JP2011120501A - Mutant gaussia luciferase - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a mutant Gaussia luciferase which is good in refolding efficiency and long in emission attenuation time. <P>SOLUTION: The mutant Gaussia luciferase comprises an amino acid sequence obtained by replacing the 59-cysteine of an amino acid sequence comprising a specific sequence and contained in Gaussia luciferase by another amino acid. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a protein having catalytic activity using luciferin as a substrate, a polynucleotide encoding the same, a recombinant vector containing the polynucleotide, a transformant containing the recombinant vector, a method for producing the protein, and the like.

  Gaussia luciferase is a secretory enzyme produced by Gaussia princeps, a deep-sea copepoda, which simply catalyzes a luminescent reaction in the presence of oxygen and a substrate (luciferin such as coelenterazine). It is an enzyme of the system.

  In the luminescence reaction catalyzed by Gaussia luciferase, the luminescence reaction is simple and emits strong blue light compared to the luminescence reaction catalyzed by firefly luciferase, which requires ATP, magnesium ions, etc. Use in applications is expected.

  Gaussia luciferase is a simple protein having a 17-residue signal peptide sequence for secretion at the amino terminus and a peptide sequence composed of 168 amino acids as a catalytic moiety. Gaussia luciferase has 10 cysteines in one molecule. By treating Gaussia luciferase with a reducing agent such as mercaptoethanol or dithiothreitol, the luminescence catalytic activity of Gaussia luciferase is completely deactivated. -It has been reported that binding is essential (Non-Patent Document 1).

  However, the position of intra-molecular S—S— bond of Gaussia luciferase is still unclear. It is also unclear whether Gaussia luciferase expressed in E. coli and animal cultured cells can form an accurate intramolecular -S-S- bond like natural Gaussia luciferase.

  When a large amount of Gaussia luciferase is expressed and purified using Escherichia coli as a host and used as a probe for luminescence assay, a method for preparing highly active Gaussia luciferase is required. There is a need to increase the efficiency of refolding in formation.

  Moreover, since more than 90% of Gaussia luciferase expressed in E. coli is expressed as an insoluble protein, efficient solubilization is also desired (Non-patent Document 1).

  Furthermore, when used as a probe for imaging in living cells, the luminescence pattern of Gaussia luciferase expressed in E. coli and animal cultured cells shows a maximum luminescence intensity after addition of the substrate, and is less than 25% within 50 seconds. It has been reported that it attenuates. Further, even when a luminescent substrate is added to the reaction solution after the decay of luminescence, there is no further enhancement of luminescence activity (Non-patent Documents 2 and 3).

  If the decay time of luminescence can be increased (the half-life of luminescence can be increased), Gaussia luciferase is more effective as a probe for luminescence assay, like firefly luciferase and Renilla luciferase.

  In order to achieve this purpose in recent years, clones having a long half-life were isolated by randomly introducing mutations into the Gaussia luciferase gene and screening from the resulting thousands of mutant Gaussia luciferases. And it is shown that the mutant Gaussia luciferase in which the 43rd methionine residue in the amino acid sequence (168 residues) of Gaussia luciferase is replaced with an isoleucine residue has a longer half-life than natural Gaussia luciferase. (Non-Patent Document 4). However, this mutant Gaussia luciferase has room for improvement in terms of refolding efficiency.

  So far, a mutant Gaussia luciferase with good refolding efficiency and long decay time of luminescence has not been obtained.

Inouye, S. & Sahara, Y. (2008) Biochem. Biophys. Res. Commun. 365, 96-101. Verhaegen, M. & Christopoulos, T. K. (2002) Anal. Chem. 74, 4378-4385. Tannous, B.T. et al. (2005) Mol. Ther. 11,435-443. Maguire, C.A. et al. (2009) Anal. Chem. 81, 7102-7106.

  In the above situation, there has been a demand for mutant Gaussia luciferase having a high refolding efficiency and a long emission decay time.

  As a result of intensive studies to solve the above problems, the present inventors have determined that the 59th cysteine residue among the 10 cysteine residues in the amino acid sequence (168 residues) of the catalytic portion of Gaussia luciferase By substituting for other amino acids, it was found that mutant Gaussia luciferase with good refolding efficiency and long decay time of luminescence was obtained, and the present invention was completed.

