such as multicloning sites are well known and reported in the art (eg, Yanisch-Perron, C., Vieira, J. and Messing, J. Gene 33 (1985) 103-119, Improved M13 phage cloning vectors and host strains: Nucleotide sequences of the M13mp18 and pUC19 vectors. See Gene 33 (1985) 103-119).

  In such an expression vector, at least one restriction enzyme site into which a second coding sequence containing a polynucleotide encoding a polypeptide selected from the group consisting of (i) to (l) can be inserted. An expression vector capable of expressing the fusion protein can be prepared by linking (inserting) a polynucleotide encoding a polypeptide selected from the group consisting of (i) to (l).

As such an expression vector, among them, an expression vector containing the following promoter sequence, coding sequence, and restriction enzyme site is preferable.
(1) Promoter sequence capable of inducing expression at low temperature;
(2) Formula (Z) ₂
(In the formula, Z represents the same meaning as described above.)
A first coding sequence comprising a polynucleotide encoding a polypeptide represented by:
(3) A polypeptide having an amino acid sequence of Gly-Pro-Glu-Phe at the N-terminus of a polypeptide selected from the group consisting of (i) to (l) when cleaved. A coding sequence comprising a polynucleotide that encodes a linker peptide that adds; and (4) a second coding sequence comprising a polynucleotide that encodes a polypeptide selected from the group consisting of (i)-(l) At least one restriction enzyme site that can be inserted.

  A coding sequence containing a polynucleotide encoding an amino acid sequence for purification may be further contained on the 5 'side of the first coding sequence. The amino acid sequence for purification is as described above.

3.4. Production of Fusion Protein and Protein of the Present Invention The present invention also provides a method for producing a fusion protein, comprising the steps of culturing the transformant to produce a fusion protein. The fusion protein is produced, for example, by culturing the transformant under conditions capable of expressing the polynucleotide (DNA) encoding the fusion protein of the present invention, generating and accumulating the fusion protein, and separating and purifying it. be able to.

Furthermore, in the present invention, the fusion protein is cleaved at the cleavage site of the linker peptide, and Gly-Pro-Glu-Phe having no polypeptide represented by the formula (Z) _n is added to (i) to (l A method for producing the protein of the present invention, comprising the step of obtaining a protein comprising the polypeptide selected from the group consisting of: The protein of the present invention can be produced, for example, by cleaving the produced fusion protein at the cleavage site of the linker peptide, and separating and purifying it.

(Culture of transformants)
The transformant of the present invention can be cultured according to a usual method used for host culture. By the culturing, the fusion protein of the present invention is produced by the transformant, and the fusion protein 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, ammonium salts of inorganic acids or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, or other nitrogen-containing compounds, peptone, meat extract, corn steep liquor, and the like are used. Examples of inorganic salts include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate. During culture, an antibiotic such as ampicillin or tetracycline may be added to the medium as necessary. When cultivating a transformant transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, when cultivating a transformant transformed with an expression vector using the Lac promoter, transformation by transforming isopropyl-β-D-thiogalactopyranoside (IPTG) with an expression vector using the trp promoter When culturing the body, indoleacrylic acid (IAA) or the like may be added to the medium.

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

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

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

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

  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 fusion protein)
The fusion protein can be obtained by separating and purifying the fusion protein from the culture. Here, the culture means any of a culture solution, cultured cells or cultured cells, or crushed cells of cultured cells or cultured cells. Separation and purification of the fusion protein 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 cultured by a usual method (for example, ultrasound, lysozyme, freeze-thawing, etc.). After crushing, a crude fusion protein extract can be obtained by conventional methods (eg, centrifugation, filtration, etc.). When the fusion protein is accumulated in the periplasmic space, an extract containing the fusion protein can be obtained by an ordinary method (for example, osmotic shock method) after completion of the culture. When the fusion protein is accumulated in the culture solution, the fusion protein is contained by separating the cells or cells from the culture supernatant by a usual method (for example, centrifugation, filtration, etc.) after completion of the culture. A culture supernatant can be obtained.

  Purification of the fusion protein contained in the extract or culture supernatant thus obtained can be performed according to a normal 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 contains the amino acid sequence for purification described above, it is preferable to purify using this. Specifically, when the fusion protein contains a histidine tag sequence, nickel chelate affinity chromatography is used, and when the fusion protein contains a binding domain to glutathione of S-transferase, affinity chromatography using a glutathione-binding gel may be used. it can.

(Cleavage of fusion protein)
The fusion protein can be cleaved by a known method.
Specifically, the fusion protein has a cleavage site and, when cleaved, an amino acid of Gly-Pro-Glu-Phe at the N-terminus of a polypeptide selected from the group consisting of (i) to (l) Since it contains a linker peptide that adds a polypeptide comprising a sequence, the polypeptide represented by the formula (Z) n is removed from the fusion protein by treating the fusion protein with a cleavage substance, and Gly- A protein containing a polypeptide selected from the group consisting of (i) to (l) to which a polypeptide consisting of the amino acid sequence of Pro-Glu-Phe is added can be obtained.
Examples of the cleavage substance include an enzymatic cleavage substance and a chemical cleavage substance. The enzymatic cleavage substance is preferably a protease. Examples of the protease include human rhinovirus 3C protease.
The linker peptide having a cleavage site is preferably a linker peptide consisting of the amino acid sequence of SEQ ID NO: 6. The linker peptide consisting of the amino acid sequence of SEQ ID NO: 6 has a human rhinovirus 3C protease cleavage site. Examples of the protease used for cleavage of the human rhinovirus 3C protease cleavage site include PreScission protein (GE-Healthcare Bio-Science, Piscataway, NJ).

