MUTATED GENES FOR THE CATALYTIC PROTEIN OF OPLOPHORUS LUCIFERASE AND USE THEREOF

Abstract

Secreted luciferases which are different from those known heretofore have been desired. The present invention provides a luciferase mutant comprising an amino acid sequence in which at least one amino acid selected from amino acids at the positions of 1 to 4 is deleted in the amino acid sequence of SEQ ID NO: 2.

Claims

1-14. (canceled)

15. A method for assaying the activity of a sequence associated with promoter regulation, which comprises using a polynucleotide encoding a luciferase mutant as a reporter gene and contacting the luciferase mutant with a luciferin, wherein the luciferase mutant is selected from (a) to (e) below: (a) a luciferase mutant consisting of an amino acid sequence in which at least one amino acid selected from amino acids at the positions of 1 to 4 is deleted in the amino acid sequence of SEQ ID NO: 2; (b) a luciferase mutant consisting of an amino acid sequence in which at least one amino acid selected from amino acids at the positions of 1 to 4 is deleted in the amino acid sequence of SEQ ID NO: 2 and an amino acid sequence excluding the amino acids at the positions of 1 to 4 is an amino acid sequence in which 1 to 17 amino acids are deleted, substituted, inserted and/or added in the amino acid sequence at the positions of 5 to 169 of SEQ ID NO: 2, and having a luciferase activity; (c) a luciferase mutant consisting of an amino acid sequence in which at least one amino acid selected from amino acids at the positions of 1 to 4 is deleted in the amino acid sequence of SEQ ID NO: 2 and an amino acid sequence excluding the amino acids at the positions of 1 to 4 has at least 90% identity to the amino acid sequence at the positions of 5 to 169 of SEQ ID NO: 2, and having a luciferase activity; (d) a luciferase mutant consisting of an amino acid sequence in which at least one amino acid selected from amino acids at the position of 1 to 4 is deleted in the amino acid sequence of SEQ ID NO: 2 and an amino acid sequence excluding the amino acids at the position of 1 to 4 is encoded by a polynucleotide which hybridizes under high stringent condition to a polynucleotide consisting of a nucleotide sequence complementary to a nucleotide sequence encoding the amino acid sequence at the positions of 5 to 169 of SEQ ID NO: 2, wherein the high stringent conditions are 5SSC, 5Denhart's solution, 0.5% (w/v) SDS, 50% (v/v) formamide and 50 C., and having a luciferase activity; and, (e) a luciferase mutant consisting of an amino acid sequence selected from SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10.

16. The method according to claim 15, wherein the luciferin is coelenterazines.

17. The method according to claim 16, wherein the coelenterazines is bis-coelenterazine or 6h-f-coelenterazine.

18-20. (canceled)

21. The method according to claim 15, wherein the luciferase mutants defined in (b) to (d) above are mutants defined in (b-1) to (d-1) below: (b-1) a luciferase mutant consisting of an amino acid sequence in which at least one amino acid selected from amino acids at the positions of 1 to 4 is deleted in the amino acid sequence of SEQ ID NO: 2 and an amino acid sequence excluding the amino acids at the positions of 1 to 4 is an amino acid sequence in which 1 to 9 amino acids are deleted, substituted, inserted and/or added in the amino acid sequence at the positions of 5 to 169 of SEQ ID NO: 2, and having a luciferase activity; (c-1) a luciferase mutant consisting of an amino acid sequence in which at least one amino acid selected from amino acids at the positions of 1 to 4 is deleted in the amino acid sequence of SEQ ID NO: 2 and an amino acid sequence excluding the amino acids at the positions of 1 to 4 has at least 95% identity to the amino acid sequence at the positions of 5 to 169 of SEQ ID NO: 2, and having a luciferase activity; and, (d-1) a luciferase mutant consisting of an amino acid sequence in which at least one amino acid selected from amino acids at the positions of 1 to 4 is deleted in the amino acid sequence of SEQ ID NO: 2 and an amino acid sequence excluding the amino acids at the positions of 1 to 4 is encoded by a polynucleotide which hybridizes under high stringent conditions to a polynucleotide consisting of a nucleotide sequence complementary to a nucleotide sequence encoding the amino acid sequence at the positions of 5 to 169 of SEQ ID NO: 2, wherein the high stringent conditions are 5SSC, 5Denhart's solution, 0.5% (w/v) SDS, 50% (v/v) formamide and 50 C., and having a luciferase activity.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0046] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0047] FIG. 1 shows the results of SDS-PAGE analysis of the crude enzyme solution (crude extracts) of E. coli in which the amino-terminal deleted nanoKAZ was expressed using a pColdII vector.

[0048] FIG. 2 shows the results of SDS-PAGE analysis of the crude enzyme solution of E. coli in which the amino-terminal deleted nanoKAZ was expressed using a pCold-ZZ-P vector.

[0049] FIG. 3 shows the secretory inhibition of nanoKAZ by brefeldin A from the stable expressed cell line, pcDNA3-GLsp-nanoKAZ/CHO-K1, which was established using the nanoKAZ gene with a secretory signal peptide sequence. Open circles and closed circles indicate the presence and absence of brefeldin A, respectively.

[0050] FIG. 4 shows the secretion of nanoKAZ from the stable expressed cell line, pcDNA3-nanoKAZ/CHO-K1, which was established using the nanoKAZ gene lacking a secretory signal peptide sequence, in the presence of brefeldin A. Open circles and closed circles indicate the presence and absence of brefeldin A, respectively.

[0051] FIG. 5 shows the bright-field image and the visualized luminescence image of nanoKAZ secretion. Labeled a and b represent the bright-field image and luminescence image of nanoKAZ from the stable expressed cell line, pcDNA3-GLsp-nanoKAZ/CHO-K1, respectively, and labeled c and d represent the bright-field image and luminescence image of nanoKAZ from the stable expressed cell line pcDNA3-nanoKAZ/CHO-K1, respectively.

MODE FOR CARRYING OUT THE INVENTION

[0052] The present invention will be described below in detail.

1. Luciferase Mutants of the Invention

[0053] The term luciferase mutant of the present invention refers to a mutant of the protein with a molecular weight of 19 kDa of Oplophorus luciferase. Specifically, the luciferase mutant of the present invention is intended to mean a luciferase mutant having substantially the same activity as the luciferase mutant comprising an amino acid sequence in which at least one amino acid selected from amino acids at the positions of 1 to 4 is deleted in the amino acid sequence of SEQ ID NO: 2.

[0054] The term substantially the same activity is intended to mean at least one activity selected from luciferase activity, activity for extracellular secretion when expressed in animal cells in the absence of any secretory signal peptide sequences, and so on.

[0055] The term luciferase activity is intended to mean the activity for catalyzing the luminescence reaction using a luciferin (e.g., coelenterazines) which serves as a substrate, namely, the reaction in which luciferin (e.g., coelenterazines) is oxidized with molecular oxygen to produce oxyluciferin in its excited state. The excited state of oxyluciferin produced emits visible light and converts to the ground state of oxyluciferin.

[0056] Luminescence activity can be determined by the method described in, e.g., Inouye, S. & Shimomura, O. (1977) Biochem. Biophys. Res. Commun. 233, 349-353. Specifically, the luciferase mutant of the present invention is mixed with a luciferin to start the luminescence reaction, and the activity of catalyzing luminescence reaction can be determined using a luminometer. Commercially available luminometers, e.g., Luminescencer-PSN AB2200 (manufactured by Atto Corp.) or Centro 960 luminometer (manufactured by Berthold Inc.) may be used as luminometers.

[0057] The luciferin used in the present invention may be any luciferin as far as it serves as a substrate for the luciferase mutants of the present invention. Specifically, the luciferin used in the present invention includes coelenterazines containing the imidazopyrazinone ring as the backbone.

[0058] The term coelenterazines are used to mean coelenterazine (also referred to as native coelenterazine) or its analogues. Coelenterazine analogues include, for example, bis-coelenterazine, 6hf-coelenterazine, deoxyfuran-coelenterazine (furimazine), h-coelenterazine, hep-coelenterazine, cp-coelenterazine, ficoelenterazine, fcp-coelenterazine, n-coelenterazine. MeO-coelenterazine, e-coelenterazine, cl-coelenterazine, ch-coelenterazine, 3iso-coelenterazine, 3meo-coelenterazine, cf3-coelenterazine, i-coelenterazine, et-coelenterazine, me-coelenterazine, 3me-coelenterazine, meh-coelenterazine, 8-(1-naphthyl)-coelenterazine, 8-(2-naphthyl)-coelenterazine, 8-(2-thienyl)-coelenterazine, 6,8-di-(2-thienyl)-coelenterazine, 8-(4-hydroxyphenyl)-coelenterazine, 8-(2-benzothienyl)-coelenterazine, 8-(b-styryl)-coelenterazine, 8-phenyl-coelenterazine, 6-deoxy-coelenterazine, 8-(3-thienyl)-coelenterazine, and 8-(3-benzo[b]thienyl)-coelenterazine. Of these coelenterazines, bis-coelenterazine or 6h-f-coelenterazine is particularly preferred in some embodiments of the present invention. In some other embodiments of the present invention, h-coelenterazine or f-coelenterazine is particularly preferred.

[0059] These coelenterazines could be synthesized by publicly known methods or may also be commercially available.

[0060] The coelenterazines could be synthesized by the methods described in, e.g., Shimomura et al. (1988) Biochem. J. 251, 405-410, Shimomura et al. (1989) Biochem. J. 261, 913-920, Shimomura et al. (1990) Biochem. J. 270, 309-312, Tetrahedron Lett, 38: 6405-6406, WO 2010/090319, Inouye et al, (2010) Anal. Biochem, 407, 247-252 or Inouye et al. (2013) Biocchem. Biophys. Res. Commun. 437, 23-28, or respective modifications thereof. Furimazine may be produced by the method described in Hall et al. (2012) ACS Chem. Biol. 16; 848-1857.

[0061] The coelenterazines which are commercially available include, for example, coelenterazine, cf3-coelenterazine and h-coelenterazine manufactured by JNC Corp.; hep-coelenterazine, cp-coelenterazine, f-coelenterazine, fcp-coelenterazine and n-coelenterazine manufactured by Biotium Inc.; and bis-coelenterazine manufactured by Prolume Ltd. and coelenterazine, furimazine and h-coelenterazine manufactured by Promega Corp.

[0062] The luminescence activity using a luciferin as a substrate refers to luminescence activity using preferably coelenterazines as a substrate. In a preferred embodiment of the invention, the luminescence activity using coelenterazines as a substrate is the luminescence activity in which bis-coelenterazine or 6h-f-coelenterazine serves as the substrate. In another preferred embodiment of the invention, the luminescence activity using coelenterazines as a substrate is the luminescence activity in which h-coelenterazine orfcoelenterazine serves as the substrate.

[0063] The activity for extracellular secretion (secreting extracellularly) when expressed in animal cells in the absence of any secretory signal peptide sequences is intended to mean that when the protein is expressed in animal cells, the expressed protein is secreted extracellularly from the endoplasmic reticulum but not via the trans-Golgi network, despite having no secretory signal peptide. The extracellular secretion (secreting extracellularly) refers specifically to extracellular secretion of the protein in an amount (by weight) of 5% or more, 10% or more, or 20% or more of the expressed protein. Specific examples of the animal cells are those later described. The secretory signal peptide is intended to mean a signal for secretion, excluding the region in which the secretion information of nanoKAZ is carried, specifically excluding the region of amino acids at the positions of 1 to 4 from the amino terminus.

[0064] The luciferase mutant having substantially the same activity as the luciferase mutant comprising an amino acid sequence in which at least one amino acid selected from amino acids at the positions of 1 to 4 is deleted in the amino acid sequence of SEQ ID NO: 2 includes, for example, a luciferase mutant selected from (a) to (d) described below.