That is, the present invention provides the following protein having catalytic activity using luciferin as a substrate, a polynucleotide encoding the same, a recombinant vector containing the polynucleotide, a transformant containing the recombinant vector, and the protein The manufacturing method etc. are provided.
[1] The protein according to any one of the following (a) to (d):
(A) a protein comprising an amino acid sequence in which the 59th cysteine is replaced with another amino acid in the amino acid sequence of SEQ ID NO: 30;
(B) In the amino acid sequence of SEQ ID NO: 30, the 59th cysteine is replaced with another amino acid, and the 52nd, 56th, 65th, 77th, 120th, 123rd, 127th, 136th and 148th positions A protein having an amino acid sequence in which one to a plurality of amino acids are substituted with other amino acids other than the position, and having a luminescence catalytic activity using luciferin as a substrate;
(C) a protein having an amino acid sequence in which the 59th cysteine in the amino acid sequence of SEQ ID NO: 30 is substituted with another amino acid and having a luminescence catalytic activity using luciferin as a substrate; and (d) of SEQ ID NO: 30 In the amino acid sequence, the 59th cysteine is substituted with another amino acid, and 1 to a plurality of amino acids other than positions 52, 56, 65, 77, 120, 123, 127, 136 and 148 A protein having an amino acid sequence in which an amino acid is substituted with another amino acid and having a luminescence catalytic activity using luciferin as a substrate.
[2] The protein according to [1] above, which is any of the following (a) to (d):
(A) a protein comprising an amino acid sequence in which the 59th cysteine is replaced with another amino acid in the amino acid sequence of SEQ ID NO: 30;
(B) In the amino acid sequence of SEQ ID NO: 30, the 59th cysteine is replaced with another amino acid, and the 52nd, 56th, 65th, 77th, 120th, 123rd, 127th, 136th and 148th positions A protein having a photocatalytic activity comprising an amino acid sequence in which 1 to 25 amino acids other than the position are substituted with other amino acids, and luciferin as a substrate;
(C) a protein having an amino acid sequence in which the 59th cysteine in the amino acid sequence of SEQ ID NO: 30 is substituted with another amino acid and having a luminescence catalytic activity using luciferin as a substrate; and (d) of SEQ ID NO: 30 In the amino acid sequence, the 59th cysteine is substituted with another amino acid, and 1 to 25 other than positions 52, 56, 65, 77, 120, 123, 127, 136 and 148 A protein having an amino acid sequence in which an amino acid is substituted with another amino acid and having a luminescence catalytic activity using luciferin as a substrate.
[3] The protein according to [1], which is the following (a) or (b):
(A) a protein comprising an amino acid sequence in which the 59th cysteine is replaced with another amino acid in the amino acid sequence of SEQ ID NO: 30; and (b) the 59th cysteine is replaced with another amino acid in the amino acid sequence of SEQ ID NO: 30. A protein having a light-emitting catalytic activity comprising the amino acid sequence and luciferin as a substrate.
[4] The protein according to any one of [1] to [3] above, wherein the other amino acid substituted for the 59th cysteine in the amino acid sequence of SEQ ID NO: 30 is serine.
[5] The protein according to [4], which is the following (a) or (b):
(A) a protein comprising the amino acid sequence of SEQ ID NO: 2, 4 or 32; and (b) a protein comprising the amino acid sequence of SEQ ID NO: 2, 4 or 32 and having a luminescence catalytic activity using luciferin as a substrate.
[5a] The protein of the above-mentioned [5], which is the following (a) or (b):
(A) a protein comprising the amino acid sequence of SEQ ID NO: 2; and (b) a protein comprising the amino acid sequence of SEQ ID NO: 2 and having a luminescence catalytic activity using luciferin as a substrate.
[6] A polynucleotide containing a polynucleotide encoding the protein according to any one of [1] to [5a] above.
[7] A recombinant vector containing the polynucleotide according to [6] above.
[8] A transformant into which the recombinant vector according to [7] is introduced.
[9] The method according to any one of [1] to [5a], comprising the step of culturing the transformant according to [8] and generating the protein according to any one of [1] to [5a]. A method for producing the protein according to the description.
[10] A kit comprising the protein according to any one of [1] to [5a] above.
[11] A kit comprising the polynucleotide described in [6] above, the recombinant vector described in [7] above, or the transformant described in [8] above.
[12] The kit according to [10] or [11] above, further comprising luciferin.
[13] The kit according to [12] above, wherein the luciferin is coelenterazines.
[14] The kit according to [13] above, wherein the coelenterazine is coelenterazine.
[15] The protein according to any one of the following (a) to (d):
(A) a protein comprising an amino acid sequence in which the 59th cysteine is replaced with another amino acid in the amino acid sequence of SEQ ID NO: 30;
(B) In the amino acid sequence of SEQ ID NO: 30, the 59th cysteine is replaced with another amino acid, and the 52nd, 56th, 65th, 77th, 120th, 123rd, 127th, 136th and 148th positions A protein having an amino acid sequence in which one to a plurality of amino acids are substituted with other amino acids other than the position, and having a luminescence catalytic activity using luciferin as a substrate;
(C) a protein having an amino acid sequence in which the 59th cysteine in the amino acid sequence of SEQ ID NO: 30 is substituted with another amino acid and having a luminescence catalytic activity using luciferin as a substrate; and (d) of SEQ ID NO: 30 In the amino acid sequence, the 59th cysteine is substituted with another amino acid, and 1 to a plurality of amino acids other than positions 52, 56, 65, 77, 120, 123, 127, 136 and 148 A method for performing a luminescent reaction, comprising contacting a luciferin with a protein having an amino acid sequence in which an amino acid is substituted with another amino acid and having a luminescence catalytic activity using luciferin as a substrate.
[15a] The method according to [15] above, wherein the protein is a protein according to any one of the following (a) to (d):
(A) a protein comprising an amino acid sequence in which the 59th cysteine is replaced with another amino acid in the amino acid sequence of SEQ ID NO: 30;
(B) In the amino acid sequence of SEQ ID NO: 30, the 59th cysteine is replaced with another amino acid, and the 52nd, 56th, 65th, 77th, 120th, 123rd, 127th, 136th and 148th positions A protein having a photocatalytic activity comprising an amino acid sequence in which 1 to 25 amino acids other than the position are substituted with other amino acids, and luciferin as a substrate;
(C) a protein having an amino acid sequence in which the 59th cysteine in the amino acid sequence of SEQ ID NO: 30 is substituted with another amino acid and having a luminescence catalytic activity using luciferin as a substrate; and (d) of SEQ ID NO: 30 In the amino acid sequence, the 59th cysteine is substituted with another amino acid, and 1 to 25 other than positions 52, 56, 65, 77, 120, 123, 127, 136 and 148 A protein having an amino acid sequence in which an amino acid is substituted with another amino acid and having a luminescence catalytic activity using luciferin as a substrate.
[15b] The method according to [15] above, wherein the protein is the protein described in the following (a) or (b):
(A) a protein comprising an amino acid sequence in which the 59th cysteine is replaced with another amino acid in the amino acid sequence of SEQ ID NO: 30; and (b) the 59th cysteine is replaced with another amino acid in the amino acid sequence of SEQ ID NO: 30. A protein having a light-emitting catalytic activity comprising the amino acid sequence and luciferin as a substrate.
[15c] The method according to any one of [15] to [15b] above, wherein the other amino acid substituted for the 59th cysteine in the amino acid sequence of SEQ ID NO: 30 is serine.
[15d] The method according to [15c] above, wherein the protein is the protein described in (a) or (b) below:
(A) a protein comprising the amino acid sequence of SEQ ID NO: 2, 4 or 32; and (b) a protein comprising the amino acid sequence of SEQ ID NO: 2, 4 or 32 and having a luminescence catalytic activity using luciferin as a substrate.
[15e] The method described in [15d] above, wherein the protein is the protein described in (a) or (b) below:
(A) a protein comprising the amino acid sequence of SEQ ID NO: 2; and (b) a protein comprising the amino acid sequence of SEQ ID NO: 2 and having a luminescence catalytic activity using luciferin as a substrate.
[15f] The method according to any one of [15] to [15e] above, wherein the luciferin is coelenterazines.
[15g] The method described in [15f] above, wherein the coelenterazine is coelenterazine.
[16] The protein according to any one of the following (a) to (d):
(A) a protein comprising an amino acid sequence in which the 59th cysteine is replaced with another amino acid in the amino acid sequence of SEQ ID NO: 30;
(B) In the amino acid sequence of SEQ ID NO: 30, the 59th cysteine is replaced with another amino acid, and the 52nd, 56th, 65th, 77th, 120th, 123rd, 127th, 136th and 148th positions A protein having an amino acid sequence in which one to a plurality of amino acids are substituted with other amino acids other than the position, and having a luminescence catalytic activity using luciferin as a substrate;
(C) a protein having an amino acid sequence in which the 59th cysteine in the amino acid sequence of SEQ ID NO: 30 is substituted with another amino acid and having a luminescence catalytic activity using luciferin as a substrate; and (d) of SEQ ID NO: 30 In the amino acid sequence, the 59th cysteine is substituted with another amino acid, and 1 to a plurality of amino acids other than positions 52, 56, 65, 77, 120, 123, 127, 136 and 148 A bioluminescence resonance energy transfer (BRET) method is performed using a protein having an amino acid sequence substituted with another amino acid and having a luminescence catalytic activity using luciferin as a substrate as a donor protein. Physiological function analysis or enzyme activity measurement method.
[16a] The method according to [16] above, wherein the protein is a protein according to any one of the following (a) to (d):
(A) a protein comprising an amino acid sequence in which the 59th cysteine is replaced with another amino acid in the amino acid sequence of SEQ ID NO: 30;
(B) In the amino acid sequence of SEQ ID NO: 30, the 59th cysteine is replaced with another amino acid, and the 52nd, 56th, 65th, 77th, 120th, 123rd, 127th, 136th and 148th positions A protein having a photocatalytic activity comprising an amino acid sequence in which 1 to 25 amino acids other than the position are substituted with other amino acids, and luciferin as a substrate;
(C) a protein having an amino acid sequence in which the 59th cysteine in the amino acid sequence of SEQ ID NO: 30 is substituted with another amino acid and having a luminescence catalytic activity using luciferin as a substrate; and (d) of SEQ ID NO: 30 In the amino acid sequence, the 59th cysteine is substituted with another amino acid, and 1 to 25 other than positions 52, 56, 65, 77, 120, 123, 127, 136 and 148 A protein having an amino acid sequence in which an amino acid is substituted with another amino acid and having a luminescence catalytic activity using luciferin as a substrate.
[16b] The method according to [16] above, wherein the protein is the protein described in the following (a) or (b):
(A) a protein comprising an amino acid sequence in which the 59th cysteine is replaced with another amino acid in the amino acid sequence of SEQ ID NO: 30; and (b) the 59th cysteine is replaced with another amino acid in the amino acid sequence of SEQ ID NO: 30. A protein having a light-emitting catalytic activity comprising the amino acid sequence and luciferin as a substrate.
[16c] The method according to any one of [16] to [16b] above, wherein the other amino acid substituted for the 59th cysteine in the amino acid sequence of SEQ ID NO: 30 is serine.
[16d] The method according to [16c] above, wherein the protein is the protein described in (a) or (b) below:
(A) a protein comprising the amino acid sequence of SEQ ID NO: 2, 4 or 32; and (b) a protein comprising the amino acid sequence of SEQ ID NO: 2, 4 or 32 and having a luminescence catalytic activity using luciferin as a substrate.
[16e] The method described in [16d] above, wherein the protein is the protein described in (a) or (b) below:
(A) a protein comprising the amino acid sequence of SEQ ID NO: 2; and (b) a protein comprising the amino acid sequence of SEQ ID NO: 2 and having a luminescence catalytic activity using luciferin as a substrate.
[16f] The method according to any one of [16] to [16e] above, wherein the luciferin is coelenterazines.
[16g] The method according to [16f] above, wherein the coelenterazine is coelenterazine.