(Separation and production of the protein of the present invention)
The protein of the present invention contained in the cleaved product can be purified according to a usual 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 contains the amino acid sequence for purification described above, it is preferable to purify using this. Specifically, when the fusion protein contains a histidine tag sequence, nickel chelate affinity chromatography is used, and when the fusion protein contains a binding domain to glutathione of S-transferase, affinity chromatography using a glutathione-binding gel may be used. it can. As a result, the protein of the present invention can be separated and purified from the uncut fusion protein and the cleaved polypeptide represented by the formula (Z) _n in the cleaved product.

4). Calcium-binding photoprotein of the present invention The calcium-binding photoprotein of the present invention is obtained by contacting a protein produced by the method for producing a protein of the present invention with coelenterazine or a coelenterazine analog to obtain a calcium-binding photoprotein. Can be manufactured or regenerated.

  Here, “contact” means that the protein of the present invention and coelenterazine or a coelenterazine analog are present in the same reaction system. For example, the protein of the present invention is contained in a container containing coelenterazine or a coelenterazine analog. , Adding coelenterazine or coelenterazine analog to a container containing the protein of the present invention, or mixing the protein of the present invention with coelenterazine or coelenterazine analog, and the like. According to one aspect of the invention, the contacting is carried out at a low temperature in the presence of a reducing agent (such as mercaptoethanol or dithiothreitol) and oxygen. More specifically, photoproteins of the present invention include, for example, Shimomura 0. et al (1988) Biochem. J. 251,405-410 and Shimomura 0. et al. Biochem. J. (1989) 261, 913-920. It can be produced by the method described in 1. In the presence of oxygen, the photoprotein of the present invention exists in a state where the protein of the present invention, coelenterazine and peroxide of coelenterazine generated from molecular oxygen form a complex. When calcium ions are bound to the complex, it emits instantaneous light and generates coelenteramide, which is an oxide of coelenterazine, and carbon dioxide. The complex may be referred to as “the photoprotein of the present invention”.

  The coelenterazine analog is, for example, selected from the group consisting of n-coelenterazine, cp-coelenterazine, hcp-coelenterazine, f-coelenterazine, fcp-coelenterazine, h-coelenterazine, bis-coelenterazine, e-coelenterazine, and methoxycoelenterazine. Things can be mentioned. According to a preferred embodiment of the present invention, the coelenterazine analog is selected from the group consisting of n-coelenterazine, cp-coelenterazine, hcp-coelenterazine, f-coelenterazine, fcp-coelenterazine, h-coelenterazine, and e-coelenterazine. According to another aspect of the invention, the coelenterazine analog is n-coelenterazine. According to yet another aspect of the invention, the coelenterazine analog is cp-coelenterazine. According to yet another aspect of the invention, the coelenterazine analog is hcp-coelenterazine. According to yet another aspect of the invention, the coelenterazine analog is f-coelenterazine. According to yet another aspect of the invention, the coelenterazine analog is fcp-coelenterazine. According to yet another aspect of the invention, the coelenterazine analog is h-coelenterazine. According to yet another aspect of the invention, the coelenterazine analog is e-coelenterazine.

5. Use of calcium binding protein of the present invention (detection or quantification of calcium ion)
As described above, the protein (apoprotein) of the present invention can be produced by forming a non-covalent bond with the coelenterazine or coelenterazine peroxide produced from molecular oxygen and the peroxide of coelenterazine analogue. A photoprotein (holoprotein) that is a protein and emits light by the action of calcium ions can be formed. Therefore, the protein of the present invention or the photoprotein of the present invention can be used for detection or quantification of calcium ions.

  When calcium ion is detected or quantified, a photoprotein comprising the protein of the present invention (apoprotein) and coelenterazine peroxide or coelenterazine analog peroxide is used. The photoprotein can be produced according to the method described above. Detection or quantification of calcium ions can be performed by directly adding the sample solution to the photoprotein solution and measuring the generated luminescence. Alternatively, calcium ions can be detected or quantified by adding a photoprotein solution to the sample solution and measuring the generated luminescence. In addition, before adding the photoprotein to a measurement system for detecting or quantifying calcium ions, the aqueous protein (apoprotein) solution of the present invention and coelenterazine or a derivative thereof (for example, h-coelenterazine, e-coelenterazine, cl -Coelenterazine, ch-coelenterazine, hcp-coelenterazine, and the like) may be used. Further, by contacting the protein of the present invention (apoprotein) with coelenterazine or a derivative thereof in a measurement system, a photoprotein composed of the protein of the present invention and a peroxide of coelenterazine or a coelenterazine analog is generated. May be. The generated photoprotein is a complex (photoprotein) of the protein of the present invention (apoprotein) and coelenterazine peroxide or coelenterazine analog peroxide, and the complex (that is, the photoprotein of the present invention) has a calcium ion concentration. It emits light in a dependent manner. Therefore, the protein of the present invention (apoprotein) or the photoprotein of the present invention can be used for detection of calcium ions. Specifically, the detection of calcium ions can be performed by adding the sample solution directly to the photoprotein solution and measuring the luminescence generated as described above. Alternatively, calcium ions can be detected by adding a photoprotein solution to the sample solution and measuring the generated luminescence.

The detection or quantification of calcium ions can be performed by measuring the luminescence of the photoprotein of the present invention by calcium ions using a luminescence measuring device. As the luminescence measuring device, a commercially available device, for example, Centro LB 960 (manufactured by Bertrand) can be used. Quantification of the calcium ion concentration can be measured by creating a luminescence standard curve for a known calcium ion concentration using a photoprotein. According to a preferred embodiment of the present invention, the calcium ions to be detected or quantified are in a high concentration, for example, a concentration range of 10 ⁻⁶ M to 10 ⁻⁴ M.