[0065] (a) a luciferase mutant comprising an amino acid sequence in which at least one amino acid selected from the 1st to 4th amino acids is deleted in the amino acid sequence of SEQ ID NO: 2;

[0066] (b) a luciferase mutant comprising an amino acid sequence in which at least one amino acid selected from amino acids at the positions of 1 to 4 is deleted in the amino acid sequence of SEQ ID NO: 2 and an amino acid sequence excluding the amino acids at the positions of 1 to 4 is an amino acid sequence in which 1 to 17 amino acids are deleted, substituted, inserted and/or added in the amino acid sequence at the positions of 5 to 169 of SEQ ID NO: 2, and having a luciferase activity;

[0067] (c) a luciferase mutant comprising an amino acid sequence in which at least one amino acid selected from amino acids at the positions of 1 to 4 is deleted in the amino acid sequence of SEQ ID NO: 2 and an amino acid sequence excluding the amino acids at the positions of 1 to 4 has at least 90% identity to the amino acid sequence at the positions of 5 to 169 of SEQ ID NO: 2, and having a luciferase activity; and,

[0068] (d) a luciferase mutant comprising an amino acid sequence in which at least one amino acid selected from amino acids at the positions of 1 to 4 is deleted in the amino acid sequence of SEQ ID NO: 2 and an amino acid sequence excluding the amino acids at the positions of 1 to 4 is encoded by a polynucleotide which hybridizes under stringent conditions to a polynucleotide consisting of a nucleotide sequence complementary to a nucleotide sequence encoding the amino acid sequence at the positions of 5 to 169 of SEQ ID NO: 2, and having a luciferase activity.

[0069] In (a) to (d) described above, the term at least one amino acid selected from amino acids at the positions of 1 to 4 is deleted in the amino acid sequence of SEQ. ID NO: 2 is intended to mean a deletion of at least one amino acid residue at the positions of 1 to 4 in the amino acid sequence of SEQ ID NO: 2. Preferably, the amino acid at the position of 1, all amino acids at the positions of 1 and 2, all amino acids at the positions of 1 to 3 or all amino acids at the positions of 1 to 4 are deleted.

[0070] Specifically, at least one in at least one amino acid is deleted refers to 1, 2, 3 or 4, preferably 1 or 2, and more preferably 1.

[0071] In (b) to (d) described above, the amino acid sequence excluding the amino acids at the positions of 1 to 4 is intended to mean an amino acid sequence corresponding to the amino acid sequence at the positions of 5 to 169 of SEQ ID NO: 2 prior to the mutation, in each of the amino acid sequences in the luciferase mutants defined in (b) to (d) described above.

[0072] In (b) above, the term 1 to 17 amino acids are deleted, substituted, inserted and/or added is intended to mean that the deletion, substitution, insertion and/or addition of 1 to 17 amino acid residues occur at an optional position(s) in the same sequence and at 1 to 17 positions in the amino acid sequence.

[0073] The range of 1 to 17 in the 1 to 17 amino acids are deleted, substituted, inserted and/or added described above is, for example, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6 (1 to several), 1 to 5, 1 to 4, 1 to 3, 1 to 2 and 1. In general, the less the number of amino acids deleted, substituted, inserted and/or added, the more preferred. Such proteins may be produced by site-directed mutagenesis described in J. Sambrook, E. F. Fritsch & T. Maniatis (Ed.), Molecular cloning, a laboratory manual (4th edition), Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2012); Ausbel F. M. et al., Current Protocols in Molecular Biology, Supplement 1-38, John Wiley and Sons (1987-1997); Nuc. Acids. Res., 10, 6487 (1982); Proc. Natl. Acad. Sci. USA, 79, 6409 (1982); Gene, 34, 315 (1985); Nuc. Acids. Res., 13, 4431 (1985); Proc. Natl. Acad. Sci. USA, 82, 488 (1985), etc.

[0074] The position(s) of the amino acid(s) which is/are substituted in the amino acid sequence at the positions of 5 to 169 of SEQ ID NO: 2 are not particularly limited so long as it is other than at the positions of 11, 18, 27, 33, 43, 68, 72, 75, 90, 115, 124 and 166, and include one or more position(s) selected from the group consisting of the positions of 13, 14, 15, 25, 30, 36, 70, 83, 106, 128, 153, 156, 157, 159, 162, 163 and 169. In particular, the substitution positions can be at least (one or more) position(s) selected from the group consisting of the positions of 13, 14, 153, 159, 163 and 169.

[0075] Examples of amino acid residues which are mutually substitutable are given below, Amino acid residues in the same group are mutually substitutable.

[0076] Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, o-methylserine, t-butylglycine, t-butylalanine and cyclohexylalanine;

[0077] Group B: aspartic acid, glutamic acid, isoaspartic acid, isoglutamic acid, 2-arninoadipic acid and 2-aminosuberic acid;

[0078] Group C: asparagine and glutamine;

[0079] Group D: lysine, arginine, ornithine, 2,4-diaminobutanoic acid and 2,3-diaminopropionic acid;

[0080] Group E: proline, 3-hydroxyproline and 4-hydroxyproline;

[0081] Group F: serine, threonine and homoserine; and,

[0082] Group G: phenylalanine and tyrosine.

[0083] In (c) described above, the range of at least 90% in the amino acid sequence having at least 90% 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, or 99.9% or more. In general, the numerical value of the identity described above is more preferable as the number becomes larger. The identity of amino acid sequences or nucleotide sequences can be determined using sequencing programs such as BLAST (see, e.g., Altzchul, S. F. et al., J. Mol. Biol., 215, 403 (1990)). When BLAST is used, the default parameters for the respective programs are employed.

[0084] In (d) described above, the polynucleotide which hybridizes under stringent conditions is intended to mean a polynucleotide (e.g., DNA) which is obtained by, for example, colony hybridization, plaque hybridization or Southern hybridization using as a probe all or part of the polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence encoding the amino acid sequence at the positions of 5 to 169 of SEQ ID NO: 2. Specific examples include polynucleotides which can be identified by performing hybridization at 65 C. in the presence of 0.7 to 1.0 mol/L NaCl using a filter on which the polynucleotide from a colony or plaque is immobilized, then washing the filter at 65 C. with an SSC (saline-sodium citrate) solution having a concentration of 0.1 to 2 times (1SSC solution is composed of 150 mmol/L sodium chloride and 15 mmol/L sodium citrate).

[0085] Hybridization may be performed in accordance with modifications of the methods described in laboratory manuals, e.g., J. Sambrook, E. F. Fritsch & T. Maniatis (Ed.), Molecular Cloning, A Laboratory Manual (4th edition), Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2012), Ausbel F. M. et al., Current Protocols in Molecular Biology, Supplement 1-38, John Wiley and Sons (1987-1997), Glover D. M. and Hames B. D., DNA Cloning 1: Core Techniques, A practical Approach, Second Edition, Oxford University Press (1995), etc.

[0086] The stringent conditions may be any of low stringent conditions, moderate stringent conditions or high stringent conditions. The low-stringent conditions are, for example, conditions of 5SSC, 5Denhart's solution, 0.5% (w/v) SDS, 50% (v/v) formamide and 32 C. The moderate stringent conditions are, for example, conditions of 5SSC, 5Denhart's solution, 0.5% (w/v) SDS, 50% (v/v) formamide and 42 C. The high-stringent conditions are, for example, 5SSC, 5Denhart's solution, 0.5% (w/v) SDS, 50% (v/v) formamide and 50 C. The more stringent the conditions are, the higher the complementarity required for double-strand formation. Specifically, under these conditions, for example, a polynucleotide (e.g., DNA) of higher homology is expected to be obtained efficiently as the temperature becomes higher, although multiple factors are involved in hybridization stringency, including temperature, probe concentration, probe length, ionic strength, time, base concentration, etc. One skilled in the art may achieve a similar stringency by appropriately choosing these factors.

[0087] When commercially available kits are used for the hybridization, for example, Alkphos Direct Labeling Reagents (manufactured by GE Healthcare Inc.) can be used. In this case, according to the protocol attached, a membrane is incubated with a labeled probe overnight, the membrane is washed with a primary wash buffer containing 0.1% (w/v) SDS under conditions at 55 C. and then the hybridized DNA can be detected.

[0088] Other hybridizable polynucleotides include, as calculated by a sequencing program such as BLAST or the like using the default parameters, DNAs having the identity of approximately 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 88% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.3% or more, 99.5% or more, 99.7% or more, 99.8% or more, or 99.9% or more, to the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2. The identity of nucleotide sequences can be determined using the method described above.

[0089] In a preferred embodiment of the invention, the luciferase mutant is a luciferase mutant comprising the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10, more preferably, a luciferase mutant comprising the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8, and most preferably, a luciferase mutant comprising the amino acid sequence of SEQ ID NO: 4.

[0090] The luciferase mutant of the present invention may further contain an additional peptide sequence at the N terminus and/or C terminus, preferably at the N terminus. The additional peptide sequence is at least one peptide sequence selected from the group consisting of a peptide sequence for purification, a peptide sequence for expressing the luciferase mutant of the present invention as a soluble protein and an epitope sequence capable of recognizing an antibody. The additional peptide sequence is preferably a peptide sequence for purification. In another preferred embodiment of the invention, the additional peptide sequence is at least one sequence selected from the group consisting of a peptide sequence for purification and a peptide sequence for expressing the luciferase mutant of the present invention as a soluble protein.

[0091] Peptide sequences employed in the art may be used as the peptide sequence for purification. The peptide sequence for purification includes, for example, a histidine tag sequence with a consecutive amino acid sequence of at least 4 histidine residues and preferably at least 6 residues, an amino acid sequence with a binding domain of glutathione S-transferase into glutathione, the amino acid sequence of Protein A, etc.

[0092] The peptide used to express the luciferase mutant of the present invention as a soluble protein includes, for example, polypeptides represented by formula (Z).sub.n. The amino acid sequences for the polypeptides represented by formula (Z).sub.n and the nucleic acid sequences encoding the same are described in, e.g., JPA KOKAI No. 2008-99669.

[0093] Peptide sequences used in the art can be used as the epitope sequence capable of recognizing an antibody.

[0094] In some embodiments of the present invention, the additional peptide sequence in the luciferase mutant does not carry a secretory signal peptide sequence.

[0095] The secretory signal peptide sequence includes a secretory peptide sequence of Gaussia luciferase, or the like.

[0096] The method for acquiring the luciferase mutant of the invention is not particularly limited. The luciferase mutant of the invention may be a protein synthesized by chemical synthesis, or a recombinant protein produced by a genetic engineering technique. When the luciferase mutant of the invention is to be chemically synthesized, synthesis may be carried out by, for example, the Fmoc (fluorenylmethyloxycarbonyl) method or the tBoc (t-butyloxycarbonyl) method. In addition, peptide synthesizers available from Advanced ChemTech, PerkinElmer, Pharmacia, Protein Technology Instrument, Synthecell-Vega, PerSeptive, Shimadzu Corporation, etc. may also be used for chemical synthesis. When the luciferase mutant of the invention is to be produced by a genetic engineering technique, the mutant may be produced by a conventional genetic recombination technique. More specifically, the luciferase mutant of the invention may be produced by inserting a polynucleotide (e.g., a DNA) encoding the luciferase mutant of the invention into a suitable expression system. The polynucleotide encoding the luciferase mutant of the invention, expression of the luciferase mutant of the invention in an expression system or the like will be later described.

2. Polynucleotide of the Invention

[0097] The present invention also provides a polynucleotide comprising a polynucleotide encoding the luciferase mutant of the invention described above. The polynucleotide of the invention may be any polynucleotide so long as it has a nucleotide sequence encoding the luciferase mutant of the invention, although a DNA is preferred. Examples of the DNA include genomic DNA, genomic DNA library, cellular or tissue cDNA, cellular or tissue cDNA library; synthetic DNA, etc. Vectors used in the libraries are not particularly limited and may be any of bacteriophages, plasmids, cosmids, phagemids, etc. Also, these vectors may be amplified directly by a Reverse Transcription Polymerase Chain Reaction (hereinafter abbreviated as RT-PCR) using the total RNA or mRNA fraction prepared from the cell or tissue described above.

[0098] The polynucleotide of the invention includes the following polynucleotides (i) to (iv).