  The protein of the present invention has a luminescent catalytic activity using luciferin as a substrate. According to a preferred embodiment of the present invention, a mutant Gaussia luciferase having good refolding efficiency and a long light emission decay time is provided.

FIG. 1 is a schematic diagram showing the expression vector pCold-ZZGL-C59S prepared in Example 1. FIG. 2 is a schematic diagram showing the expression vector pCold-hGL-C59S prepared in Example 2. Figure 3 shows the time course of luminescence regeneration of mutant Gaussia luciferase (solid line: pCold-ZZGL-C59S) in which the 59th cysteine residue is replaced with serine residue and natural Gaussia luciferase (dotted line: pCold-ZZGL). It is a graph to show. Fig. 4 shows the time course of luminescence regeneration of mutant Gaussia luciferase (solid line: pCold-hGL-C59S) in which the 59th cysteine residue is replaced with serine residue and natural Gaussia luciferase (dotted line: pCold-hGL). It is a graph to show. FIG. 5 is a graph showing luminescence patterns of mutant Gaussia luciferase (solid line: pCold-ZZGL-C59S) in which the 59th cysteine residue is substituted with a serine residue and natural Gaussia luciferase (dotted line: pCold-ZZGL). is there. FIG. 6 is a graph showing the luminescence patterns of mutant Gaussia luciferase (solid line: pCold-hGL-C59S) in which the 59th cysteine residue is substituted with a serine residue and natural Gaussia luciferase (dotted line: pCold-hGL). is there.

  Hereinafter, embodiments of the present invention will be described in detail.

1． Protein of the Present Invention The protein of the present invention is a protein comprising an amino acid sequence in which the 59th cysteine is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 30, and a protein having substantially the same activity or function as the protein. Means.

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

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

  The luciferin used in the present invention may be luciferin that serves as a substrate for the protein of the present invention. Specific examples of luciferin used in the present invention include coelenterazines.

  In the present specification, coelenterazines mean coelenterazine and coelenterazine derivatives. Examples of the coelenterazine derivatives include h-coelenterazine, hcp-coelenterazine, cp-coelenterazine, f-coelenterazine, fcp-coelenterazine, n-coelenterazine, Bis-coelenterazine, MeO-coelenterazine, e-coelenterazine, cl-coelenterazine, etc. Can be given. Among these coelenterazines, coelenterazine is particularly preferable in the present invention. These coelenterazines may be synthesized by a known method, or commercially available products can be obtained.

As a method for synthesizing coelenterazines, for example, Shimomura et al. (1988) Biochem. J. 251 , 405-410, Shimomura et al. (1989) Biochem. J. 261 , 913-920, Shimomura et al. 1990) Biochem. J. 270 , 309-312 and the like, or a method analogous thereto.

  As for the coelenterazine commercial products, for example, coelenterazine and h-coelenterazine manufactured by Chisso Corporation; hcp-coelenterazine, cp-coelenterazine, f-coelenterazine, fcp-coelenterazine and n-coelenterazine manufactured by Sigma Can do.

As the protein of the present invention, more specifically, for example,
(A) a protein comprising an amino acid sequence in which the 59th cysteine is replaced with another amino acid in the amino acid sequence of SEQ ID NO: 30;
(B) In the amino acid sequence of SEQ ID NO: 30, the 59th cysteine is replaced with another amino acid, and the 52nd, 56th, 65th, 77th, 120th, 123rd, 127th, 136th and 148th positions A protein having an amino acid sequence in which one to a plurality of amino acids are substituted with other amino acids other than the position, and having a luminescence catalytic activity using luciferin as a substrate;
(C) a protein having an amino acid sequence in which the 59th cysteine in the amino acid sequence of SEQ ID NO: 30 is substituted with another amino acid and having a luminescence catalytic activity using luciferin as a substrate; and (d) of SEQ ID NO: 30 In the amino acid sequence, the 59th cysteine is substituted with another amino acid, and 1 to a plurality of amino acids other than positions 52, 56, 65, 77, 120, 123, 127, 136 and 148 Examples thereof include a protein having an amino acid sequence in which an amino acid is substituted with another amino acid and having a luminescence catalytic activity using luciferin as a substrate.

  The other amino acid substituted for the 59th cysteine in the amino acid sequence of SEQ ID NO: 30 is, for example, serine, threonine, alanine, glycine or proline, preferably serine, threonine or alanine, more preferably serine. It is.

As a protein of a preferred embodiment of the present invention, for example,
(A) an amino acid sequence in which the 59th cysteine is replaced with serine in the amino acid sequence of SEQ ID NO: 30, that is, a protein consisting of the amino acid sequence of SEQ ID NO: 32;
(B) a protein containing the amino acid sequence of SEQ ID NO: 32, and the like.

As a protein containing the amino acid sequence of SEQ ID NO: 32, for example,
(A) a protein consisting of the amino acid sequence of SEQ ID NO: 2, 4 or 32;
(B) a protein containing the amino acid sequence of SEQ ID NO: 2, 4 or 32.

  The above “one to a plurality of amino acids are substituted” means that there is substitution of one or a plurality of amino acid residues at any position in one or a plurality of amino acid sequences in the same sequence.

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

  The range of “1 to plural” in the above “amino acid sequence in which one or more amino acids are substituted with other amino acids” is, for example, 1 to 50, 1 to 30, 1 to 25, 1 to 22 1-20 pieces, 1-15 pieces, 1-10 pieces, 1-9 pieces, 1-8 pieces, 1-7 pieces, 1-6 pieces (1 to several pieces), 1-5 pieces, 1-4 One, three, one, two, one. Generally, the smaller the number of substituted amino acids, the better. Such proteins are described in “Sambrook J. et al., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press (2001)”, “Ausbel FM et al., Current Protocols in Molecular Biology, Supplement 1- 38, John Wiley and Sons (1987-1997) ”,“ Nuc. Acids. Res., 10, 6487 (1982) ”,“ Proc. Natl. Acad. Sci. USA, 79, 6409 (1982) ”,“ Gene , 34, 315 (1985) ”,“ Nuc. Acids. Res., 13, 4431 (1985) ”,“ Proc. Natl. Acad. Sci. USA, 82, 488 (1985) ”, etc. It can be obtained using a mutagenesis method.