  The protein of the present invention is prepared by preparing a photoprotein comprising the protein of the present invention and a coelenterazine peroxide or a coelenterazine analog peroxide, and directly introducing the photoprotein into a cell by a technique such as microinjection. It can also be used to detect changes in intracellular calcium ion concentration under dynamic conditions.

  The protein of the present invention can be produced in the cell by expressing the apoprotein gene (polynucleotide encoding the protein of the present invention) in the cell, in addition to being introduced into the cell by a technique such as microinjection. Also good. Furthermore, a photoprotein may be produced by adding coelenterazine or a derivative thereof from outside the cell to the produced protein of the present invention (apoprotein).

  Using the photoprotein of the present invention thus introduced into the cell or produced in the cell, changes in the intracellular calcium ion concentration in response to external stimuli (for example, stimulation with drugs involved in receptors) are measured. You can also

(Use as reporter protein)
The protein of the present invention can also be used as a reporter protein for measuring transcriptional activity of a promoter and the like. A vector is constructed in which a polynucleotide encoding the 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). The activity of a target promoter or other expression control sequence can be measured by introducing the vector into a host cell and detecting luminescence derived from the protein of the present invention (that is, luminescence of the photoprotein of the present invention). it can.
The polynucleotide of the present invention can be used as a reporter gene as described above.

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

(Materials for amusement supplies)
A complex composed of the protein of the present invention and a coelenterazine peroxide or a coelenterazine analog peroxide (the photoprotein of the present invention) emits light only by binding to a trace amount of calcium ions. Therefore, the protein of the present invention, the photoprotein of the present invention, and the like can be suitably used as a light-emitting base material for amusement article 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 photoprotein of the present invention can be used in analysis methods such as analysis of physiological functions and measurement of enzyme activity using the principle of intermolecular interaction by the bioluminescence resonance energy transfer (BRET) method.
For example, when the photoprotein of the present invention is used as a donor protein and an organic compound or a fluorescent protein is used as an acceptor protein, the interaction between the proteins is detected by causing bioluminescence resonance energy transfer (BRET) between them. can do. In one embodiment of the present invention, the organic compound used as the acceptor protein is Hoechist3342, Indo-1, DAP1, or the like. In another embodiment of the present invention, the fluorescent protein used as the acceptor protein is a green fluorescent protein (GFP), a blue fluorescent protein (BFP), a mutant GFP fluorescent protein, phycobilin, or 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 Nat Methods 2006, 3: 165-174 or Biotechnol J. 2008, 3: 311-324. Can do.

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

[Method and material]
Materials Recombinant Aequorin Shimomura & Inouye, Protein Express. Purif. 16 (1999) 91-95, Krytin-I Inouye & Sahara, Protein Express. Purif. 53 (2007) 384-389, and Krytin II Prepared as described in Inouye, J. Biochem. 143 (2008) 711-717.
Ethylenediaminetetraacetic acid disodium (EDTA · 2Na), CaCl ₂ · 2H ₂ O, 100 mM CaCO ₃ standard solution, tris (hydroxymethyl) aminomethane, (±) -dithiothreitol (DTT), 2-mercaptoethanol, imidazole, NiSO ₄ .6H ₂ O, (NH ₄ ) ₂ SO ₄ , and n-coelenterazine (n-CTZ) were obtained from Wako Pure Chemical Industries, Ltd.
Chelate Sepharose Fast Flow and PreScience protease were obtained from GE-Healthcare Bio-Science (Piscataway, NJ).
cp-coelenterazine (cp-CTZ), hcp-coelenterazine (hcp-CTZ), f-coelenterazine (f-CTZ), and fcp-coelenterazine (fcp-CTZ) were obtained from Promega (madison, WI).
Coelenterazine (CTZ), h-coelenterazine (h-CTZ), and Bis-coelenterazine (Bis-CTZ) were obtained from Chisso Corporation.
e-Coelenterazine (e-CTZ) and methoxycoelenterazine (MeO-CTZ) were provided by Dr. Katsunori Teranishi (Mie University) (for synthesis method, Shimomura et al., Biochem. J. 251 (1988)). (See, for example, 405-410.)

Construction of pCold-ZZ-PX is a vector capable of inducing expression at a low temperature, a histidine tag sequence for nickel chelate affinity chromatography, a ZZ domain sequence, and a coding sequence of the target protein. A cleavage sequence of human schistovirus 3C protease (PreScience protease) and multiple cloning sites (EcoRI / XhoI / HindIII / SalI / PstI / XbaI). pCold-ZZ-PX was constructed from pCold-ZZ-X (Inouye & Sahara, Biochem. Biophys. Res. Commun. 376 (2008) 448-453) by the following procedure.
After digesting pCold-ZZ-X with restriction enzymes BamHI / EcoRI, a synthetic linker (PreScience B / E-F (SEQ ID NO: 13, 5 ′ GA TCT CTG GAA GTT CTG TTC CAG GGG CCC) having a cleavage recognition sequence of PreScission protease. G3 ′) and PreScission B / ER (SEQ ID NO: 14, 5 ′ AA TTC GGG CCC CTG GAA CAG AAC TTC CAG A 3 ′)) are synthesized and inserted into the BamHI-EcoRI site of pCold-ZZ-X As a result, pCold-ZZ-PX was obtained (FIGS. 2A and 2B). This vector contains the cold shock protein A (cspA) promoter (Qing et al., Nature Biotechnol. 22 (2004) 877-882) controlled by the lac operator.