[0099] (i) A polynucleotide comprising a polynucleotide encoding the luciferase mutant described in (a) above;

[0100] (ii) A polynucleotide comprising a polynucleotide encoding the luciferase mutant described in (b) above;

[0101] (iii) A polynucleotide comprising a polynucleotide encoding the luciferase mutant described in (c) above; and,

[0102] (iv) A polynucleotide comprising a polynucleotide encoding the luciferase mutant described in (d) above.

[0103] A polynucleotide encoding a protein having a given amino acid sequence, in which one or more amino acids are substituted in the amino acid sequence, can be obtained by using a site-specific mutagenesis technique (see, e.g., Gotoh, T. et al., Gene 152, 271-275 (1995), Zoller, Mi., and Smith, M., Methods Enzymol. 100, 468-500 (1983), Kramer, W. et al., Nucleic Acids Res. 12, 9441-9456 (1984), Kramer W, and Fritz Hi., Methods. Enzymol, 154, 350-367 (1987), Kunkel, T. A., Proc. Natl. Acad. Sci. USA. 82, 488-492 (1985), Kunkel, Methods Enzymol. 85, 2763-2766 (1988); etc.), the methods using amber mutation (see, e.g., the gapped duplex method, Nucleic Acids Res., 12, 9441-9456 (1984), etc.), etc.

[0104] Alternatively; mutations may also be introduced into the polynucleotide by PCR (cf, e.g., Ho S. N. et al., Gene, 77, 51 (1989), etc.) using a pair of primers bearing on the respective 5 ends a sequence in which the targeted mutation (deletion, addition, substitution and/or insertion) has been introduced.

[0105] Also, a polynucleotide encoding a partial fragment of protein, which is one type of the deletion mutant, can be obtained using as the primers an oligonucleotide having a sequence which matches the nucleotide sequence at the 5 end of the region encoding the partial fragment to be produced in the polynucleotide encoding the target protein and an oligonucleotide having a sequence complementary to the nucleotide sequence at the 3 end thereof, and performing PCR in which the polynucleotide encoding the target protein is used as a template.

[0106] The polynucleotide of the present invention includes preferably a polynucleotide comprising a polynucleotide encoding the luciferase mutant comprising the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10, more preferably, a polynucleotide comprising a polynucleotide encoding the luciferase mutant comprising the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8, and most preferably, a polynucleotide comprising a polynucleotide encoding the luciferase mutant comprising the amino acid sequence of SEQ ID NO: 4.

[0107] The polynucleotide encoding the luciferase mutant comprising the amino acid sequence of SEQ ID NO: 2 includes a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1. The polynucleotide encoding the luciferase mutant comprising the amino acid sequence of SEQ ID NO: 4 includes a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 3. The polynucleotide encoding the luciferase mutant comprising the amino acid sequence of SEQ ID NO: 6 includes a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 5. The polynucleotide encoding the luciferase mutant comprising the amino acid sequence of SEQ ID NO: 8 includes a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 7. The polynucleotide encoding the luciferase mutant comprising the amino acid sequence of SEQ ID NO: 10 includes a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 9.

[0108] In some embodiments of the present invention, the polynucleotide is preferably a polynucleotide comprising a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ED NO: 9, more preferably, a polynucleotide comprising a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, and most preferably, a polynucleotide comprising a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3.

[0109] The polynucleotide of the present invention may further contain a polynucleotide encoding an additional peptide sequence at the 5 end and/or 3 end, preferably at the 5 end. The polynucleotide encoding the additional peptide sequence includes a polynucleotide encoding at least one peptide sequence selected from the group consisting of a peptide sequence for purification, a peptide sequence for expressing the luciferase mutant of the present invention as a soluble protein, an epitope sequence capable of recognizing an antibody, and the like.

[0110] Polynucleotides comprising nucleotide sequences encoding the peptide sequence for purification employed in the art can be used as the polynucleotide encoding the peptide sequence for purification. Examples of the peptide sequence for purification include those as described above.

[0111] The polynucleotide encoding the peptide sequence used to express the luciferase mutant of the present invention as a soluble protein includes, for example, polypeptides represented by formula (Z).sub.n. The amino acid sequences for the polypeptides represented by formula (Z).sub.n and the nucleic acid sequences encoding the same are those as described above.

[0112] Polynucleotides comprising nucleotide sequences encoding the epitope sequence capable of recognizing antibodies which are used in the art can be used as the polynucleotide encoding the antibody-recognizing epitope sequence.

[0113] In some embodiments of the present invention, a polynucleotide as the polynucleotide encoding the additional peptide sequence does not include polynucleotides encoding a secretory signal peptide sequence. The secretory signal peptide sequence includes those as described above, and the like.

3. Recombinant Vector and Transformant of the Invention

[0114] The present invention further provides recombinant vectors and transformants comprising the polynucleotides of the present invention described above.

Preparation of Recombinant Vector

[0115] The recombinant vector of the invention can be obtained by ligating (inserting) the polynucleotide (DNA) of the invention to (into) an appropriate vector. More specifically, the recombinant vector can be obtained by digesting the purified polynucleotide (DNA) with a suitable restriction enzyme, then inserting into a suitable vector at the restriction enzyme site or multiple-cloning site, and ligating to the vector. The vector for inserting the polynucleotide of the invention is not particularly limited as long as it is replicable in a host, and includes plasmids, bacteriophages, animal viruses, etc. Examples of plasmids include plasmids from E. coli (e.g., pBR322, pBR325, pUC118, pUC119, etc.), plasmids from Bacillus subtilis (e.g., pUB110, pTP5, etc.) and plasmids from yeast (e.g., YEp13, YEp24, YCp50, etc.). Examples of bacteriophages include, e.g., phage. Examples of animal viruses include retroviruses, vaccinia viruses and insect viruses (e.g., baculoviruses). In addition, a pCold I vector, a pCold II vector, a pCold III vector and a pCold IV vector (all manufactured by Takara Bio Inc.), a pcDNA3 vector, a PICZa vector (manufactured by Invitrogen Inc.) and the like may also be suitably used.

[0116] The polynucleotide of the present invention is generally ligated in an expressible manner downstream of a promoter in a suitable vector. When the host used for transformation is an animal cell, the promoter is preferably an SV40-derived promoter, retrovirus promoter, metallothionein promoter, heat shock promoter, cytomegalovirus promoter, SRa promoter, and so on. When the host is a bacterium of the genus Escherichia, Trp promoter, T7 promoter, lac promoter, recA promoter, PL promoter, 1pp promoter, etc. are preferred. When the host is a bacterium of the genus Bacillus, SPO1 promoter, SPO2 promoter, penP promoter, etc. are preferred. When the host is yeast, PHO5 promoter, PGK promoter, GAP promoter, ADH 1 promoter, GAL promoter, etc. are preferred. When the host is an insect cell, polyhedrin promoter, P10 promoter, etc. are preferred.

[0117] A low-temperature expression-inducible promoter may also be suitably used. Examples of the low-temperature expression-inducible promoter include promoter sequences for cold shock genes. The cold shock gene includes, for example, E. coli cold shock genes (e.g., cspA, cspB, cspG, cspl and csdA), Bacillus caldolyticus cold shock genes (e.g., Bc-Csp), Salmonella enterica cold shock genes (e.g., cspE) and Erwinia carotovora cold shock genes (e.g., cspG). Among others, cspA promoter, cspB promoter, cspG promoter, cspi promoter, csdA promoter and the like can be advantageously used as the low-temperature expression-inducible promoter.

[0118] In addition to the foregoing, the recombinant vector of the invention may further contain, if desired, an enhancer, a splicing signal, a polyA addition signal, a ribosome binding sequence (SD sequence), a selection marker, etc., and provided for use. The selection marker includes, for example, a dihydrofolate reductase gene, an ampicillin resistance gene, a neomycin resistance gene, etc.

Preparation of Transformant

[0119] The thus obtained recombinant vector comprising the polynucleotide of the invention introduced into an appropriate host to prepare the transformant. The host is not particularly limited as long as it is capable of expressing the polynucleotide (DNA) of the invention, and may be bacteria of the genera Escherichia, Bacillus, Pseudomonas and Rhizobium, yeast, animal cells or insect cells, etc. Bacteria of the genus Escherichia include Escherichia coli, etc. Bacteria of the genus Bacillus include Bacillus subtilis, etc. Bacteria of the genus Pseudomonas include Pseudomonas putida, etc. Bacteria of the genus Rhizobium include Rhizobium meliloti, etc. Yeast includes Saccharomyces cerevisiae, Schizosaccharomyces pombe, etc. Animal cells include primary cell cultures, iPS cells, cultured cell lines (CHO cells, HEK293 cells, HL-60 cells, HeLa cells, MDCK cells, NIH3T3cells. PC12 cells), etc. Insect cells include Sf9, Sf21, etc.

[0120] The method of transfecting the recombinant vector into the host and the method of transformation by the same can be performed according to various general methods. The method for transfecting the recombinant vector into the host cell includes the calcium phosphate method (Virology, 52, 456-457 (1973)), the lipofection method (Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)), the electroporation method (EMBO J., 1, 841-845 (1982)), etc. The method for transformation of the bacteria of the genus Escherichia includes the methods described in, e.g., Proc. Natl. Acad. Sci. USA, 69, 2110 (1972), Gene, 17, 107 (1982), etc. The method for transformation of the bacteria of the genus Bacillus includes the method described in Molecular & General Genetics, 168, 111 (1979), etc. The method for transforming yeast includes the method described in Proc. Natl. Acad. Sci. USA, 75, 1929 (1978), etc. The method for transformation of animal cells includes the method described in Virology, 52, 456 (1973), etc. The method for transformation of insect cells includes the method described in Bio/Technology, 6, 47-55 (1988), etc. Thus, the transformant transformed with the recombinant vector comprising the polynucleotide encoding the luciferase mutant of the invention (the polynucleotide of the invention) can be obtained.

[0121] Expression Vector and Transformant Comprising Low-Temperature Expression-Inducible Promoter Sequence

[0122] An expression vector comprising the low-temperature expression-inducible promoter sequence is preferred as the expression vector among others.

[0123] Specifically, the expression vector comprising the low-temperature expression-inducible promoter sequence is intended to mean an expression vector comprising the following promoter sequence and coding sequence:

(1) a low-temperature expression-inducible promoter sequence; and,
(2) a coding sequence comprising the polynucleotide of the invention.

[0124] The low-temperature expression-inducible promoter sequence is intended to mean a promoter sequence which is capable of inducing expression of the protein by lowering the temperature from the culture conditions under which host cells can grow. Examples of the low-temperature expression-inducible promoter are promoters for genes encoding cold shock proteins (cold shock genes). Examples of the cold shock gene promoters include those as described above.

[0125] The temperature at which the low-temperature expression-inducible promoter used in the invention is capable of inducing expression is generally 30 C. or less, preferably 25 C. or less, more preferably 20 C. or less, and most preferably 15 C. or less. In order to induce the expression more efficiently, however, the expression induction is generally performed at 5 C. or more, preferably at 10 C. or more, and most preferably at approximately 15 C.

[0126] In preparing the expression vector of the invention comprising the low-temperature expression-inducible promoter sequence, the pCold I vector, pCold II vector, pCold III vector and pCold IV vector (all manufactured by Takara Bio Inc.) can be suitably used as the vector for insertion of the polynucleotide of the invention. The protein can be produced as a soluble protein in the cytoplasm in a host cell when expression is performed in a prokaryotic host cell using these vectors.

[0127] Prokaryotic cells are preferred as the host into which the expression vector comprising the low-temperature expression-inducible promoter sequence is introduced, more preferably E. coli, and particularly preferably, the BL21 and JM109 strains. Among others, the BL21 strain is most preferred. The BL21 and JM109 strains are available from, e.g., Novagen.

[0128] Temperatures for incubation at which the transformant carrying the expression vector comprising the low-temperature expression-inducible promoter sequence grows are generally 25 to 40 C. and preferably 30 to 37 C. Temperatures for inducing the expression are generally 4 to 25 C., preferably 10 to 20 C., more preferably 12 to 18 C., and most preferably 15 C.