  The amino acid substitution position in the amino acid sequence of SEQ ID NO: 30 is particularly limited as long as it is a position other than the 52nd, 56th, 65th, 77th, 120th, 123th, 127th, 136th and 148th positions. For example, 26th, 28th, 35th, 43rd, 61st, 66th, 69th, 72nd, 73th, 80th, 82th, 86th, 99th, 101st, 108th, One to a plurality of positions selected from the group consisting of 112th, 116th, 134th, 139th, 142th, 145th, 146th, 153rd, 155th, 159th, etc. can be mentioned. Specifically, 35th, 43rd, 61st, 66th, 69th, 72th, 73rd, 80th, 82nd, 86th, 108th, 112th, 116th, 134th, 139th, 142th , 145th, 146th, 153rd, 155th, 159th, etc., one to a plurality of positions selected from the group.

The protein of a particularly preferred embodiment of the present invention is:
(A) a protein comprising the amino acid sequence of SEQ ID NO: 2,
(B) a protein having the amino acid sequence of SEQ ID NO: 2 and having a luminescence catalytic activity using luciferin as a substrate.

  The protein of the present invention may further contain other peptide sequences at the N-terminus and / or C-terminus, preferably at the N-terminus, like the amino acid sequences of SEQ ID NOs: 2 and 4. The other peptide sequence is selected from the group consisting of, for example, a peptide sequence for purification, a secretory signal peptide sequence, a peptide sequence for expressing the protein of the present invention as a soluble protein, and an epitope sequence capable of recognizing an antibody. There may be mentioned at least one peptide sequence. The other peptide sequence is preferably a peptide sequence for purification and / or a secretory signal peptide sequence. In another preferred embodiment of the invention, the other peptide sequence is at least one selected from the group consisting of a peptide sequence for purification, a secretory signal peptide sequence, and a sequence for expressing the protein of the invention as a soluble protein. Is an array.

  The peptide sequence used in this technical field can be used as the peptide sequence for purification. Examples of the peptide sequence for purification include a histidine tag sequence having an amino acid sequence of 4 or more, preferably 6 or more histidine residues, an amino acid sequence of a binding domain of glutathione S-transferase to glutathione, or Examples include protein A amino acid sequence.

  The secretory signal peptide means a peptide region that plays a role of allowing a protein bound to the secretory signal peptide to permeate the cell membrane. The amino acid sequences of such secretory signal peptides and the nucleic acid sequences encoding them are well known and reported in the art (eg von Heijine G (1988) Biochim. Biohys. Acra 947: 307-333, von Heijine G (1990) J. Membr. Biol. 115: 195-201). More specifically, examples of the secretory signal peptide include a secretory signal peptide (OmpA) derived from outer membrane protein A of E. coli (Ghrayeb, J. et al. (1984) EMBO J. 3: 2437-2442), cholera. Examples include secretory signal peptides derived from fungal cholera toxin.

Examples of the peptide for expressing the protein of the present invention as a soluble protein include a polypeptide represented by the formula (Z) _n . The amino acid sequence of the polypeptide represented by the formula (Z) _n and the nucleic acid sequence encoding it are described in, for example, JP-A-2008-99669.

More specifically, when expressed as a fusion protein with the protein of the present invention, the polypeptide represented by the formula (Z) _n has an activity or function that allows the fusion protein to be expressed as a soluble protein.

Z represents a polypeptide selected from the group consisting of the following (a) to (d):
(A) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 34;
(B) When the amino acid sequence of SEQ ID NO: 34 comprises an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added and is expressed as a fusion protein with the protein of the present invention. A polypeptide having an activity or function that allows the fusion protein to be expressed as a soluble protein;
(C) When the fusion protein comprises an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 34 and is expressed as a fusion protein with the protein of the present invention, the fusion protein is regarded as a soluble protein. A polypeptide having an activity or function that can be expressed; and (d) an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 33 And a polypeptide having an activity or function capable of being expressed as a soluble protein when expressed as a fusion protein with the protein of the present invention.

The range of “one to plural” in the “amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added” in the polypeptide represented by formula (Z) _n is, for example, 1 to 20 Pieces, 1-15 pieces, 1-10 pieces, 1-9 pieces, 1-8 pieces, 1-7 pieces, 1-6 pieces (1 to several pieces), 1-5 pieces, 1-4 pieces, 1- Three, 1-2, and one. In general, the smaller the number of amino acids deleted, substituted, inserted or added, the better. Two or more of the amino acid residue deletions, substitutions, insertions and additions may occur simultaneously. Such regions include Sambrook J. et al., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press (2001), Ausbel FM et al., Current Protocols in Molecular Biology, Supplement 1-38, John. Wiley and Sons (1987-1997), “Nuc. Acids. Res., 10, 6487 (1982)”, “Proc. Natl. Acad. Sci. USA, 79, 6409 (1982)”, “Gene, 34, 315” (1985) ”,“ Nuc. Acids. Res., 13, 4431 (1985) ”,“ Proc. Natl. Acad. Sci. USA, 82, 488 (1985) ”and the like. And can be obtained.

The range of “90% or more” in the “amino acid sequence having 90% or more identity” in the polypeptide represented by the formula (Z) _n is, for example, 90% or more, 91% or more, 92% or more 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4 % Or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, 99.9% or more. In general, the larger the numerical value of identity, the better. The identity of amino acid sequences and base sequences can be determined using an analysis program such as BLAST (see, for example, Altzshul SF et al., J. Mol. Biol. 215, 403 (1990)). When using BLAST, the default parameters of each program are used.

The “polynucleotide hybridizing under stringent conditions” in the polypeptide represented by the above formula (Z) _n is a polynucleotide or sequence comprising a base sequence complementary to the base sequence of SEQ ID NO: 33 The polynucleotide (for example, DNA) obtained by using colony hybridization method, plaque hybridization method, Southern hybridization method, etc., using all or part of the polynucleotide encoding the amino acid sequence of No. 34 as a probe . Specifically, hybridization was performed at 65 ° C. in the presence of 0.7 to 1.0 mol / L NaCl using a filter on which a polynucleotide derived from colony or plaque was immobilized, and then 0.1 to 2 Wash the filter under 65 ° C conditions using a double-concentration SSC (Saline-sodium citrate) solution (the composition of the single-concentration SSC solution consists of 150 mmol / L sodium chloride and 15 mmol / L sodium citrate). Polynucleotides that can be identified by:

  Hybridization is described in Sambrook J. et al., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press (2001), Ausbel FM et al., Current Protocols in Molecular Biology, Supplement 1-38, John Wiley and Sons (1987-1997), Glover DM and Hames BD, DNA Cloning 1: Core Techniques, A practical Approach, Second Edition, Oxford University Press (1995), etc. .