Construction of Fusion Protein Expression Vector (pCold-ZZ-P-MI) pCold-ZZ-P-MI is a vector that expresses a fusion protein of apomytrocomin and ZZ domain in E. coli. pCold-ZZ-P-MI was constructed as follows.
Using pMI-17 (Fagan et al., FEBS Lett., 333 (1993) 301-305) as a template, MI-5N / EcoRI (SEQ ID NO: 15, 5 ′ ggc GAA TTC GTC AAG CTT ACG ACT GAC TTT 3 ′ Underlined EcoRI site) and pCold-R primer (SEQ ID NO: 16, 5 ′ GGC AGG GAT CTT AGA TTC TG 3 ′) primer set using Ex-Taq polymerase (25 cycles; 94 ° C. For 1 minute, 50 ° C. for 1 minute, and 72 ° C. for 1 minute) to obtain a fragment containing the coding region of recombinant mitrocomin. The obtained fragment was digested with EcoRI and XhoI and inserted into the EcoRI-XhoI site of pCold-ZZ-P-X to obtain pCold-ZZ-P-MI (FIG. 2 (A)). The base sequence of pCold-ZZ-P-MI was confirmed using Applied Biosystems model 310 DNA Sequencer and BigDye Terminator v1.1 cycle sequence kit.

Expression of fusion protein in E. coli cells and purification of recombinant apomitrocomin Recombinant apomitrocomin was purified according to the procedure of FIG. First, the fusion protein was expressed as a soluble protein in E. coli cells. The expressed fusion protein has a histidine tag containing 6 histidine residues, a ZZ domain, a protease cleavage site, and a target protein (ie, recombinant apomytrocomine). Next, the soluble fraction containing the fusion protein expressed in E. coli cells was applied to the first nickel chelate column. Then, the fraction adsorbed on the column was collected as a fraction containing the fusion protein. Next, the fusion protein eluted from the column was cleaved with protease. Further, the cleavage fragment of the fusion protein was applied to the second nickel chelate column, and the fraction passed through the column was recovered. The passage fraction contains recombinant apomitrocomin which is the target protein. Uncleaved fusion protein and cleaved histidine-tagged ZZ domain were adsorbed to the gel. Hereinafter, these procedures will be described in detail.

(I) Expression of His-ZZ-P-apomitrocomin (His-ZZ-P-apoMI) as a soluble protein BL21 (Novagen, Madison, WI) was used as a host E. coli strain. The BL21 strain containing pCold-ZZ-P-MI was cultured in 10 ml of Luria-Bertani medium containing ampicillin (50 μg / ml) at 37 ° C. for 18 hours. This seed culture was inoculated into a 3 L flask containing 400 ml of LB medium, cultured for 3 hours, and then cooled with cold water for 1 hour. IPTG was added to the medium so that the final concentration was 0.2 mM, and further cultured at 15 ° C. for 17 hours. E. coli was recovered from the 800 ml culture by centrifugation at 3000 × g for 5 minutes, suspended in 80 ml of 50 mM Tris-HCl (pH 7.6), performed using Branson model 250 sonifier (Danbury, CT), cooled However, Escherichia coli was destroyed by sonication three times for 3 minutes. After centrifugation at 12000 × g for 20 minutes, the supernatant containing ZZ-P-apomitrocomine was applied to a nickel chelate column (column size; 2.5 × 6 cm) equilibrated with 20 mM Tris-HCl (pH 7.6). Provided. After washing the column with 250 ml of 50 mM Tis-HCl (pH 7.6), ZZ-P-apomitrocomine adsorbed with 0.1 M imidazole was eluted. The yield of the fusion protein His-ZZ-P-apoMI from 800 ml of cultured cells was 51.2 mg, and the purity was 95% or more by SDS-PAGE analysis.

(Ii) Digestion of His-ZZ-P-ApoMI by PreScience Protease In order to examine the optimal digestion conditions, His-ZZ-P-apoMI (2.2 mg) eluted from a nickel chelate column was mixed with 150 mM NaCl, 1 mM EDTA, Digestion was carried out at 4 ° C. for 18 hours in various amounts (1, 3, 10, and 20 ug) of PreScission protease in 1 ml of 50 mM Tris-HCl (pH 7.0) containing 1 mM DTT. Digestion of the fusion protein His-ZZ-P-apoMI was analyzed by SDS-PAGE.
For purification of the following recombinant apomitrocomin, 6 μg of PreScission protease was used for 9.3 mg of His-ZZ-P-apoMI.

(Iii) Purification of Recombinant Apomitrocomin In the digestion solution of His-ZZ-P-ApoMI by PreScission protease, the ZZ domain generated by cleavage, the recombinant apomytrocomin, and the uncut His-ZZ- P-apoMI is included. In order to separate the recombinant apomytrocomin from the cleaved ZZ domain and the uncleaved His-ZZ-P-apoMI, a PreScission protease treated solution was equilibrated with 20 mM Tris-HCl (pH 7.6) (0.5 × 6 cm). ) Directly. And the passage fraction containing recombinant apomitrocomin was collected.

Measurement of luminous activity of recombinant and semi-synthetic recombinant mitrocomin Regeneration of recombinant or semi-synthetic recombinant mitrocomin is performed in 1 ml of 30 mM Tris-HCl (pH 7.6) -10 mM EDTA containing 1 μl of 2-mercaptoethanol. 1 μg coelenterazine or coelenterazine analog (1 μg / μl in methanol) and recombinant apomitrocomine (1 ug) were left at 4 ° C. for 2 hours. Recombinant mitrocomin is regenerated from recombinant apomaitrocomin and coelenterazine, and semi-synthetic recombinant mitrocomin is regenerated from recombinant apomytrocomin and coelenterazine analogs. Luminescence measurement was performed using an AB2200 luminometer provided by Hamamatsu R4220P photomultiplier. 100 μl of 50 mM Tris-HCl (pH 7.6) containing 50 mM CaCl ₂ was injected into the regenerated recombinant mitrocomin or semi-synthetic recombinant mitrocomin (1 μl) and measured for 10 seconds. Initial luminescence intensity (Imax), luminescence capacity, and half decay time were determined.