4. Production of Luciferase Mutant of the Invention

[0129] The present invention further provides a method for producing the luciferase mutant of the invention, which comprises the steps of culturing the transformant described above to produce the luciferase mutant of the invention. The luciferase mutant of the invention can be produced, for example, by culturing the transformant described above under conditions where the polynucleotide (DNA) encoding the luciferase mutant of the invention can be expressed, producing/accumulating and then separating/purifying the luciferase mutant of the invention.

Incubation of Transformant

[0130] The transformant of the invention can be incubated in a conventional manner used for incubation of a host. By the incubation, the luciferase mutant of the invention is produced by the transformant and accumulated within the transformant or in the culture medium.

[0131] The medium used for culturing the transformant using bacteria of the genus Escherichia or the genus Bacillus as a host may be any of a natural medium and a synthetic medium as far as it is a medium which contains carbon sources, nitrogen sources, inorganic salts, etc. necessary for growth of the transformant, and in which the transformant can efficiently grow. Examples of carbon sources which can be used are carbohydrates such as glucose, fructose, sucrose, starch, etc.; organic acids such as acetic acid, propionic acid, etc.; alcohols such as ethanol, propanol, and the like. Examples of nitrogen sources which can be used include ammonia, ammonium salts of inorganic or organic acids such as ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate, etc., and other nitrogen-containing compounds, and further include peptone, meat extracts, corn steep liquor, and the like. Examples of inorganic salts include monobasic potassium phosphate, dibasic potassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, etc. If necessary, antibiotics such as ampicillin or tetracycline can be added to the medium during incubation. Where the transformant transformed by the expression vector using an inducible promoter as the promoter is cultured, an inducer may also be added to the medium, if necessary. For example, when the transformant transformed by an expression vector using a Lac promoter is cultured, isopropyl--D-thiogalactopyranoside (IPTG), etc. may be added to the medium and indoleacrylic acid (IAA), etc. may be added to the medium in culturing the transformant transformed by an expression vector using a tip promoter.

[0132] When the host is bacteria of the genus Escherichia, incubation is performed generally at approximately 15 to 43 C. for approximately 3 to 24 hours. If necessary, aeration and agitation may be applied. When the host is bacteria of the genus Bacillus, incubation is performed generally at approximately 30 to 40 C. for approximately 6 to 24 hours. If necessary, aeration and agitation may be applied.

[0133] Media for incubation of the transformant when the host is yeast include Burkholder's minimal medium (Proc. Natl. Acad. Sci. USA, 77, 4505 (1980)) and an SD medium containing 0.5% (w/v) Casamino acids (Proc. Natl. Acad, Sci. USA, 81, 5330 (1984)). Preferably, the pH of the medium is adjusted to approximately 5 to 8. Incubation is performed generally at approximately 20 to 35 C. for approximately 24 to 72 hours. If necessary, aeration and agitation may be applied.

[0134] Media for culturing the transformant when the host is an animal cell include MEM medium supplemented with approximately 5 to 20% (v/v) fetal calf serum (Science, 122, 501 (1952)), DMEM medium (Virology, 8, 396 (1959)), etc. Preferably, the pH of the medium is adjusted to approximately 6 to 8. Incubation is performed generally at approximately 30 to 40 C. for approximately 15 to 60 hours. If necessary, aeration and agitation may be applied.

[0135] Media for culturing the transformant when the host is an insect cell include Grace's insect medium (Nature, 195, 788 (1962)) to which additives such as 10% (v/v) immobilized bovine serum are suitably added. Preferably, the pH of the medium is adjusted to approximately 6.2 to 6.4. Incubation is performed generally at approximately 27 C. for approximately 3 to 5 days. If necessary, aeration and agitation may be applied.

[0136] Temperatures for incubation at which the transformant transformed by the expression vector comprising the low-temperature expression-inducible promoter sequence and temperatures for expression induction are as described above.

Separation/Purification of Luciferase Mutant of the Invention

[0137] The luciferase mutant of the invention can be obtained by separating/purifying the luciferase mutant of the invention from the culture described above. As used herein, the culture is intended to mean any one of a culture broth, cultured cells or cultured bacteria and a cell lysate of the cultured cells or cultured bacteria. The luciferase mutant of the invention can be separated/purified in a conventional manner.

[0138] Specifically, when the luciferase mutant of the invention accumulates in the cultured bacteria or cultured cells, after completion of the incubation the bacteria or cells are disrupted in a conventional manner (e.g., ultrasonication, lysozyme, freezing and thawing, etc.,) and then a crude extract of the luciferase mutant of the invention can be obtained in a conventional manner (e.g., centrifugation, filtration, etc.). When the luciferase mutant of the invention accumulates in the periplasmic space, after completion of the incubation the extract containing the luciferase mutant of the invention can be obtained in a conventional manner (e.g., the osmotic shock method, etc.). When the luciferase mutant of the invention accumulates in the culture broth, after completion of the incubation the culture supernatant containing the luciferase mutant of the invention can be obtained by separating the bacteria or cells and the culture supernatant in a conventional manner (e.g., centrifugation, filtration, etc.).

[0139] The luciferase mutant of the invention contained in the extract or culture supernatant thus obtained can be purified by conventional methods of separation and purification. Examples of these methods for separation and purification which may be used include ammonium sulfate precipitation, gel filtration chromatography, ion-exchange chromatography, affinity chromatography, reversed-phase high-performance liquid chromatography, dialysis, ultrafiltration, etc., alone or in a suitable combination thereof. When the luciferase mutant of the invention contains the peptide sequence for purification described above, it is preferred to perform purification using the same, Specifically, when the luciferase mutant of the invention contains a histidine tag sequence, nickel chelate affinity chromatography may be used; when the luciferase mutant of the invention contains the binding domain of S-transferase to glutathione, affinity chromatography with a glutathione-binding gel may be used; when the luciferase mutant of the invention contains the amino acid sequence of Protein A, antibody affinity chromatography may be used.

5. Use of Luciferase Mutant of the Invention

[0140] The luciferase mutant of the invention can be used as a detection marker which emits luminescence in the presence of a luciferin (hereinafter detection marker of the present invention). The detection marker of the present invention can be utilized for detection of the target substance in, e.g., an immunoassay, a hybridization assay, etc.

[0141] The luciferase mutant of the invention can be expressed, e.g., as a fusion protein with a target protein, and introduced into cells by means of the microinjection method, etc., and the resulting product can be used to determine distribution of the target protein described above. The distribution of such a target protein or the like can be determined by using detection methods such as luminescence imaging. In addition to the introduction into cells by means of the microinjection method or the like, the luciferase mutant of the invention can be expressed in cells to provide for use. The luminescence substrate (luciferin) used is preferably coelenterazines, and particularly preferably, his-coelenterazine or 6h-f-coelenterazine, as described above, Bis-coelenterazine or 6h-f-coelenterazine displays the luminescence activity approximately 10 times higher than that of coelenterazine and emits glow.

Use as Reporter Protein

[0142] The luciferase mutant of the invention may also be used as a reporter protein to assay the transcription activity of promoters, etc. In this case, the polynucleotide of the invention is used as a reporter gene and the luciferase mutant encoded by the reporter gene is contacted with luciferin. As used herein, the term contact is intended to mean that the luciferase mutant of the invention and a luciferin are allowed to be present in the same reaction system or culture system, which includes, for example, addition of a luciferin to a culture container charged with cells expressing the luciferase mutant of the invention, mixing the cells with a luciferin, and incubation of the cells in the presence of a luciferin. The polynucleotide encoding the luciferase mutant of the invention (i.e., the polynucleotide of the invention) is fused to a target promoter or some other expression control sequence (e.g., an enhancer, etc.) to construct a vector. By introducing the vector into a host cell and detecting the luminescence from the luciferase mutant of the invention in the presence of a luciferin (luminescence substrate), the activity of the target promoter or some other expression control sequence can be assayed. Furthermore, the expressed luciferase mutant is reacted with coelenterazines and the luminescence generated may also be visualized in pictures by using a high-sensitive detector.

[0143] The luciferin used is preferably coelenterazines, and particularly preferably, bis-coelenterazine or 6h-f-coelenterazine, as described above. Bis-coelenterazine or 6h-f-coelenterazine displays the luminescence activity approximately 10 times higher than that of coelenterazine and emits glow.

[0144] The cells used are preferably animal cells. In the case of animal cells, the luciferase mutant in a preferred embodiment of the invention is secretion outside of cells.

[0145] The polynucleotide of the invention can be used as a reporter gene in such a manner as described above.

Method for Visualizing Luminescence Reaction

[0146] The luciferase mutant of the present invention can be used in the method for visualizing luminescence activities. By visualizing luminescence activities, for example, the pattern of secretion can be observed when the luciferase mutant of the present invention is secreted extracellularly. The luciferase mutant of the invention is secreted extracellularly without passing through the trans-Golgi network, as will be demonstrated in EXAMPLES described below.

[0147] Specifically, the method for visualizing a luminescence reaction comprises contacting the luciferase mutant of the invention with a luciferin. As used herein, the term contact is intended to mean that the luciferase mutant of the invention and a luciferin are allowed to be present in the same reaction system or culture system, which includes, for example, addition of a luciferin to a culture container charged with cells expressing the luciferase mutant of the invention, mixing the cells with a luciferin and incubation of the cells in the presence of a luciferin. A vector comprising the polynucleotide encoding the luciferase mutant of the invention (i.e., the polynucleotide of the invention) is constructed. By introducing the vector into a host cell and detecting the luminescence from the luciferase mutant of the invention in the presence of a luciferin (luminescence substrate), the luminescence reaction can be visualized. In this case, the luminescence is detected by using, e.g., a high-sensitive detector.

[0148] The luciferin used is preferably coelenterazines, and particularly preferably, h-coelenterazine orf-coelenterazine, as described above. H-coelenterazine or f-coelenterazine displays the luminescence activity 10 times higher than that of coelenterazine and its light decays fast.

[0149] The cells used are preferably animal cells. Even in animal cells, the luciferase mutant in a preferred embodiment of the invention is secreted extracellularly,

[0150] The polynucleotide of the invention can be used in the method for visualizing a luminescence reaction in such a manner as described above.

Material for Amusement Supplies

[0151] The luciferase mutant of the invention has the activity of catalyzing the reaction where a luciferin is oxidized with oxygen molecules to form oxyluciferin in its excited state. The oxyluciferin in the excited state emits visible light to decay to the ground state. Accordingly, the luciferase mutant of the invention can be used preferably as a luminescent material for amusement supplies. Examples of such amusement supplies are luminescent soap bubbles, luminescent ice bars, luminescent candies, luminescent color paints, etc. These amusement supplies of the invention can be prepared in a conventional manner.

[0152] The luciferin used is preferably coelenterazines, and particularly preferably, bis-coelenterazine or 6h-f-coelenterazine, as described above. Bis-coelenterazine or 6h-f-coelenterazine displays the luminescence activity approximately 10 times higher than that of coelenterazine and emits glow.

Bioluminescence Resonance Energy Transfer (BRET) Method

[0153] By utilizing the principle of interaction between molecules by the bioluminescence resonance energy transfer (BRET) method, the luciferase mutant of the invention is available for analytical methods such as analysis of physiological functions, assay of enzyme activities, etc.

[0154] For instance, when the luciferase mutant of the invention of the invention is used as a donor and the fluorescent substance (e.g., an organic compound, a fluorescent protein, etc.) is used as an acceptor, the interactions between the donor and acceptor above can be detected by inducing bioluminescence resonance energy transfer (BRET) between them.

[0155] In an embodiment of the present invention, the organic compound used as an acceptor includes Hoechist3342, indo-1, DAPI, etc. In another embodiment of the present invention, the fluorescent protein used as an acceptor includes a green fluorescent protein (GFP), a blue fluorescent protein (BFP), a muted GFP fluorescent protein, phycobilin, etc. These organic compounds and fluorescent proteins are commercially available.

[0156] In a preferred embodiment of the present invention, the physiological functions to be analyzed include an orphan receptor (especially, a G protein-coupled receptor), apoptosis, transcription regulation by gene expression, etc. In a further preferred embodiment of the present invention, the enzyme to be analyzed is protease, esterase, kinase, or the like.