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

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

  As other polynucleotides that can be hybridized, a polynucleotide encoding the amino acid sequence of SEQ ID NO: 34 is approximately 60% or more and 65% when calculated using the default parameters by an analysis program such as BLAST. 70% or more, 75% or more, 80% or more, 85% or more, 88% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.3% or more, Examples include DNA having 99.5% or more, 99.7% or more, 99.8% or more, and 99.9% or more identity. The identity of amino acid sequences and base sequences can be determined using the method described above. In one embodiment of the present invention, the base sequence identity value is determined using BLAST.

  n represents an integer of 1 to 5, preferably 2 or 3, and particularly preferably 2.

In the polypeptide represented by the formula (Z) _n , each Z may be the same or different.

As the polypeptide represented by the formula (Z) _n , the polypeptide represented by the formula (Z) ₂ is particularly preferable.

The polypeptide represented by formula (Z) ₂ has, for example, substantially the same activity or function as the polypeptide consisting of the amino acid sequence of SEQ ID NO: 36 or the polypeptide consisting of the amino acid sequence of SEQ ID NO: 36. A polypeptide. In the present specification, a polypeptide consisting of the amino acid sequence of SEQ ID NO: 36 or a polypeptide having substantially the same activity or function as the polypeptide consisting of the amino acid sequence of SEQ ID NO: 36 is referred to as a “ZZ domain”. is there.

  The “substantially the same quality of activity or function” means, for example, an activity or function that allows the fusion protein to be expressed as a soluble protein when expressed as a fusion protein with the protein of the present invention. Such activity or function, for example, the IgG binding ability of the ZZ domain can be measured by an IgG binding assay.

As a polypeptide represented by the formula (Z) ₂ , more specifically, for example,
(E) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 36;
(F) When the amino acid sequence of SEQ ID NO: 36 comprises an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added, and is expressed as a fusion protein with the polypeptide of the present invention And a polypeptide having an activity or function that allows the fusion protein to be expressed as a soluble protein;
(G) When the fusion protein comprises an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 36 and is expressed as a fusion protein with the protein of the present invention, the fusion protein becomes a soluble protein. A polypeptide having activity or function that can be expressed; and (h) an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence of SEQ ID NO: 35 And a polypeptide selected from the group consisting of polypeptides having an activity or function that can be expressed as a soluble protein when expressed as a fusion protein with the protein of the present invention. .

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

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

The polynucleotide of the present invention includes
(A) a polynucleotide containing a polynucleotide encoding a protein consisting of an amino acid sequence in which the 59th cysteine is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 30;
(B) In the amino acid sequence of SEQ ID NO: 30, the 59th cysteine is replaced with another amino acid, and the 52nd, 56th, 65th, 77th, 120th, 123rd, 127th, 136th and 148th positions A polynucleotide comprising a polynucleotide encoding a protein having a light-emitting catalytic activity comprising an amino acid sequence in which one to a plurality of amino acids are substituted with other amino acids at positions other than luciferin;
(C) a polynucleotide containing a polynucleotide encoding a protein having an amino acid sequence in which the 59th cysteine is replaced with another amino acid in the amino acid sequence of SEQ ID NO: 30 and having luminescence catalytic activity using luciferin as a substrate; And (d) in the amino acid sequence of SEQ ID NO: 30, the 59th cysteine is substituted with another amino acid, and the 52nd, 56th, 65th, 77th, 120th, 123rd, 127th, 136th and 148th And a polynucleotide containing a polynucleotide encoding a protein having a luminescence catalytic activity using luciferin as a substrate, wherein the polynucleotide contains an amino acid sequence in which one or more amino acids are substituted with other amino acids other than the second position. .

  The other amino acid sequence that substitutes the 59th cysteine in the amino acid sequence of SEQ ID NO: 30 is as described above.

  The above “amino acid sequence substituted with one or more amino acids” is as described above.

  A polynucleotide encoding a protein having an amino acid sequence in which one or more amino acids are substituted with respect to a certain amino acid sequence is obtained by site-directed mutagenesis (for example, Gotoh, T. et al., Gene 152, 271-2 275 (1995), Zoller, MJ, and Smith, M., Methods Enzymol. 100, 468-500 (1983), Kramer, W. et al., Nucleic Acids Res. 12, 9441-9456 (1984), Kramer W , and Fritz HJ, Methods. Enzymol. 154, 350-367 (1987), Kunkel, TA, Proc. Natl. Acad. Sci. USA. 82, 488-492 (1985), Kunkel, Methods Enzymol. 85, 2763- 2766 (1988), etc.), a method using amber mutation (see, for example, Gapped duplex method, Nucleic Acids Res. 12, 9441-9456 (1984), etc.) and the like.

  PCR (for example, HoS. N. et al., Gene 77, using a pair of primers each having a 5′-end of a sequence into which the target mutation (deletion, addition, substitution and / or insertion) has been introduced) 51 (1989), etc.) can also introduce mutations into a polynucleotide.

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

As a polynucleotide of a preferred embodiment of the present invention, for example,
(A) a polynucleotide containing a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 32, ie, the amino acid sequence of SEQ ID NO: 30 in which the 59th cysteine is replaced with serine;
(B) a polynucleotide comprising a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 32,
Etc.

  Examples of the “polynucleotide encoding the protein consisting of the amino acid sequence of SEQ ID NO: 32” include a polynucleotide consisting of the base sequence of SEQ ID NO: 31.

As a polynucleotide containing a polynucleotide encoding a protein containing the amino acid sequence of SEQ ID NO: 32, for example,
(A) a polynucleotide comprising a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2, 4 or 32;
(B) a polynucleotide comprising a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 2, 4 or 32,
Etc.

  Examples of the “polynucleotide encoding the protein consisting of the amino acid sequence of SEQ ID NO: 2” include a polynucleotide consisting of the base sequence of SEQ ID NO: 1. Examples of the “polynucleotide encoding the protein consisting of the amino acid sequence of SEQ ID NO: 4” include a polynucleotide consisting of the base sequence of SEQ ID NO: 3.

A particularly preferred embodiment of the polynucleotide of the present invention is:
(A) a polynucleotide containing a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2; and (b) a polynucleotide containing a polynucleotide consisting of the base sequence of SEQ ID NO: 1.

  The polynucleotide of the present invention further comprises a polynucleotide encoding another peptide sequence, such as the nucleotide sequences of SEQ ID NOs: 1 and 3, at the 5 ′ end and / or 3 ′ end, preferably at the 5 ′ end. May be. Examples of the polynucleotide encoding another peptide sequence include a peptide sequence for purification, a secretory signal peptide sequence, a peptide sequence for expressing the protein of the present invention as a soluble protein, and an epitope sequence capable of recognizing an antibody. Mention may be made of a polynucleotide encoding at least one peptide sequence selected from the group.

  As a polynucleotide encoding a peptide sequence for purification, a polynucleotide containing a base sequence encoding a peptide sequence for purification used in the art can be used. Examples of the peptide sequence for purification include those described above.

  As a polynucleotide encoding a secretory signal peptide, a polynucleotide containing a nucleic acid sequence encoding a secretory signal peptide known in the art can be used. Examples of the secretory signal peptide include those described above.

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

3． Recombinant vector and transformant of the present invention Furthermore, the present invention provides a recombinant vector and a transformant containing the above-described polynucleotide of the present invention.