Measurement of Ca2 ⁺ detection sensitivity of recombinant mitrocomin 50 mM Tris-HCl (pH 7.6) containing 0.01 mM EDTA, 0.1% bovine albumin, and 150 mM NaCl as recombinant mitrocomin regenerated from recombinant apomytrocomin Dissolved in 10 μl of recombinant mitrocomin (5 ng) was injected into 50 mM Tris-HCl (pH 7.6) solution containing 50 μl of various Ca ²⁺ concentrations (10 ^−8.5 to 10 ⁻⁴ M). . 9124B photomultiplier (Electron tube limited, UK) equipped with Berthold technologies (Bad Wildbad, Germany) model Centro 960 luminometer was used to measure the luminescence intensity of recombinant mitrocomin.

Measurement of bioluminescence emission spectrum of recombinant photoproteins The emission spectra of recombinant Mytrocomin, Kleitin-I, Kleitin-II, and aequorin by Ca ²⁺ were measured by Inouye & Sasaki FEBS Lett. 580 (2006) 1977-1982. The excitation light source at 22 to 25 ° C. with a fluorescence spectrometer FP6500 (manufactured by JASCO Corporation) (emission bandwidth, 10 nm; response, 0.5 seconds; sensitivity, medium; scanning speed, 1000 nm / min) The measurement was performed with the mark turned off.

Protein analysis SDS-PAGE analysis was performed under reducing conditions using 12% separation gels as described in Laemmli, Nature 227 (1970) 680-685. The electrophoresis conditions were performed at a current of 25 mA for 90 minutes. After electrophoresis, the gel was stained using a colloid CBB staining kit (TEFCO). The protein concentration was determined by Bradford's dye binding method (Bradford, Anal. Biochem. 72 (1976) 248-) as a bovine serum albumin (Pierce, Rockford, IL) standard substance using a commercially available protein measurement kit (Bio-Rad, Richmond, CA). 254).

Mass spectrometry The mass spectrometry of the protein was performed by matrix-assisted laser desorption / ionization mass spectrometry (MALDI-TOF-MS) using Voyager DE Pro mass spectrometer (PerSeptive Biosystems Inc., Framingham, Mass.) (Inouye et al. Anal, Biochem. 316 (2003) 216-222.).

[Results and discussion]
Construction of a vector for expressing a fusion protein (ZZ domain-binding apomtrocomine) in E. coli cells Recently, in the low-temperature induction system using Escherichia coli, the present inventors used a target protein having a ZZ domain as a soluble partner as a soluble protein. (Inouye & Sahara, Biochem. Biophys. Res. Commun. 376 (2008) 448-453).
In order to prepare a target protein excluding the ZZ domain, it is necessary to remove the ZZ domain from the fusion protein. The present inventors achieved this by introducing a cleavage site for human rhinovirus 3C protease (PreScission protease) between the ZZ domain and the target protein (FIG. 1). For this purpose, we constructed a novel expression vector pCold-ZZ-PX (FIG. 2). This is a vector derived from pCold-ZZ-X (Inouye & Sahara, Biochem. Biophys. Res. Commun. 376 (2008) 448-453) (FIG. 1).
For expression as a fusion protein in the cytoplasm of E. coli cells, the apomytrocomine coding region of pMI-17 (Fagan et al., FEBS Lett. 333 (1993) 301-305) is used as the EcoRI of pCold-ZZ-PX. -It inserted in the XhoI site, and pCold-ZZ-P-MI was obtained (FIG. 2 (A)). This expression vector comprises a histidine tag sequence containing 6 histidines, a ZZ domain sequence, a pre-cision protease cleavage sequence, a cspA promoter, and a part of apomytrocomine cDNA under the control of the lac operator (SEQ ID NO: 7 th 42nd to 614th base sequence). Fusion protein expression was induced using low temperature at 15 ° C. and 0.2 mM IPTG. The base sequence of the fusion protein is shown in SEQ ID NO: 11, and the amino acid sequence is shown in SEQ ID NO: 12 (FIG. 8).

Recombinant recombinant apomytrocomin from ZZ domain-bound apomytrocomin (histidine-tagged ZZ domain-bound apomytrocomin: His-ZZ-P-apoMI) is expressed as a soluble protein in the cytoplasm of E. coli cells, Purification by nickel chelate affinity chromatography (Figure 3, lane 2). From 800 ml of cultured cells, 51.7 mg of purified His-ZZ-P-apoMI was obtained (Table 1). In order to remove the ZZ domain from His-ZZ-P-apoMI, the cleavage conditions of the PreScience protease were examined using various concentrations of PreScience protease (lanes 3 to 6 in FIG. 3). As a result, a recombinant apomytrocomin without a ZZ domain could be obtained under conditions where the ratio of His-ZZ-P-apoMI to PreScission protease exceeds 2000 times (lane 6 in FIG. 3). After the fusion protein was digested with PreScission protease, the digested product was directly applied to a nickel column to separate the recombinant apomitrocomin from the uncut His-ZZ-P-apoMI and the cleaved histidine-tagged ZZ domain. In this way, a fraction containing the recombinant apomitrocomin was obtained (FIG. 4, lane 5). In SDS-PAGE analysis under reducing conditions, recombinant apomitrocomin showed a molecular weight of 24 kDa with a purity of 95% or more (FIG. 4, lane 5). Nickel chelate chromatography yielded 5.9 mg of recombinant apomitrocomin (Table 1).
In order to confirm that the fusion protein was correctly cleaved by cleavage of the PreScience proteinase, MALDI-TOF-MS analysis was performed, and mass values m / z22, 245.3 were observed. This mass value was in good agreement with the calculated average mass [M + H] ⁺ 22,261.9 of purified recombinant apomitrocomin. From this, it was confirmed that the ZZ domain could be correctly cleaved by digestion with PreScience proteinase. That is, the recombinant apomitrocomine is a recombinant protein consisting of 194 amino acid residues containing an additional amino acid sequence of Gly-Pro-Glu-Phe at the N-terminus. The base sequence of recombinant apomitrocomine is shown in SEQ ID NO: 9, and the amino acid sequence is shown in SEQ ID NO: 10.