[0157] Analysis of the physiological functions by the BRET method can be performed by known methods, for example, by modifications of the method described in Biochem. J. 2005, 385, 625-637 or Expert Opin. Ther Tarets, 2007 11: 541-556. Enzyme activities may also be assayed by known methods, for example, by modifications of the method described in Nature Methods 2006, 3:165-174 or Biotechnol. J. 2008, 3:311-324.

[0158] The luminescence substrate (luciferin) used is preferably coelenterazines, and particularly preferably, his-coelenterazine or 6h-f-coelenterazine, as described above. Bis-coelenterazine or 6h-f-coelenterazine displays the luminescence activity approximately 10 times higher than that of coelenterazine and emits glow.

6. Kit of the Invention

[0159] The present invention also provides a kit comprising any one selected from the luciferase mutant of the invention, the polynucleotide of the invention, the recombinant vector of the invention and the transformant of the invention. The kit of the invention may further contain a luciferin.

[0160] The luciferin is preferably coelenterazines, as described above. In some embodiments of the present invention, bis-coelenterazine or 6h-f-coelenterazine is particularly preferred. In some other embodiments of the present invention, h-coelenterazine or f-coelenterazine is particularly preferred in some embodiments of the present invention.

[0161] The kit of the present invention may be prepared with conventional materials by conventional methods. The kit of the present invention may further contain, e.g., sample tubes, plates, instructions for the kit user, solutions, buffers, reagents, and samples suitable for standardization or control samples. The kit of the present invention may further contain salts including halide ions.

[0162] The kit of the present invention can be used for the aforesaid measurement using a reporter protein or a reporter gene, the detection marker with luminescence, or the analysis of physiological functions or measurement of enzyme activities by the BRET method. The kit can also be used for the method for luminescence reaction as described below.

7. Method for Luminescence Reaction

Luminescence Activity

[0163] The luciferase mutant of the invention has the ability of catalyzing the reaction which involves oxidization of a luciferin with oxygen molecules to form an oxyluciferin in its excited state. The oxyluciferin in the excited state emits light on returning to the ground state. That is, the luciferase mutant of the invention catalyzes the luminescence reaction in which a luciferin serves as a substrate to cause luminescence. This activity is sometimes referred to as the luminescence activity in the specification.

Luminescence Reaction

[0164] The luminescence reaction using the luciferase mutant of the invention in which a luciferin serves as a substrate can be performed by contacting the luciferase mutant of the invention with the luciferin. As used herein, the term contact is intended to mean that the luciferase mutant of the invention and a luciferin are allowed to be present in the same reaction system, which includes, for example, addition of the luciferase mutant of the invention to a container charged with a luciferin, addition of a luciferin to a container charged with the luciferase mutant of the invention and mixing the luciferase mutant of the invention with a luciferin. The reaction can be carried out under conditions conventionally used for the luminescence reaction using Oplophorus luciferase or under conditions modified therefrom.

[0165] Specifically, solvents for the reaction which are employed are, for example, a buffer solution such as Tris-HCl buffer, sodium phosphate buffer, etc., water, and the like.

[0166] Temperatures for the reaction are generally approximately 4 C. to 40 C. and preferably approximately 4 C. to 25 C.

[0167] In the reaction solution, pH is generally approximately 5 to 10, preferably approximately 6 to 9, more preferably approximately 7 to 8 and most preferably approximately 7.5.

[0168] The luciferin is preferably coelenterazines, as described above. In some embodiments of the present invention, bis-coelenterazine or 6h-f-coelenterazine is particularly preferred. In some other embodiments of the present invention, h-coelenterazine or Lcoelenterazine is particularly preferred.

[0169] The luciferin may also be added to the reaction system in the form of a solution in a polar solvent such as dimethylformamide, dimethylsulfoxide, etc., or in an alcohol such as methanol, ethanol, butanol, etc.

Activation of Luminescence Activity

[0170] The luminescence activity of the luciferase mutant of the invention can be activated by halide ions, nonionic surfactants, etc.

[0171] Examples of the halide ions are fluorine ions, chlorine ions, bromine ions and iodine ions; preferred are chlorine ions, bromine ions and iodine ions.

[0172] The concentration of halide ions is generally approximately 10 M to 100 mM, preferably approximately 100 M to 50 mM and particularly preferably approximately 1 mM to 20 mM.

[0173] The addition of the halide ions to the reaction system is performed by a method which comprises adding them in a salt form. The salts used are alkali metal salts such as sodium salts, potassium salts, etc.; alkaline earth metal salts such as calcium salts, magiesium salts, barium salts, etc. More specific examples are NaF, NaCl, NaBr, NaI, KF, KCl, KBr, KI, CaF.sub.2, CaCl.sub.2, CaBr.sub.2, CaI.sub.2, MgF.sub.2, MgCl.sub.2, MgBr.sub.2, MgI.sub.2, etc.

[0174] Examples of nonionic surfactants which are commercially available (trade name) include Tween 20 (polyoxyethylene sorbitan monolaurate), Tween 80 (polyoxyethylene sorbitan monooleate), Triton X-100 (polyethylene glycol-p-isooctylphenyl ether), Briji-58 (polyoxyethylene (20) cetyl ether), Nonidet P-40 (ethylphenolpoly(ethylene glycol ether) n), etc., and preferably, Tween 20, Triton X-100, etc. These surfactants are commercially available from, e.g., Wako Pure Chemical Industries, Ltd., Tokyo Chemical Industry Co., Ltd. and Sigma Aldrich.

[0175] Concentration of the nonionic surfactant is generally approximately 0.0002% (w/v) to 0.2% (w/v), preferably, approximately 0.001% (w/v) to 0.1% (w/v), and particularly preferably, approximately 0.05% (w/v) to 0.02% (w/v).

[0176] Regardless of their purposes, all of the documents and publications described in the specification are incorporated herein by reference, each in its respective entirety.

[0177] Unless otherwise indicated with respect to the embodiments and working examples, the methods described in standard sets of protocols such as J. Sambrook, E. F. Fritsch & T. Maniatis (Ed.), Molecular cloning, a laboratory manual (4th edition), Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2012); F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, K. Struhl (Ed.), Current Protocols in Molecular Biology, John Wiley & Sons Ltd., etc. or modifications or variations thereof are used. When commercially available reagent kits or measuring apparatuses are used, protocols attached to them are used unless otherwise indicated.

[0178] The objects, characteristics and advantages of the present invention as well as the idea thereof are apparent to those skilled in the art from the descriptions given herein. Based on the description given herein, those skilled in the art can easily reproduce the present invention.

[0179] It can be understood that the embodiments of the invention, specific working examples, etc. are disclosed as preferred embodiments of the present invention. These descriptions are only for illustrative and explanatory purposes and are not intended to restrict the invention thereto. It is further apparent to those skilled in the art that various modifications may be made based on the descriptions given herein within the intent and scope of the present invention disclosed herein.

EXAMPLES

[0180] Hereinafter, the present invention will be described with reference to specific examples but is not deemed to be limited thereto.

Example 1: Preparation of Mutated 19kOLase Gene (dnKAZ) with 16 Mutations

[0181] Gene amplification was perfoimed by PCR using a template of pCold-ZZ-P-nanoKAZ described in Inouye et al (2013) Biochem. Biophys. Res. Commun. 437: 23-28 and the following primers.

TABLE-US-00001 PrimernanoKAZ-1N/EcoRI(SEQIDNO:19:5 gcgGAATTCTTCACCCTGGAGGACTTCGTCGGC3:EcoRI sequenceunderlined) PrimernanoKAZ-3C/XbaI(SEQIDNO:20:5 gccTCTAGATTAGGCCAGGATTCTCTCGCACAGTCT3:XbaI sequenceunderlined)

[0182] Herein, the nucleotide sequence and amino acid sequence of pCold-ZZ-P-nanoKAZ are shown by SEQ ID NO: 11 and SEQ ID NO: 12, respectively.

Example 2: Secretory Expression Vectors for dnKAZ Using the Secretory Signal Peptide Sequence of Gaussia Luciferase

[0183] The expression vector for dnKAZ was constructed as follows. Firstly, a novel expression vector pcDNA3-GLsp in animal cultured cells was constructed. Specifically, the secretory signal peptide sequence of Gaussia luciferase was obtained from pcDNA3-GLuc vector (manufactured by Prolume Ltd.) by PCR using the following primers.

TABLE-US-00002 PrimerGLsp-1R/EcoRI(SEQIDNO:21:5 ggcGAA TTCGGTGGGCTTGGCCTCGGCCAC3,EcoRI sequenceunderlined) T7primer(SEQIDNO:22:5 TAATACG ACTCACTATAGGG3)

[0184] After digestion with HindIII/EcoRI, the resultant fragment was inserted into the HindIII-EcoRI site of pcDNA3 vector (manufactured by Invitrogen Inc.) to construct a novel expression vector pcDNA3-GLsp. That is, the novel expression vector is under the control of the CMV promoter, followed by the Kozak sequence, the secretory signal pepetide sequence of Gaussia luciferase and a multiple-cloning site sequence.

[0185] Next, the expression vector for dnKAZ was constructed as follows, using the novel expression vector pcDNA3-GLsp. The DNA fragment obtained in EXAMPLE 1 was digested with the restriction enzymes of EcoRI/XbaI in a conventional manner and then ligated to the EcoRI-Xbat site of pcDNA3-GLsp to construct the expression vector pcDNA3-GLsp-dnKAZ. The gene sequence inserted was confirmed by sequencing using a DNA Sequencer (manufactured by ABI Inc.)

Example 3: Preparation of nanoKAZ Gene Fragments with the Deletion of Amino Acid Sequences at the Amino Terminal Region

[0186] The gene fragments of nanoKAZ with the deletion of the amino acids at the amino terminal region were prepared in the following manner.

[0187] PCR (cycle conditions: 25 cycles of 1 min/94 C., 1 min/50 C. and 1 min/72 C.) was performed with a PCR kit (manufactured by Takara Bio Inc.) using pcDNA3-GLsp-dnKAZ obtained in EXAMPLE 2 as a temperate with two PCR primers.

[0188] In preparing, e.g., one amino acid-deleted N2T-nKAZ mutant gene, PCR was performed using a pcDNA3-GLsp-dnKAZ as a template and the following two primers.

TABLE-US-00003 (SEQIDNO:23) D2-nKAZ-15N/EcoRI(5gccGAATTCAAGCTTGGTACCAC CATGGTCACCCTGGAGGACTTCGTCGGCGAC3:EcoRI sequenceandKpnIsequenceunderlined) (SEQIDNO:20) nanoKAZ-3C/XbaI(5gccTCTAGATTAGGCCAGGATTCTCTCGC ACAGTCT3)

[0189] As a result, the gene (N2T-nKAZ) encoding the amino acid sequence of nanoKAZ (SEQ ID NO: 2) with the deletion of the phenylalanine residue at the first amino acid was amplified. The nucleotide sequence of nanoKAZ gene is shown by SEQ ID NO: 1. The nucleotide sequence and amino acid sequence of N2T-nKAZ are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

[0190] The nanoKAZ genes deleted at the amino-terminal regions were obtained in a similar manner except for using the templates and primers described in TABLE 2. The amino-terminal sequences of the amino-terminal deleted nanoKAZ genes obtained are shown in TABLE 1.

[0191] Herein, the nucleotide sequence and amino acid sequence of N3L-nKAZ are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively. The nucleotide sequence and amino acid sequence of N4E-nKAZ are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively. The nucleotide sequence and amino acid sequence of N5D-nKAZ are shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively.