Production of Recombinant Vector The recombinant vector of the present invention can be obtained by ligating (inserting) the polynucleotide (DNA) of the present invention into an appropriate vector. More specifically, it can be obtained by cleaving a purified polynucleotide (DNA) with an appropriate restriction enzyme, inserting it into a restriction enzyme site or a multiple cloning site of an appropriate vector, and ligating the vector. The vector for inserting the polynucleotide of the present invention is not particularly limited as long as it can replicate in the host, and examples thereof include plasmids, bacteriophages, animal viruses and the like. Examples of plasmids include plasmids derived from E. coli (eg, pBR322, pBR325, pUC118, pUC119, etc.), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, etc.), and plasmids derived from yeast (eg, YEp13, YEp24, YCp50, etc.). can give. Examples of the bacteriophage include λ phage. Examples of animal viruses include retroviruses, vaccinia viruses, insect viruses (eg, baculoviruses) and the like. Moreover, a pCold I vector, a pCold II vector, a pCold III vector, a pCold IV vector (manufactured by Takara Bio Inc.) and the like can also be suitably used.

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

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

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

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

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

As an expression vector and a transformant expression vector containing a promoter sequence capable of inducing expression at a low temperature, an expression vector containing a promoter sequence capable of inducing expression at a low temperature is particularly preferable.
The expression vector containing a promoter sequence capable of inducing expression at a low temperature specifically means an expression vector containing the following promoter sequence and coding sequence:
(1) a promoter sequence capable of inducing expression at low temperature; and (2) a coding sequence containing the polynucleotide of the present invention.

Of these, expression vectors containing the following promoter sequences and coding sequences are preferred:
(1) Promoter sequence capable of inducing expression at low temperature;
(2) Formula (Z) _n
(In the formula, n and Z have the same meaning as described above.)
And a first code comprising a polynucleotide encoding a polypeptide having an activity or function that can be expressed as a soluble protein when expressed as a fusion protein with the protein of the present invention. A sequence; and (3) a second coding sequence containing a polynucleotide of the invention.

In addition, expression vectors containing the following promoter sequences and coding sequences are most preferred:
(1) Promoter sequence capable of inducing expression at low temperature;
(2) Formula (Z) ₂
(In the formula, Z represents the same meaning as described above.)
A first coding sequence containing a polynucleotide encoding a polypeptide represented by: and (3) a second coding sequence containing a polynucleotide of the invention.

  The promoter sequence capable of inducing expression at a low temperature means a promoter sequence capable of inducing the expression of the fusion protein by lowering the temperature from the culture conditions for growing the host cells. Examples of the promoter capable of inducing expression at a low temperature include a promoter of a gene encoding a cold shock protein (cold shock gene). Examples of the cold shock gene promoter include those described above.

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

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

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

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

Four. Production of the protein of the present invention The present invention also provides a method for producing the protein of the present invention, comprising the steps of culturing the transformant to produce the protein of the present invention. The protein of the present invention can be obtained, for example, by culturing the transformant under conditions capable of expressing the polynucleotide (DNA) encoding the protein of the present invention, producing and accumulating the protein of the present invention, and separating and purifying the protein. Can be manufactured by.

Culturing of the transformant The transformant of the present invention can be cultured according to a usual method used for culturing a host. By the culture, the protein of the present invention is produced by the transformant, and the protein of the present invention is accumulated in the transformant or in the culture solution.

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

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

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

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

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

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

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

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

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

Five. Use of the protein of the present invention
Use as Reporter Protein The protein of the present invention can also be used as a reporter protein for measurement of transcriptional activity of a promoter or the like. A vector is constructed in which a polynucleotide encoding the protein of the present invention (ie, the polynucleotide of the present invention) is fused to a target promoter or other expression control sequence (for example, an enhancer). By introducing the vector into a host cell and detecting luminescence derived from the protein of the present invention in the presence of luciferin, the activity of the target promoter or other expression control sequence can be measured.
The polynucleotide of the present invention can be used as a reporter gene as described above.

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

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

Bioluminescence Resonance Energy Transfer (BRET) Method The protein of the present invention is used for analysis methods such as analysis of physiological function and measurement of enzyme activity using the principle of intermolecular interaction by bioluminescence resonance energy transfer (BRET) method. be able to.
For example, when the photoprotein of the present invention is used as a donor protein and an organic compound or fluorescent protein is used as an acceptor, the interaction between the proteins is detected by causing bioluminescence resonance energy transfer (BRET) between them. be able to. In one embodiment of the present invention, the organic compound used as an acceptor is Hoechist3342, Indo-1, DAP1, or the like. In another embodiment of the present invention, the fluorescent protein used as an acceptor is green fluorescent protein (GFP), blue fluorescent protein (BFP), mutant GFP fluorescent protein, phycobilin, and the like. In a preferred embodiment of the present invention, the physiological function to be analyzed is orphan receptor (especially G protein-coupled receptor), apoptosis, or transcriptional regulation by gene expression. In a preferred embodiment of the present invention, the enzyme to be analyzed is a protease, esterase or phosphorylase.

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

6． Kit of the present invention The present invention also provides a kit comprising any one selected from the protein of the present invention, the polynucleotide of the present invention, the recombinant vector of the present invention and the transformant of the present invention. The kit of the present invention may further contain luciferin (for example, coelenterazines). The kit of the present invention can be produced by commonly used materials and methods. The kits of the invention may include, for example, sample tubes, plates, instructions for kit users, solutions, buffers, reagents, samples suitable for standardization or control samples. The kit of the present invention may further contain a salt containing a halide ion.
The kit of the present invention can be used for measurement using the above-described reporter protein or reporter gene, detection marker by luminescence, analysis of physiological function by BRET method, measurement of enzyme activity, and the like.

7． Luminescent reaction method
Luminescent Catalytic Activity The protein of the present invention has an activity of catalyzing a reaction in which luciferin (for example, coelenterazines) is oxidized with oxygen molecules to produce excited oxyluciferin. Excited oxyluciferin emits visible light when it reaches the ground state. That is, the protein of the present invention catalyzes a luminescence reaction using luciferin (for example, coelenterazines) as a substrate and has an activity of generating luminescence. This activity is sometimes referred to herein as “luminescence catalytic activity”.

Luminescence Reaction The luminescence reaction using the protein of the present invention and luciferin (for example, coelenterazines) as a substrate can be performed by contacting the protein of the present invention with luciferin. As the reaction conditions, it can be carried out under conditions usually used for luminescence reaction using Gaussia luciferase or conditions based thereon (for example, WO99 / 49019, J. Biol. Chem. 279, 3212-3217 (2004), And their references).

  Specifically, as the reaction solvent, for example, a buffer such as a Tris-HCl buffer or a sodium phosphate buffer, water, or the like is used.

  The reaction temperature is generally about 4 ° C to about 40 ° C, preferably about 4 ° C to about 25 ° C.

  The pH of the reaction solution is usually about 5 to about 10, preferably about 6 to about 9, more preferably about 7 to about 8, and particularly preferably about 7.5.

  As luciferin, coelenterazines are preferable, and coelenterazine is particularly preferable.

  Luciferin may be added to the reaction system as a polar solvent such as dimethylformamide or dimethylsulfoxide, or an alcohol solution such as methanol, ethanol or butanol.

Activation of Luminescent Activity The luminescent activity (luminescent catalytic activity) of the protein of the present invention using luciferin (for example, coelenterazines) as a substrate is activated by halide ions.
Examples of the halide ion include fluorine ion, chlorine ion, bromine ion and iodine ion, and chlorine ion, bromine ion and iodine ion are preferable.