Emission spectrum analysis of recombinant mitrocomin Similar to the method for the preparation of recombinant aequorin (Inouye et al., J. Biochem. 105 (1989) 473-477), coelenterazine in the presence of 2-mercaptoethanol at 4 ° C. Alternatively, recombinant mitrocomin or semi-synthetic recombinant mitrocomin was regenerated by incubating recombinant apomytrocomin with coelenterazine analogs. When left for 2 hours, 95% or more of the recombinant mitrocomin or semi-synthetic recombinant mitrocomin could be regenerated. The emission spectrum of the recombinant mitrocomin with Ca ²⁺ was measured and compared with the emission spectra of other photoproteins (FIG. 5). The recombinant mitrocomin bioluminescence spectrum showed an emission peak at 456 nm, with a full width at half maximum (FWHM) of 72 nm (FIG. 5). This spectrum is shown for natural mitrocomin (Halistaurin) and crude preparations of mitrocomin (Fagan et al., FEBS Lett. 333 (1993) 301-305 and Shimomura et al., Biochem. J. 228 (1985) 745-749. ) But not the same. Under the same conditions, aequorin (Inouye & Sasaki, FEBS Lett. 580 (2006) 1977-1982) and Klytin (Inouye, J. Biochem. 143 (2008) 711-717 and Inouye & Sahara, Protein Express. Purif. 53 (2007) 384-389) have a maximum emission wavelength of 460 nm with a FWHM of 84 nm and a maximum emission wavelength of 470 nm with a FWHM of 76 nm. These results suggest that the environment of coelenteramide, which became the luminescence source, is different from that of apoaequorin and apocrytin in recombinant apomytrocomin.

Luminescence characteristics of recombinant mitrocomin and semi-synthetic recombinant mitrocomin The luminescence pattern of recombinant mitrocomin by addition of Ca ²⁺ is shown in FIG. The half decay time was 1.33 seconds, indicating a slower decay time than that of all other photoproteins.
Recombinant apomei can be prepared by procedures similar to those for the preparation of semisynthetic aequorin (Shimomura et al., Biochem. J. 270 (1990) 309-312 and Shimomura et al., Cell Calcium, 14 (1993) 373-378). Semi-synthetic recombinant mitrocomin was prepared by incubating trocomine and coelenterazine analogs. Excess Ca ²⁺ is added to semisynthetic recombinant mytrocomin, and the initial luminescence intensity, luminescence amount, and semi-decay time are measured, and those of aequorin, criticin-I, and criticin-II described in other papers Compared with. The comparison results are shown in Table 2. FIG. 6A shows the result of comparing the light emission patterns. Furthermore, the light emission pattern of semisynthetic recombinant mitrocomin is shown in FIG. Interestingly, the recombinant n-mytrocomine obtained from n-coelenterazine showed a slowly increasing emission pattern with a half decay time of over 10 seconds. Moreover, the semi-synthetic recombinant mitrocomin obtained from hcp-coelenterazine or fcp-coelenterazine showed better luminescence intensity than other photoproteins. It is considered that the cyclophenyl (cp) group at the 8-position of the imidazopyrazinone structure in coelenterazine significantly affects the emission intensity of mitrocomine.

Recombinant Mytrocomin Ca ²⁺ Detection Sensitivity By injecting recombinant mitrocomin into a standard Ca ²⁺ solution, the detection sensitivity of the recombinant mitrocomin to Ca ²⁺ was measured, and Inouye, J. Biochem. -717 or other photoproteins described in Inouye & Sahara, Protein Express. Purif. 53 (2007) 384-389. When the Ca ²⁺ concentration of the standard Ca ²⁺ solution is varied from 10 ^−8.5 to 10 ⁻⁴ M, recombinant mitrocomin is detected with Ca ²⁺ at a high concentration of 10 ⁻⁶ to 10 ⁻⁴ M. I was able to. This result indicates that the sensitivity of recombinant mitrocomin is not limited to natural photoproteins including natural mitrocomin (Shimomura et al., Biochem. J. 228 (1985) 745-749) and other recombinant photoproteins (Inouye). , J. Biochem. 143 (2008) 711-717, Inouye & Sahara, Protein Express. Purif., 53 (2007) 384-389, and Markova et al., Biochemistry, 41 (2002) 2227-2236). It became clear that it was lower than twice (FIG. 7). This recombinant protein can be used to detect and measure high concentrations of Ca ²⁺ .