TABLE-US-00004 TABLE1 Aminoacidsequenceofamino-terminaldeleted nanoKAZgene Number of deleted Deletion amino mutant acids Amino-terminalsequence nanoKAZ 0 FTLEDFVGDWRQTAGYNLDQVLEQG... N2T-nKAZ 1 TLEDFVGDWRQTAGYNLDQVLEQG... N3L-nKAZ 2 LEDFVGDWRQTAGYNLDQVLEQG... N4E-nKAZ 3 EDFVGDWRQTAGYNLDQVLEQG... N5D-nKAZ 4 DFVGDWRQTAGYNLDQVLEQG... N6F-nKAZ 5 FVGDWRQTAGYNLDQVLEQG... N7V-nKAZ 6 VGDWRQTAGYNLDQVLEQG... N8G-nKAZ 7 GDWRQTAGYNLDQVLEQG... N9D-nKAZ 8 DWRQTAGYNLDQVLEQG... N10W-nKAZ 9 WRQTAGYNLDQVLEQG... N15G-nKAZ 14 GYNLDQVLEQG... N20Q-nKAZ 19 QVLEQG...

TABLE-US-00005 TABLE2 TemplatesandPCRprimersusedfordeletionoftheamino-terminal sequenceofnanoKAZ. Deletion mutant Template Primer Sequence N2T-nKAZ pcDNA3-GLsp- a D9nanoKAZ-15N/ 5 gccGAATTCAAGCTTGGTACCACCATGGTCACCCTGGAGGACTTCGTCGG dnKAZ EcoRI CGAC3(SEQIDNO:23) b nanoKAZ-3C/XbaI 5 gccTCTAGATTAGGCCAGGATTCTCTCGCACAGTCT3(SEQID NO:20) N3L-nKAZ pcDNA3-GLsp- a D3-nKAZ-16N/ 5 gccGAATTCAAGCTTGGTACCACCATGGTCCTGGAGG dnKAZ ECoRI ACTTCGTCGGCGACTGG3(SEQIDNO:24) b nanoKAZ-3C/XbaI 5 gccTCTATTAGGCCAGGATTCTCTCGCACAGTCT3(SEQID NO:20) N4E-nKAZ pcDNA3-GLsp- a D4-nKAZ-17N/ 5 gccGAATTCAAGCTTGGTACCACCATGGTCGAGG dnKAZ ECoRI ACTTCGTCGGCGACTGGAGA3(SEQIDNO:25) b nanoKAZ-3C/XbaI 5 gccTCTAGATTAGGCCAGGATTCTCTCGCACAGTCT3(SEQID NO:20) N5D-nKAZ pcDNA3-GLsp- a D5nanoKAZ-4N/ 5 gccGAATTCAAGCTTGGTACCACCATGGTCGACTTCGTCGG dnKAZ EcoRI CGACTGGAGACAGA3(SEQIDNO:26) b nanoKAZ-3C/XbaI 5 gccTCTAGATTAGGCCAGGATTCTCTCGCACAGTCT3(SEQID NO:20) N6F-nKAZ pcDNA3-GLsp- a D6nanoKAZ-8N/ 5 gccGAATTCAAGCTTGGTACCACCATGGTCTTCGTCGG dnKAZ EcoRI CGACTGGAGACAGACC3(SEQIDNO:27) b nanoKAZ-3C/XbaI 5 gccTCTAGATTAGGCCAGGATTCTCTCGCACAGTCT3(SEQID NO:20) N7V-nKAZ pcDNA3-GLsp- a D7nanoKAZ-9N/ 5 gccGAATTCAAGCTTGGTACCACCATGGTCGTCGG dnKAZ EcoRI CGACTGGAGACAGACCGCC3(SEQIDNO:28) b nanoKAZ-3C/XbaI 5 gccTCTAGATTAGGCCAGGATTCTCTCGCACAGTCT3(SEQID NO:20) N8G-nKAZ pcDNA3-GLsp- a D8nanoKAZ-10N/ 5 gccGAATTCAAGCTTGGTACCACCATGGTCGG dnKAZ EcoRI CGACTGGAGACAGACCGCCGGC3(SEQIDNO:29) b nanoKAZ-3C/XbaI 5 gccTCTAGATTAGGCCAGGATTCTCTCGCACAGTCT3(SEQID NO:20) N9D-nKAZ pcDNA3-GLsp- a D9nanoKAZ-11N/ 5 gccGAATTCAAGCTTGGTACCAC dnKAZ EcoRI CATGGTCGACTGGAGACAGACCGCCGGCTAC3(SEQIDNO:30) b nanoKAZ-3C/XbaI 5 gccTCTAGATTAGGCCAGGATTCTCTCGCACAGTCT3(SEQID NO:20) N10W-nKAZ pcDNA3-GLsp- a D10nanoKAZ-5N/ 5 gccGAATTCAAGCTTGGTACCAC dnKAZ EcoRI CATGGTCTGGAGACAGACCGCCGGCTACAACC3(SEQIDNO:31) b nanoKAZ-3C/XbaI 5 gccTCTAGATTAGGCCAGGATTCTCTCGCACAGTCT3(SEQID NO:20) N15G-nKAZ pcDNA3-GLsp- a D15nanoKAZ-6N/ 5 gccGAATTCAAGCTTGGTACCACCATGGTCGGCTACAACCT dnKAZ EcoRI GGACCAGGTCCTGG3(SEQIDNO:32) b nanoKAZ-3C/XbaI 5 gccTCTAGATTAGGCCAGGATTCTCTCGCACAGTCT3(SEQID NO:20) N20Q-nKAZ pcDNA3-GLsp- a D20nanoKAZ-7N/ 5 gccGAATTCAAGCTTGGTACCACCATGGTCCAGGTC dnKAZ EcoRI CTGGAGCAGGGCGGCGTCA3(SEQIDNO:33) b nanoKAZ-3C/XbaI 5 gccTCTAGATTAGGCCAGGATTCTCTCGCACAGTCT3(SEQID NO:20)

Example 4: Construction of E. coli Expression Vectors for Amino-Terminal Deleted nanoKAZ using a pColdII Vector

[0192] The DNA fragment obtained in EXAMPLE 3 was purified with a PCR purification kit (manufactured by QIAGEN Inc.), digested with the restriction enzymes of EcoRI and XbaI and then ligated to the EcoRI-XbaI site in the expression vector of pColdII (Takara Bio Inc.) to construct the expression vectors, pCold-N5D-nKAZ, pCold-N6F-nKAZ, pCold-N7V-nKAZ, pCold-N8G-nKAZ, pCold-N9D-nKAZ and pCold-N10W-nKAZ for the amino-terminal deleted nanoKAZ.

[0193] Also, the pCold-ZZ-P-nanoKAZ described in EXAMPLE 1 was digested with the restriction enzymes of EcoRI and XbaI in a conventional manner and then ligated to the EcoRI-XbaI site in the expression vector of pColdII to construct the pCold-rianoKAZ vector. The nucleotide sequence of the inserted DNA was confirmed by sequencing using a DNA Sequencer (manufactured by ABI Inc.). The nucleotide sequence and amino acid sequence of pCold-nanoKAZ are shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively.

Example 5: Expression of Amino-Terminal Deleted nanoKAZ in E. coli and Preparation of Crude Enzyme Solution

[0194] In order to express the amino-terminal deleted nanoKAZ in E. coli, the recombinant plasmid obtained in EXAMPLE 4 was used. The E. coli BL21. strain (Novagen, Madison, Wis.) was used as a host cell. The BL21 strain carrying the recombinant plasmid was incubated in 5 mL of Luria-Bertani medium (hereinafter designated as LB medium) containing ampicillin (50 g/mL) at 37 C. for 18 hours. The seed culture of 0.1 mL was inoculated to 10 mL of LB medium and incubated for 3 hours, followed by cooling in an ice-water bath for 1 hour. IPTG was added to the culture medium at a final concentration of 0.1 mM, followed by incubation at 15 C. for further 17 hours. After completion of the incubation, 1 mL of the culture medium was collected by centrifugation at 10,000 rpm for 2 minutes. The collected E. coli cells were suspended in 0.5 mL of 30 mM Tris-HCl (pH 7.6)-10 mM EDTA (manufactured by Wako Pure Chemical Industries, Ltd.) (hereinafter designated as TE). The E. coli cells were disrupted by sonication for 3 seconds using a Branson model 250 sonifire (Danbury, Conn.) to give a crude enzyme solution. Then, 5 L of the crude enzyme solution was analyzed by SDS-PAGE to confirm the protein expression (FIG. 1).

[0195] In FIG. 1, labeled M and 1 to 7 represent as follows: M: molecular weight markers; 1: nanoKAZ, 2: N5D-nKAZ, 3: N6F-nKAZ, 4: N7V-nKAZ, 5: N8G-nKAZ, 6: N9D-nKAZ and 7: N10W-nKAZ.

Example 6: Construction of E. coli Expression Vectors for ZZ-Fused Amino-Terminal Deleted nanoKAZ

[0196] To express the amino-terminal deleted nanoKAZ as a soluble protein, the expression vector of pCold-ZZ-X (described in Inouye & Sahara, Protein Express. Purif. (2009) 66: 52-57) was used. The DNA fragment obtained in EXAMPLE 3 was digested with the restriction enzyme of EcoRI and XbaI and ligated to the EcoRI-XbaI site of pCold-ZZ-X to construct the following 11 expression vectors for the ZZ-fused amino-terminal deleted nanoKAZ: pCold-ZZ-P-N2T-nKAZ, pCold-ZZ-P-N3L-nKAZ, pCold-ZZ-P-N4E-nKAZ, pCold-ZZ-P-N5D-nKAZ, pCold-ZZ-P-N6F-nKAZ, pCold-ZZ-P-N7V-nKAZ, pCold-ZZ-P-N8G-nKAZ, pCold-ZZ-P-N9D-nKAZ, pCold-ZZ-P-AN10W-nKAZ, pCold-ZZ-P-N15G-nKAZ and pCold-ZZ-P-N20Q-nKAZ.

Example 7: Expression of ZZ-Fused Amino-Terminal Deleted nanoKAZ in E. coli and Preparation of Crude Enzyme Solution

[0197] To express the ZZ-fused amino-terminal deleted nanoKAZ in E. coli, the recombinant plasmid obtained in EXAMPLE 6 was used. Crude enzyme solutions were prepared using a similar manner to EXAMPLE 5 using the E. coli BL21 strain (Novagen, Madison, Wis.) as a host cell. Then, 5 L of the crude enzyme solution obtained was subjected to SDS-PAGE analysis to confirm the protein expression (FIG. 2).

[0198] In FIG. 2, labeled M and 1 to 12 represent as follows. M: molecular weight size markers; 1: ZZ-P-nanoKAZ, 2: ZZ-P-N2T-nKAZ, 3: ZZ-P-N3L-nKAZ, 4: ZZ-P-N4E-nKAZ, 5: ZZ-P-N5D-nKAZ, 6: ZZ-P-N6F-nKAZ, 7: ZZ-P-N7V-nKAZ, 8: ZZ-P-N8G-nKAZ, 9: ZZ-P-N9D-nKAZ, 10: ZZ-P-N10G-nKAZ, 11: ZZ-P-N15G-nKAZ and 12: ZZ-P-N20Q-nKAZ.

Example 8: Assay for Luminescence Activity of Amino-Terminal Deleted nanoKAZ and ZZ-Fused Amino-Terminal Deleted nanoKAZ in Crude Enzyme Solution

[0199] The crude enzyme solutions obtained in EXAMPLE 5 and EXAMPLE 7 were allowed to stand in an ice-water for over 1 hour and then diluted to 50-fold in TE. By addition of 1 L each of the crude enzyme solutions to 100 L of TE containing 1 g of coelenterazine (manufactured by JNC Corp.), a luminescence reaction was started. The luminescence activity was measured for 60 seconds using a luminometer (manufactured by Atto AB2200); the maximum intensity of luminescence (I.sub.max) is given as a percentage (%).

[0200] The luminescence activities of nanoKAZ, when expressed in E. coli, were markedly reduced by deletion of 8 or more amino acid residues at the amino-terminus of nanoKAZ. As the deletions up to 5 amino acid residues at the amino-terminus showed 40 to 60% luminescence activity, it was clear that the amino-terminal region of nanoKAZ had no direct effects on the luminescence activity.