  The concentration of halide ions is usually about 10 μM to about 100 mM, preferably about 100 μM to about 50 mM, particularly preferably about 1 mM to about 20 mM.

Examples of the method of adding halide ions to the reaction system include a method of adding as a salt. Examples of the salt used include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salt, magnesium salt and barium salt. More specifically, NaF, NaCl, NaBr, NaI, KF, KCl, KBr, KI, CaF ₂ , CaCl ₂ , CaBr ₂ , CaI ₂ , MgF ₂ , MgCl ₂ , MgBr ₂ , MgI _{2 and the} like can be mentioned.

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

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

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

Outline When Gaussia luciferase is expressed in E. coli, 90% or more is expressed as an insoluble protein, and it is known that this insoluble protein does not exhibit luminescence activity. Therefore, the present inventors first expressed Gaussia luciferase as a fusion protein with 116 amino acid residues (ZZ-domain) having IgG binding ability of protein A in order to express Gaussia luciferase as a soluble protein. Was established and a solubilized Gaussia luciferase was successfully expressed (Japanese Patent Laid-Open No. 2008-99669).

  This time, the present inventors have determined which of the 10 cysteine residues in the amino acid sequence of the catalytically active portion of natural Gaussia luciferase can be mutated without affecting the luminescent catalytic activity. I decided to confirm. For this purpose, the present inventors sequentially substituted all the amino acids of 10 cysteine residues into serine residues structurally similar to cysteine residues by site-specific mutagenesis. The luminescence activity of Gaussia luciferase and the effect of substitution with serine were investigated.

  As a result, the ZZ-domain fusion mutant Gaussia luciferase in which the 59th cysteine residue in the amino acid sequence of the catalytic part of Gaussia luciferase was replaced with a serine residue is a luminescent catalytic activity that exceeds the activity of natural Gaussia luciferase. Moreover, it was revealed that the regeneration (refolding) to luciferase having a luminescent catalytic activity is faster than that of natural Gaussia luciferase. On the other hand, the luminescence catalytic activity of the serine residue substitution mutant luciferase of each of the other nine cysteine residues alone was 3% or less when the luminescence catalytic activity of natural Gaussia luciferase was taken as 100%.

  Furthermore, as a result of measuring the luminescence pattern of the ZZ-domain fusion mutant Gaussia luciferase in which the 59th cysteine residue was substituted with a serine residue, the luminescence half-life was 3 minutes or more. This revealed that the mutant Gaussia luciferase is a slow decay mutant.

  Next, in order to confirm the effect of the presence or absence of the ZZ-domain on the luminescence catalytic activity, a mutant Gaussia luciferase having no ZZ-domain and having the 59th cysteine residue substituted with a serine residue was prepared. As a result, the mutant Gaussia luciferase showed a luminescence pattern similar to that of the ZZ-domain fusion mutant Gaussia luciferase.

Example 1 Construction of ZZ domain fusion mutant Gaussia luciferase expression vector Gaussia luciferase derived from Gausia princess, a deep-sea co-poda, is expressed in Escherichia coli as a soluble protein in the secretion signal of Gaussia luciferase gene (hGL gene) Construction of 10 ZZ domain fusion mutant Gaussia luciferase expression vectors by replacing the cysteine residues in the translation region excluding the sequence with serine residues and inserting them into the expression vector pCold-ZZ-X (manufactured by Chisso) did.

(1) Construction of vector in which cysteine residue of Gaussia luciferase is substituted with serine residue The site-directed mutagenesis was performed by PCR according to the method of Ho et al. Described in Gene (1989) 77, 51-59. .
Specifically, using the following two PCR primer pairs using pcDNA3-hGL having a Gaussia luciferase gene (Prolmi) as a template, PCR using a PCR kit (Takara Bio) (cycle conditions: 25 cycles) 1 min / 94 ° C., 1 min / 50 ° C., 1 min / 72 ° C.) to amplify the two DNA regions.

Primer pair:
pCold-F (5 ′ ACG CCA TAT CGC CGA AAG G 3 ′) (SEQ ID NO: 5); and
GL-C59S_R (5 ′ CTT GAT GTG GGA C AA GCT T AT CAG 3 ′) (SEQ ID NO: 14).

Primer pair:
GL-C59S_F (5 ′ CTG AT A AGC TT G TCC CAC ATC AAG 3 ′) (SEQ ID NO: 13); and
pCold-R (5 ′ GGC AGG GAT CTT AGA TTC TG 3 ′) (SEQ ID NO: 6).

  Using the obtained two fragments as templates, the following two kinds of PCR primers were used, and PCR (cycle condition 25 cycles; 1 minute / 94 ° C., 1 minute / 50 ° C., 1 min / 72 ° C.) was performed to amplify the desired DNA region.

Primer:
GL6-N / EcoRI (5 'gcc GAA TTC AAG CCC ACC GAG AAC AAC GAA 3') (SEQ ID NO: 7); and
GL7-C / XbaI (5 ′ gcc TCT AGA TTA GTC ACC ACC GGC CCC CTT 3 ′) (SEQ ID NO: 8).

  The obtained fragment was purified with a PCR purification kit (Qiagen) and digested with the restriction enzyme EcoRI / XbaI by a conventional method, and then placed on the restriction enzyme EcoRI / XbaI site of pCold-ZZ-X (Chizo). By ligating, an expression vector pCold-ZZGL-C59S was constructed (FIG. 1).

  According to the same method, the mutant Gaussia luciferase expression vector pCold-ZZGL-C52S, pCold-ZZGL-C56S, pCold-ZZGL-C65S, pCold-ZZGL-C77S, pCold-ZZGL-C120S, pCold-ZZGL-C123S , pCold-ZZGL-C127S, pCold-ZZGL-C136S, and pCold-ZZGL-C148S were constructed respectively. At this time, for the construction of each expression vector, the following primers were used in place of the primers GL-C59S_F (SEQ ID NO: 13) and GL-C59S_R (SEQ ID NO: 14).

pCold-ZZGL-C52S: primers of SEQ ID NO: 9 and SEQ ID NO: 10
pCold-ZZGL-C56S: primer of SEQ ID NO: 11 and SEQ ID NO: 12
pCold-ZZGL-C65S: primer of SEQ ID NO: 15 and SEQ ID NO: 16
pCold-ZZGL-C77S: SEQ ID NO: 17 and SEQ ID NO: 18 primers
pCold-ZZGL-C120S: primers of SEQ ID NO: 19 and SEQ ID NO: 20
pCold-ZZGL-C123S: SEQ ID NO: 21 and SEQ ID NO: 22 primers
pCold-ZZGL-C127S: primers of SEQ ID NO: 23 and SEQ ID NO: 24
pCold-ZZGL-C136S: SEQ ID NO: 25 and SEQ ID NO: 26 primers
pCold-ZZGL-C148S: SEQ ID NO: 27 and SEQ ID NO: 28 primers

  The insert DNA was confirmed by determining the base sequence with a DNA sequencer (manufactured by ABI).

  Here, the DNA sequence encoding ZZGL-C59S inserted into the expression vector pCold-ZZGL-C59S is shown in SEQ ID NO: 1. The amino acid sequence of the ZZGL-C59S is shown in SEQ ID NO: 2.