It is a figure which shows the purification method of the target protein from the ZZ domain fusion protein expressed in E. coli cells. It is a figure which shows the expression vector of ZZ domain fusion protein which has a cutting site. (A) is a plasmid map of the expression vector pCold-ZZ-PX. cpsA: cold shock protein A promoter, TEF: translational enhancing element, His_Tag: histidine tag sequence containing 6 histidines. (B) It is a figure which shows the amino acid sequence and base sequence of N terminal area | region of pCold-ZZ-PX containing a ZZ domain and a multicloning site (MCS). It is a photograph which shows the result of the SDS-PAGE analysis after the enzyme digestion process by various amounts of PreScission Protease of His-ZZ-P-apoMI. Lane 1: molecular weight marker (manufactured by Tefco) (β-galactosidase (116 kDa), phosphorylase b (97.4 kDa), bovine serum albumin (96.0 kDa), glutamate dehydrogenase (55.0 kDa), lactate dehydrogenase Enzyme (36.5 kDa), carbonic anhydrase (29.0 kDa), and trypsin inhibitor (20.1 kDa)), lane 2: His-ZZ-P-apoMI (2.2 μg protein) eluted from nickel column, lane 3: His-ZZ-P-apoMI / PreScience protein = 2.2 mg / 20 μg (6.6 μg protein), Lane 4: His-ZZ-P-apoMI / PreScience protein = 2.2 mg / 10 μg (6.6 μg protein) , Lane 5 His-ZZ-P-apoMI / PreScission protease = 2.2mg / 3μg (6.6μg protein), lane 6: His-ZZ-P-apoMI / PreScission protease = 2.2mg / 1μg (6.6μg protein). It is a photograph which shows the result of the SDS-PAGE analysis of the protein in each step of recombinant apomitrocomin purification. Lane 1: molecular weight marker, lane 2: crude extract of E. coli (16.5 μg protein), lane 3: purified His-ZZ-P-apoMI (2.2 μg protein) eluted from nickel chelate column, lane 4: PreScission. Purified His-ZZ-P-apoMI (2.5 μg protein) eluted from the nickel chelate column after cleavage with protease, Lane 5: Passage fraction of purified recombinant apomytrocomin eluted from the nickel chelate column (1 .6 μg protein). It is the spectrum figure which compared the luminescence spectrum of bioluminescence with recombinant mytrocomin (MI), aequorin (AQ), criticin-I (CL-I), and criticin-II (CL-II). The emission spectra of aequorin, clytin-I, and clytin-II are those described in Inouye, J. Biochem. 143 (2008) 711-717. It is a figure which shows the luminescent reaction by Ca2 ⁺ addition of recombinant mytrocomin and semisynthetic recombinant mytrocomin. . (A) It is the figure which compared the light emission pattern of recombinant mitrocomin (MI), aequorin (AQ), Kleitin-I (CL-I), and Kleitin-II (CL-II). (B) It is the figure which compared the light emission pattern of the semisynthetic recombinant mytrocomin by Ca2 ⁺ addition. It is a figure which shows the relationship between the initial luminescence intensity and Ca ^<2+ > density ^| concentration of recombinant mitrocomin (MI), aequorin (AQ), Krytin-I (CL-I), and Krytin-II (CL-II). . The data for aequorin and clytin are described in Inouye, J. Biochem. 143 (2008) 711-717 and Inouye & Sahara Protein Express. Purif. 53 (2007) 384-389, respectively. It is a figure which shows the base sequence and amino acid sequence of a fusion protein.

[SEQ ID NO: 1] This shows the base sequence of DNA encoding the amino acid sequence of the polypeptide represented by formula Z.
[SEQ ID NO: 2] This shows the amino acid sequence of the polypeptide represented by formula Z.
[SEQ ID NO: 3] This shows the base sequence of DNA encoding the amino acid sequence of the polypeptide represented by formula (Z) ₂ .
[SEQ ID NO: 4] This shows the amino acid sequence of the polypeptide represented by formula (Z) ₂ .
[SEQ ID NO: 5] This shows an example of the base sequence of DNA encoding linker peptide.
[SEQ ID NO: 6] This shows an example of amino acid sequence of linker peptide.
[SEQ ID NO: 7] This shows an example of the base sequence of DNA encoding apomytrocomin (Accession No L31623).
[SEQ ID NO: 8] This shows an example of the amino acid sequence of apomytrocomin.
[SEQ ID NO: 9] This shows the base sequence of DNA encoding the amino acid sequence of recombinant apomitrocomin prepared in the Example.
[SEQ ID NO: 10] This shows the base sequence of DNA encoding the amino acid sequence of recombinant apomitrocomin prepared in the Example.
[SEQ ID NO: 11] This shows the base sequence of DNA encoding the amino acid sequence of the fusion protein prepared in the Example.
[SEQ ID NO: 12] This shows the amino acid sequence of the fusion protein prepared in the example.
[SEQ ID NO: 13] This shows the base sequence (F) of the synthetic linker DNA used in the examples.
[SEQ ID NO: 14] This shows the base sequence (R) of the synthetic linker DNA used in the examples.
[SEQ ID NO: 15] This shows the base sequence of the primer used in the examples.
[SEQ ID NO: 16] This shows the base sequence of the primer used in the examples.

Claims (22)