TABLE-US-00006 TABLE 3 Luminescence activity of amino-terminal deleted nanoKAZ in crude enzyme solution Relative luminescence activity (%, I.sub.max) Deletion mutant pCold- pCold-ZZ-P- nanoKAZ 100 100 N2T-nKAZ 93.4 N3L-nKAZ 82.0 N4E-nKAZ 74.0 N5D-nKAZ 87.0 73.6 N6F-nKAZ 40.0 59.8 N7V-nKAZ 1.6 5.8 N8G-nKAZ 3.9 10 N9D-nKAZ 0.01 0.3 N10W-nKAZ 0.03 1.6 N15G-nKAZ 0 N20Q-nKAZ 0

Example 9: Secretory Expression Vectors for Amino-Terminal Deleted nanoKAZ Mutants Using a Secretory Signal Peptide Sequence of Gaussia Luciferase

[0201] From the results of expression of the amino-terminal deleted nanoKAZ in E. coli, detectable luminescence activities were observed in the mutants with deletions up to 9 amino acid residues from the amino terminus (N10W-nKAZ). In order to confirm that these deletion mutants could be secreted from animal cultured cells, the expression vectors for the amino-terminal deleted nanoKAZ in animal cultured cells were constructed.

[0202] Specifically, the amino-terminal deleted nanoKAZ gene fragment obtained in EXAMPLE 3 was digested with the restriction enzymes of EcoRI and XbaI in a conventional manner and then ligated to the EcoRI-XbaI site of pcDNA3-GLsp obtinaed in EXAMPLE 2 to construct the expression vectors as follows; pcDNA3-GLsp-N2T-nKAZ, pcDNA3-GLsp-N3L-nKAZ, pcDNA3-GLsp-N4E-nKAZ, peDNA3-GLsp-N5D-nKAZ, pcDNA3-GLsp-N6F-nKAZ, pcDNA3-GLsp-N7V-IIKAZ, pcDNA3-GLsp-N8G-nKAZ, peDNA3-GLsp-N9D-nKAZ, pcDNA3-GLsp-N10W-nKAZ and pcDNA3-GLsp-N15G-nKAZ and peDNA3-GLsp-N20Q-nKAZ.

[0203] Also, the pCold-ZZ-P-nanoKAZ vector obtained in EXAMPLE 1 was digested with the restriction enzymes of EcoRI and XbaI in a conventional manner and then ligated to the EcoRI-XbaI site in the expression vector of pcDNA3-GLsp to construct the pcDNA3-GLsp-nanoKAZ vector.

[0204] The gene sequences inserted were confirmed by sequencing using a DNA sequencer (manufactured by ABI Inc.). The nucleotide sequence and amino acid sequence of peDNA3-GLsp-nanoKAZ are shown in SEQ ID NO: 15 and SEQ ID NO: 16, respectively.

Example 10: Transfection of Vectors in Animal Culture Cells and Preparation of Enzyme for Assay

(1) Purification of Expression Plasmid

[0205] The following experiment was performed using the recombinant plasmids obtained in EXAMPLE 9. The recombinant plasmid was purified from E. coli JM83 strain using a plasmid purification kit (manufactured by QIAGEN) and dissolved in sterilized water. The firefly luciferase vector (pGL4.13 [Luc2/sv40]: manufactured by Promega Corp.) was similarly preapred and used as an internal standard.

(2) Transfection and Preparation of Enzyme for Assay

[0206] Chinese hamster ovary cell line CHO-K1 was cultured in Ha F-12 medium (manufactured by Wako Pure Chemical Industries, Ltd.) supplemented with 10% (v/v) fetal bovine serum (manufactured by Biowest Inc.) (hereinafter sometimes referred to as Ham's F-12 medium). The CHO-K1 cells were seeded in a 6-well plate in 110.sup.5 cells/well/2 mL medium (n=2) and cultured in an incubator at 37 C. in 5% (v/v) CO.sub.2. After 24 hours, the purified recombinant plasmid was transfected to CHO-K1 cells using a FuGene HD transfection kit (manufactured by Promega Corp.) and the cells were provided for subsequent experiment. Specifically, 1 g of the recombinant plasmid, 0.1 g of the internal standard vector pGL4.13 [Luc2/sv40] and 3 L of FuGene HD were added to 100 L of the medium and allowed to stand at room temperature for 15 minutes. Subsequently, 100 L, of the DNA-FuGene complex was added to the cells in the 6-well plate. After incubation for 44 hours, the culture medium was collected. On the other hand, the cells expressed the KAZ mutants were washed 3 times with 3 mL of 1PBS, then suspended in 1 mL of 1PBS and disrupted by sonication on ice. The resultant cell extracts of nanoKAZ deletion mutants was used as enzyme solutions.

Example 11: Construction of Vectors for the Amino-Terminal Deleted nanoKAZ Lacking the Secretory Signal Peptide Sequence in Animal Cultured Cells

[0207] After digestion of the gene fragment of amino-terminal deleted nanoKAZ obtained in EXAMPLE 3 with the restriction enzymes of Asp718 and XbaI, the fragment was inserted into the Asp718-XbaI site of a pcDNA3 vector (manufactured by Invitrogen Inc.) to construct the vectors, pcDNA3-N2T-nKAZ, pcDNA3-N3L-nKAZ, pcDNA3-N4E-nKAZ, pcDNA3-N5D-nKAZ, pcDNA3-N6F-nKAZ, pcDNA3-N7V-nKAZ, pcDNA3-N8G-nKAZ, pcDNA3-N9D-nKAZ, pcDNA3-N10W-nKAZ, pcDNA3-N15G-nKAZ and pcDNA3-N20Q-nKAZ.

[0208] Also, the pCold-ZZ-P-nanoKAZ obtained in EXAMPLE 1 was digested with the restriction enzymes of Asp718/XbaI in a conventional manner and then similarly ligated to the Asp718-XbaI site of a pcDNA3 vector to construct the pcDNA3-nanoKAZ vector. The nucleotide sequence and amino acid sequence of nanoKAZ in pcDNA3-nanoKAZ are shown in SEQ ID NO: 17 and SEQ ID NO: 18, respectively.

Example 12: Transfection of Vectors in Animal Cultured Cells and Preparation of Enzyme for Assay

(1) Purification of Expression Plasmids

[0209] The recombinant plasmids obtained in EXAMPLE 11 were purified in a similar manner to EXAMPLE 10 and dissolved in sterilized water. The firefly luciferase vector (pGL4.13 [Luc2/sv40]: manufactured by Promega Corp.) was similarly prepared and used as an internal standard.

(2) Transfection and Preparation of Enzyme for Assay

[0210] Culture media containing the secreted amino-terminal deleted nanoKAZ and cell extracts of amino-terminal deleted nanoKAZ as the enzyme solutions were prepared in the same manner as in EXAMPLE 10.

Example 13: Assay for Luminescence Activity of Amino-Terminal Deleted nanoKAZ Expressed in Animal Cultured Cells

[0211] After adding of 5 L each of the culture media and cell extracts obtained in EXAMPLE 10 and EXAMPLE 12, respectively, to 100 L of TE containing 1 g of coelenterazine (manufactured by JNC Corp.), a luminescence reaction was started. The luminescence activity was measured for 60 seconds using a luminometer (manufactured by Atto Inc.: AB2200), and the maximum intensity of luminescence (I.sub.max) was given as a percentage (%).

[0212] As a result, luminescence activities of the amino-terminal deleted nanoKAZ mutants with the secretory signal peptide sequence of Gaussia luciferase were observed in the cytoplasm of N2T-nKAZ to N8G-nKAZ and in the culture media of N2T-nKAZ to N6F-nKAZ.

[0213] On the other hand, in the amino-terminal deleted nanoKAZ lacking the secretory signal peptide sequence, the mutants including those up to N5-nKAZ with 4 amino acids deletion from the amino terminus were found to be secreted into the culture medium, but no secretion from the cells was observed in the nanoKAZ mutants with the deletion of 5 or more amino acid residues from the amino terminus. Thus, it became clear that the nanoKAZ mutants with the deletion of at least one to at most 4 amino acids at the amino terminal region could be secreted extracellularly in the absence of the secretory signal peptide sequence.

[0214] Regarding firefly luciferase used as an internal standard to confirm the efficiency of transfection, 5 L each of cell extracts obtained in EXAMPLES 10 and 12 were added to 100 L of a reagent for enzyme assay (manufactured by Promega Corp.) to start a luminescence reaction. The luminescence activity was measured for 10 seconds using a luminometer (manufactured by Atto Inc.: AB2200), The results reveal that the transfection efficiencies were almost the same.

TABLE-US-00007 TABLE 4 Luminescence activities of amino-terminal deleted nanoKAZ in culture medium and cell extracts Relative luminescence activity (%, I.sub.max) pcDNA3-GLsp- pcDNA3- Culture Cell Culture Cell Deletion mutant medium extracts medium extracts nanoKAZ 100 4.8 100 29.4 N2T-nKAZ 97.0 3.4 93.4 23.3 N3L-nKAZ 87.7 4.4 50.6 16.2 N4E-nKAZ 60.5 2.9 4.6 9.7 N5D-nKAZ 56.2 1.7 0.1 1.5 N6F-nKAZ 0.5 0.3 less than 0.06 0.01 N7V-nKAZ 0 0.1 0 less than 0.01 N8G-nKAZ 0 0 0 0.01 N9D-nKAZ 0 0 0 0 N10W-nKAZ 0 0 0 0 N15G-nKAZ 0 0 0 0 N20Q-nKAZ 0 0 0 0

[0215] From the confirmed results of the activity for the KAZ mutants expressed in E. coli in EXAMPLE 8 and of the secretion from animal cultured cells in EXAMPLE 13, the nanoKAZ mutants with 1, 2, 3 and 4 amino acid residues deletion from the amino terminus were secreted into the culture medium from the animal cultured cells The results reveal that the sequence(s) of at least one to at most 4 amino acid residues from the amino terminus are related to the extracellular secretion of nanoKAZ.

Example 14: Preparation of CHO-K1 Cell Line Stably Expressing nanoKAZ Having the Secretory Signal Peptide Sequence of Gaussia Luciferase

[0216] Chinese hamster ovary cell line CHO-K1 was cultured in Ham's F-12 medium. The CHO-K1 cells were seeded in a 6 cm petri dish in 210.sup.5 cells/well/2 triL medium (n=2) and cultured in an incubator at 37 C. in 5% (v/v) CO.sub.2. After 24 hours, the purified pcDNA3-GLsp-nanoKAZ described in EXAMPLE 10 was transfected to CHO-K1 cells using a FuGene HD transfection kit (manufactured by Promega Corp.).

[0217] The CHO-K1 cells transfected were treated with trypsin and were resuspended in 5 mL of Ham's F-12 medium. The cell suspension of 0.510.sup.5 cells were plated in six petri dishes of 10 cm with 10 mL of Ham's F-12 medium. After overnight incubation at 37 C. in a 5% CO.sub.2 incubator, 160 L (a final concentration of 800 g/mL) of 50 mg/mL G418 sulfate (manufactured by Calbiochem, Inc.) was added thereto, followed by incubation at 37 C. in a 5% CO, incubator for 7 days.

[0218] Seven days later, 48 colonies formed were picked with a sterilized toothpick and suspended in 50 L of trypsin solution in a 96-well plate. To 50 L each of the 48 trypsinized-cell suspensions was added 100 L of Ham's F-12 medium. Each mixture was added to a 24-well plate with 1 mL of Ham's F-12 medium containing G418. The cells were incubated at 37 C. in a 5% CO.sub.2 incubator for about 7 days until the cells grew.

[0219] After the cells grew, 5 L of the culture medium was recovered and added to 100 L of TE containing 0.5 g of coelenterazine, and the luminescence activity was determined. The cell lines showing a high luminescence activity were treated with trypsin and suspended in 1 mL of Ham's F-12 medium containing G418. After 50 L of the cell suspension was added to a 24-well plate with 1 nit, of Ham's F-12 medium containing G418, the cells were incubated at 37 C. in a 5% CO.sub.2 incubator for about 5 days until the cells grew.

[0220] After the cells grew, the luminescence activity was determined in a similar manner. The cell lines showing a high luminescence activity were treated with trypsin and suspended in 1 mL of Ham's F-12 medium containing G418. After 200 L of the cell suspension was added to a 6-well plate with 3 mL of Ham's F-12 medium containing G418, the cells were incubated at 37 C. in a 5% CO.sub.2 incubator for about 5 days until the cells grew.

[0221] After the cells grew, the luminescence activity was determined in a similar manner. The cell lines showing a high luminescence activity were treated with trypsin and suspended in 1 mL of Ham's F-12 medium containing G418 After 500 L of the cell suspension was added to a 10 cm petri dish with 10 mL of Ham's F-12 medium containing G418, the cells were incubated at 37 C. in a 5% CO.sub.2 incubator for approximately 3 to 5 days until the cells grew.

[0222] After the cells grew, the luminescence activity was determined in a similar manner to confirm that the cells showed a high luminescence activity. The CHO-K1 cell line stably expressing nanoKAZ having a secretory signal peptide sequence of Gaussia luciferase was obtained.

Example 15: Preparation of CHO-K1 Stably Expressing nanoKAZ Lacking the Secretory Signal Peptide Sequence

[0223] The CHO-K1 cell line stably expressing nanoKAZ in the absence of the secretory signal peptide sequence was obtained in a similar manner to EXAMPLE 14, except that pcDNA3-nanoKAZ purified in EXAMPLE 13 as a plasmid DNA for transfection was used.

Example 16: Comparison of Secretory Inhibition by Brefeldin a

[0224] Using the stable expressed cell lines established in EXAMPLE 14 and EXAMPLE 15, the effect of brefeldin A that inhibits exocytosis through the trans-Golgi network by the vesicle-mediated transport was compared. Specifically, each stable expressed cell lines with 210.sup.5 cells were seeded to a 6-well plate in 3 mL of Ham's F-12 medium containing 10% FBS, respectively; followed by incubation at 37 C. in a 5% CO.sub.2 incubator for 48 hours. After washing twice with 3 mL of Ham's F-12 medium containing 10% FBS, brefeldin A (manufactured by Wako Pure Chemical Industries, Ltd.) was added to the culture medium at a final concentration of 5 g/mL. As a control experiment, cells were cultured in the absence of brefeldin A. The luminescence activity in the culture medium was determined at each incubation times at 0, 1, 3 and 6 hours, using coelenterazine as a substrate. The results are shown in FIGS. 3 and 4.

[0225] As shown in FIG. 3, the secretion of nanoKAZ into the culture medium was markedly inhibited by brefeldin A in the pcDNA3-GLsp-nanoKAZ/CHO-K1 stable expressed cell line established using the nanoKAZ gene with the secretory signal peptide sequence, as compared to the cells in the absence of brefeldin A. This indicates that nanoKAZ having the secretory signal peptide sequence was secreted normally from a endoplasmic reticulum via the trans-Golgi network.

[0226] On the other hand, as shown in FIG. 4, the nanoKAZ was secreted from the pcDNA3-nanoKAZ/CHO-K1 stable expressed cell line established using the nanoKAZ gene without the secretory signal peptide sequence in the presence or absence of brefeldin A. This indicates that the protein was secreted through another pathway(s), not through a general trans-Golgi network from the endoplasmic reticulum.

[0227] From the foregoing results and the results of the experiments on the amino-terminal deleted nanoKAZ gene expression described in EXAMPLES 8 and 13, it was demonstrated that the amino-terminal region at the positions of 1 to 4 from the amino terminus of nanoKAZ, has different secretion information from a general signal peptide sequence for secretion.

Example 17: Visualization of nanoKAZ Secretion

[0228] Using the stable expressed cell lines established in EXAMPLES 14 and 15, the visualization of nanoKAZ secretion was performed. The 10.sup.4 cells were cultured in a 35 mm glass bottom plate (manufactured by Iwaki Corp.) in 3 mL of MEM-alpha medium (manufactured by Wako Pure Chemical Industries, Ltd.) containing 10% FBS at 37 C. in a 5% CO.sub.2 incubator for 48 hours. After washing 3 times with 3 mL of HBSS (manufactured by Wako Pure Chemical industries, Ltd.), h-coelenterazine dissolved in HBSS at a final concentration of 3 g/mL was added to cells. The visualized images were captured using an IX81-ZDC microscope (manufactured by Olympus Optical Co.) equipped with an EM-CCD camera (ImagEM 1K: manufactured by Hamamatsu Photonics K.K.) under image acquisition conditions (11 binning, fast scanning, EM-gain level 255, photon-counting level=1, image acquisition time, 0.5 sec.). After the addition of h-coelenterazine in 15 seconds, the captured images are shown in FIG. 5. Labeled a and b designate the bright-field image and the luminescence image of nanoKAZ from pcDNA3-GLsp-nanoKAZ/CHO-K1 stable expressed cells, respectively. Labeled c and d designate the bright-field image and the luminescence image of nanoKAZ from pcDNA3-nanoKAZ/CHO-K1 stable expression cells, respectively. Comparison between the luminescence images b and d obtained by the luminescence imaging indicate that, unlike nanoKAZ from the pcDNA3-GLsp-nanoKAZ/CHO-K1 cells secreted from the endoplasmic reticulum through the trans-Golgi network, nanaoKAZ from the pcDNA3-nanoKAZ/CHO-K1 cells were uniformly distributed on the cell surface and were secreted outside of cells, showing a different mode of secretion. More specifically, based on the results of experiments of amino-terminal deleted nanoKAZ gene expression in EXAMPLES 8 and 13, it was revealed that different secretion information from that of general secretory signal peptide sequences was found in the amino acid region at the positions of 1 to 4 from the amino terminus of nanoKAZ and, therefore, the visualized images of secretion were different as well.

Example 18: Substrate Specificities for Amino-Terminal Deleted nanoKAZ

[0229] The enzyme solutions of amino-terminal deleted nanoKAZ used for substrate specificity studies were prepared by the method described in EXAMPLE 12, using pcDNA3 vectors (pcDNA3-N2T-nKAZ, pcDNA3-N3L-nKAZ, pcDNA3-N4E-nKAZ, pcDNA3-N5D-nKAZ and pcDNA3-N6F-nKAZ) for expression of the amino-terminal deletions, which were obtained by the method described in EXAMPLE 11. After transfection to CHO-K1 cells and then incubation for 48 hours, the culture medium were collected and stored in a frozen state. The coelenterazine analogues used for substrate specificity studies were synthesized by the methods described in publications, respectively. Specifically, coelenterazine (CTZ), h-coelenterazine (h-CTZ) andf-coelenterazine (f-CTZ), 6h-f-coelenterazine (6h-f-CTZ) were synthesized by the method described in Inouye et al (2013) Biochem. Biophys. Res. Commun. 437: 23-28, and bis-coelenterazine (bis-CTZ) was synthesized by the method described in Nakamura et al, (1997) Tetrahedron Lett. 38: 6405-6406.

[0230] A luminescence reaction was started by the addition of 2 L of the freeze-thawed culture medium on ice to 100 L of TE containing 1 g of coelenterazine or its analogue. The luminescence activities were measured for 10 seconds using a luminorneter (manufactured by Atto Inc.: AB2200), and the maximum intensity of luminescence (I.sub.max) of nanoKAZ was shown as a relative luminescence activity using coelenterazine as a substrate. The results are shown in TABLE 5.

TABLE-US-00008 TABLE 5 Relative luminescence activity (I.sub.max) Deletion mutant CTZ bis-CTZ 6h-f-CTZ h-CTZ f-CTZ nanoKAZ 1.00 6.93 7.00 11.76 12.65 N2T-nKAZ 1.03 8.99 8.02 13.38 15.04 N3L-nKAZ 0.50 5.39 5.25 9.39 9.88 N4E-nKAZ 0.05 0.35 0.36 0.58 0.70 N5D-nKAZ 0.01 0.01 0.01 0.01 0.02 N6F-nKAZ less less than less than less than less than than 0.01 0.01 0.01 0.01 0.01

[0231] As shown in TABLE 5, when bis-CTZ, 6h-f-CTZ, h-CTZ andfiCTZ were used as substrates, the nanoKAZ deletion mutants of N2T-nKAZ, N3L-nKAZ and N4E-nKAZ showed approximately 8 to 15-fold higher activity than coelenterazine (CTZ) used as the substrate. Particularly, h-CTZ andf-CTZ. showed over 10-fold higher activity than coeleneterazine. In addition, the nanoKAZ deletion mutant of N2T-nKAZ was found to show a higher activity than nanoKAZ when bis-CTZ, 6h-f-CTZ, h-CTZ andf-CTZ were used.

[Sequence Listing Free Text]

[0232] [SEQ ID NO: 1] Nucleotide sequence of nanoKAZ
[SEQ ID NO: 2] Amino acid sequence of nanoKAZ
[SEQ ID NO: 3] Nucleotide sequence of N2T-nKAZ
[SEQ ID NO: 4] Amino acid sequence of N2T-nKAZ
[SEQ ID NO: 5] Nucleotide sequence of N3L-nKAZ
[SEQ ID NO: 6] Amino acid sequence of N3L-nKAZ
[SEQ ID NO: 7] Nucleotide sequence of N4E-nKAZ
[SEQ ID NO: 8] Amino acid sequence of N4E-nKAZ
[SEQ ID NO: 9] Nucleotide sequence of N5D-nKAZ
[SEQ ID NO: 10] Amino acid sequence of N5D-nK
[SEQ ID NO: 11] Nucleotide sequence of pCold-ZZ-P-nanoKAZ
[SEQ ID NO: 12] Amino acid sequence of pCold-ZZ-P-nanoKAZ
[SEQ ID NO: 13] Nucleotide sequence of pCold-nanoKAZ
[SEQ ID NO: 14] Amino acid sequence of pCold-nanoKAZ
[SEQ ID NO: 15] Nucleotide sequence of pcDNA3-GLsp-nanoKAZ
[SEQ ID NO: 16] Amino acid sequence of peDNA3-GLsp-nanoKAZ
[SEQ ID NO: 17] Nucleotide sequence of pcDNA3-nanoKAZ
[SEQ ID NO: 18] Amino acid sequence of peDNA3-nanoKAZ
[SEQ ID NO: 19] Nucleotide sequence of the primer used in EXAMPLES (nanoKAZ-1N/EcoRI)
[SEQ ID NO: 20] Nucleotide sequence of the primer used in EXAMPLES (nanoKAZ-3C/XbaI)
[SEQ ID NO: 21] Nucleotide sequence of the primer used in EXAMPLES (GLsp-1R/EcoRI)
[SEQ ID NO: 22] Nucleotide sequence of the primer used in EXAMPLES (T7 primer
[SEQ ID NO: 23] Nucleotide sequence of the primer used in EXAMPLES (D2-nKAZ-15N/EcoRI)
[SEQ ID NO: 24] Nucleotide sequence of the primer used in EXAMPLES (D3-nKAZ-16N/ECoRI)
[SEQ ID NO: 25] Nucleotide sequence of the primer used in EXAMPLES (D4-6KAZ-17N/ECoRI)
[SEQ ID NO: 26] Nucleotide sequence of the primer used in EXAMPLES (D5nanoKAZ-4N/EcoRI)
[SEQ ID NO: 27] Nucleotide sequence of the primer used in EXAMPLES (D6nanoKAZ-8N/EcoRI)
[SEQ ID NO: 28] Nucleotide sequence of the primer used in EXAMPLES (D7nanoKAZ-9N/EcoRI)
[SEQ ID NO: 29] Nucleotide sequence of the primer used in EXAMPLES (D8nanoKAZ-10N/EcoRI)
[SEQ ID NO: 30] Nucleotide sequence (D9nanoKAZ-11N/EcoRI) of the primer used in EXAMPLES
[SEQ ID NO: 31] Nucleotide sequence of the primer used in EXAMPLES (D10nanoKAZ-5N/EcoRI)
[SEQ ID NO: 32] Nucleotide sequence of the primer used in EXAMPLES (D15nanoKAZ-6N/EcoRI)
[SEQ ID NO: 33] Nucleotide sequence of the primer used in EXAMPLES (D20nanoKAZ-7N/EcoRI)