(1) a promoter sequence capable of inducing expression;
(2) 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 comprising the amino acid sequence of SEQ ID NO: 2,
(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: 2.
(C) a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 2, and (d) a polynucleotide comprising a base sequence complementary to the base sequence of SEQ ID NO: 1. A polypeptide comprising an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions)
And a polyprotein having a function that allows the fusion protein to be expressed as a soluble protein when expressed as a fusion protein of a linker peptide and a polypeptide selected from the group consisting of (i) to (l). A first coding sequence containing a polynucleotide encoding a peptide;
(3) A polypeptide having an amino acid sequence of Gly-Pro-Glu-Phe at the N-terminus of a polypeptide selected from the group consisting of (i) to (l) when cleaved. A coding sequence comprising a polynucleotide that encodes a linker peptide that adds; and (4) a second code comprising a polynucleotide that encodes a polypeptide selected from the group consisting of: Sequence;
(I) a polypeptide comprising the ninth to 198th amino acid sequence of SEQ ID NO: 8,
(J) consisting of an amino acid sequence in which one to a plurality of amino acids are deleted, substituted, inserted and / or added in the ninth to 198th amino acid sequences of SEQ ID NO: 8, and the calcium-binding photoprotein A polypeptide having apoprotein activity,
(K) a polypeptide comprising an amino acid sequence having 90% or more identity to the 9th to 198th amino acid sequences of SEQ ID NO: 8, and having an apoprotein activity of a calcium-binding photoprotein, And (l) an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a base sequence complementary to the 42nd to 614th base sequences of SEQ ID NO: 7, And a polypeptide having an apoprotein activity of a calcium-binding photoprotein,
Expressing the fusion protein as a soluble protein using an expression vector containing
Cleaving the fusion protein at the cleavage site of the linker peptide, and obtaining a protein comprising a polypeptide selected from the group consisting of (i) to (l), wherein Gly-Pro-Glu-Phe is added to the N-terminus. A method for producing a protein, comprising: The method according to claim 1, wherein the polypeptide represented by the formula (Z) _n is a polypeptide represented by (Z) ₂ . The polypeptide represented by the formula (Z) ₂ is:
(E) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 4,
(F) consisting of 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: 4, and from the group consisting of a linker peptide and (i) to (l) A polypeptide having a function of allowing the fusion protein to be expressed as a soluble protein when expressed as a fusion protein with the selected polypeptide;
(G) Fusion of a linker peptide and a polypeptide selected from the group consisting of (i) to (l), comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 4. A polypeptide having a function capable of expressing the fusion protein as a soluble protein when expressed as a protein, and (h) a polynucleotide comprising a base sequence complementary to the base sequence of SEQ ID NO: 3 and stringent conditions A fusion protein comprising an amino acid sequence encoded by a polynucleotide that hybridizes underneath and expressed as a fusion protein of a linker peptide and a polypeptide selected from the group consisting of (i) to (l) A polypeptide selected from the group consisting of polypeptides having the function of allowing a protein to be expressed as a soluble protein That method of claim 2 wherein.   The method according to any one of claims 1 to 3, wherein the promoter sequence capable of inducing expression is a promoter sequence capable of inducing expression at a low temperature.   The method according to claim 4, wherein the promoter sequence capable of inducing expression at a low temperature is a promoter sequence of a cold shock gene.   6. The method according to claim 5, wherein the cold shock gene promoter sequence is an E. coli cold shock gene promoter sequence.   The method according to claim 6, wherein the promoter sequence of the E. coli cold shock gene is a promoter sequence of the E. coli cold shock gene cspA, cspB, cspG, cspI or csdA.   The method according to any one of claims 1 to 7, further comprising a coding sequence containing a polynucleotide encoding an amino acid sequence for purification on the 5 'side of the first coding sequence.   The method according to claim 8, wherein the amino acid sequence for purification is a histidine tag sequence.   A method for producing a calcium-binding photoprotein, which comprises contacting a protein produced by the method according to any one of claims 1 to 9 with coelenterazine or a coelenterazine analog to obtain a calcium-binding photoprotein. .   The coelenterazine analog is selected from the group consisting of n-coelenterazine, cp-coelenterazine, hcp-coelenterazine, f-coelenterazine, fcp-coelenterazine, h-coelenterazine, Bis-coelenterazine, e-coelenterazine, and methoxycoelenterazine. 10. The method according to 10.   The method of claim 11, wherein the coelenterazine analog is selected from the group consisting of n-coelenterazine, cp-coelenterazine, hcp-coelenterazine, f-coelenterazine, fcp-coelenterazine, h-coelenterazine, and e-coelenterazine. A protein selected from the group consisting of the following (m) to (p):
(M) a protein consisting of the amino acid sequence of SEQ ID NO: 10;
(N) the amino acid sequence of SEQ ID NO: 10, consisting of an amino acid sequence in which one or more amino acids other than the first to fourth amino acids are deleted, substituted, inserted and / or added, and calcium binding A protein having an apoprotein activity of a type photoprotein;
(O) 90% or more identity to the amino acid sequence of SEQ ID NO: 10, consisting of an amino acid sequence in which the first to fourth amino acids are Gly-Pro-Glu-Phe, and calcium A protein having an apoprotein activity of a binding photoprotein; and (p) 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: 9; A protein comprising an amino acid sequence in which the first to fourth amino acids are Gly-Pro-Glu-Phe and having apoprotein activity of a calcium-binding photoprotein.   A calcium-binding photoprotein comprising the protein according to claim 13 and a peroxide of coelenterazine or a coelenterazine analog.   The coelenterazine analog is selected from the group consisting of n-coelenterazine, cp-coelenterazine, hcp-coelenterazine, f-coelenterazine, fcp-coelenterazine, h-coelenterazine, Bis-coelenterazine, e-coelenterazine, and methoxycoelenterazine. 14. The protein according to 14.   16. The protein of claim 15, wherein the coelenterazine analog is selected from the group consisting of n-coelenterazine, cp-coelenterazine, hcp-coelenterazine, f-coelenterazine, fcp-coelenterazine, h-coelenterazine, and e-coelenterazine.   A method for detecting or quantifying calcium ions, comprising using the calcium-binding photoprotein produced by the method according to any one of claims 10 to 12.   A method for detecting or quantifying calcium ions, comprising using the calcium-binding photoprotein according to any one of claims 13 to 15. The method according to claim 17 or 18, wherein the calcium ion to be detected or quantified is in a concentration range of 10 ^-6 M to 10 ^-4 M.   A method for analyzing a physiologically active substance, comprising performing bioluminescence resonance energy transfer using the calcium-binding photoprotein produced by the method according to any one of claims 10 to 12 as a donor protein.   A bioluminescent resonance energy transfer (BRET) method using the calcium-binding photoprotein according to any one of claims 13 to 15 as a donor protein.   A method for measuring enzyme activity, comprising performing a bioluminescence resonance energy transfer (BRET) method using the calcium-binding photoprotein according to any one of claims 13 to 15 as a donor protein.