(2) Construction of a vector in which two cysteines are substituted with serine Using the expression vector pCold-ZZGL-C59S constructed in Example 1 (1) as a template and using the following two PCR primer pairs, a PCR kit (Takara) PCR (cycle conditions: 25 cycles; 1 minute / 94 ° C., 1 minute / 50 ° C., 1 minute / 72 ° C.) was carried out by Biotechnology, and two DNA regions were amplified.

Primer pair:
pCold-F (5 ′ ACG CCA TAT CGC CGA AAG G 3 ′) (SEQ ID NO: 5); and
GL-C120S R (5 ′ AGT TGT GCA GTC GAC GGA CAG ATC 3 ′) (SEQ ID NO: 20).

Primer pair:
GL-C120S F (5 ′ GAT CTG TCC GTC GAC TGC ACA ACT 3 ′) (SEQ ID NO: 19); and
pCold-R (5 ′ GGC AGG GAT CTT AGA TTC TG 3 ′) (SEQ ID NO: 6).

  Using the obtained two fragments as templates, PCR (cycle conditions 25 cycles; 1 minute / 94 ° C., 1 minute / 50 ° C.) using a PCR kit (manufactured by Takara Bio Inc.) using the following two kinds of PCR primers: 1 min / 72 ° C.) was performed to amplify the desired DNA region.

Primer:
GL6-N / EcoRI (5 'gcc GAA TTC AAG CCC ACC GAG AAC AAC GAA 3') (SEQ ID NO: 7); and
GL7-C / XbaI (5 ′ gcc TCT AGA TTA GTC ACC ACC GGC CCC CTT 3 ′) (SEQ ID NO: 8).

  The obtained fragment was purified with a PCR purification kit (Qiagen), digested with restriction enzymes EcoRI / XbaI by a conventional method, and then pCold-ZZ-X (Protein Express. Purif. 2009 66: 52-57) The expression vector pCold-ZZGL-C59, 120S was constructed by ligation to the restriction enzyme EcoRI / XbaI site.

Similarly, expression vectors pCold-ZZGL-C59, 127S were constructed. At this time, for the construction of the expression vector pCold-ZZGL-C59, 127S, instead of the primers GL-C120S_F (SEQ ID NO: 19) and GL-C120S_R (SEQ ID NO: 20), SEQ ID NO: 23 and SEQ ID NO: 24 primers were used.
The insert DNA was confirmed by determining the base sequence with a DNA sequencer (manufactured by ABI).

Example 2 Construction of Gaussia luciferase-C59S expression vector A vector pCold-hGL-C59S that expresses Gaussia luciferase-C59S except the ZZ domain of the expression vector pCold-ZZGL-C59S prepared in Example 1 was constructed.
The expression vector pCold-ZZGL-C59S prepared in Example 1 is digested with the restriction enzyme EcoRI / XbaI by a conventional method and then ligated to the restriction enzyme EcoRI / XbaI site of pCold II (manufactured by Takara). pCold-hGL-C59S was constructed (FIG. 2).
Here, the DNA sequence encoding hGL-C59S inserted into the expression vector pCold-hGL-C59S is shown in SEQ ID NO: 3. The amino acid sequence of hGL-C59S is shown in SEQ ID NO: 4.

Example 3 Method for Measuring Luminescent Activity The 12 expression vectors prepared in Example 1 (pCold-ZZGL-C52S, pCold-ZZGL-C56S, pCold-ZZGL-C59S, pCold-ZZGL-C65S, pCold-ZZGL-C77S, pCold−ZZGL−C120S, pCold−ZZGL−C123S, pCold−ZZGL−C127S, pCold−ZZGL−C136S, pCold−ZZGL−C148S, pCold−ZZGL−C59, 120S and pCold−ZZGL−C59, 127S) and pCold−ZZ -HGL (prepared by the method described in Example 2 of JP-A-2008-99669) was introduced into Escherichia coli BL21 by a conventional method. The resulting transformant was added to 10 ml of LB liquid medium containing ampicillin (50 μg / ml) (10 g of bactotryptone, 5 g of yeast extract, 5 g of sodium chloride, pH 7.2 per liter of water). Inoculated and cultured at 37 ° C. for 3 hours. Thereafter, the culture solution is cooled on ice water, and isopropyl-β-D (−)-thiogalactopyranoside (IPTG, manufactured by Wako Pure Chemical Industries, Ltd.) is added to the culture solution to a final concentration of 0.2 mM. Furthermore, the cells were cultured at 15 ° C. for 18 hours.

  Collect 1 ml of the culture broth by centrifugation (10,000 rpm, 5 minutes), suspend the collected bacteria in 50 mM Tris-HCl (pH 7.6) containing 500 μl of 10 mM EDTA, and cool under ice cooling. Ultrasonic crushing (Branson, Sonifier model cycle 250) was performed for 5 seconds. Thereafter, the cell disruption solution was centrifuged at 10,000 rpm (12,000 × g) for 1 minute. The obtained soluble fraction was used as a Gaussia luciferase enzyme solution.

  The soluble fraction obtained above was allowed to stand at 4 ° C. for 25 hours, and then the luminescent catalytic activity of Gaussia luciferase was measured as follows. The substrate coelenterazine (manufactured by Chisso) (0.5 μg / μl) dissolved in ethanol was dissolved in 0.1 ml of PBS containing 0.01% Tween20 and 10 mM EDTA (manufactured by Sigma), and 1 μl of Gaussia luciferase was mixed and mixed. The luminescence reaction was started at ° C., and the luminescence catalytic activity was measured for 30 seconds with a luminescence measuring device Luminescencer-PSN AB2200 (manufactured by Atto). The luminescence catalytic activity of each mutant Gaussia luciferase was expressed as relative activity (%) when the maximum luminescence intensity (Imax) of Gaussia luciferase (natural Gaussia luciferase) from pCold-ZZ-hGL was 100. The results are shown in Table 1.

  Table 1 shows the results of measuring the luminescence activity of mutant Gaussia luciferase and natural Gaussia luciferase in which each cysteine residue is substituted with a serine residue.

  As shown in Table 1, the mutant Gaussia luciferase in which cysteine 59 was substituted with serine 59 (cysteine 59 → serine 59 substitution) had 297% activity compared to natural Gaussia luciferase. In addition, cysteine 59 → serine 59, cysteine 120 → serine 120 substitution mutant Gaussia luciferase showed 25.1% activity compared to natural Gaussia luciferase. On the other hand, other cysteine residue-substituted mutant Gaussia luciferases showed almost no luminescent catalytic activity.

  Based on the above, the 59th cysteine residue in the amino acid sequence (168 residues) of the protein of the catalytic portion of Gaussia luciferase retains the luminescent catalytic activity even without forming an -S-S- bond. It became clear. That is, it was found that the 59th cysteine residue can be substituted with another amino acid.

  On the other hand, the cysteine 59 → serine 59 substitution mutant Gaussia luciferase has a luminous intensity nearly three times higher than that of the natural Gaussia luciferase, and thus the substitution Gaussia luciferase has a faster -S-S-bond formation speed. It may be a thing. Therefore, in order to confirm the efficiency and speed of refolding of cysteine 59 → serine 59 substitution mutant Gaussia luciferase, the soluble fraction obtained above was placed at 4 ° C. for 0, 1, 3, 7, 25 or 54 hours. The luminescence activity was measured in the same manner as described above.

  The results are shown in FIG. Natural Gaussia luciferase gradually increases in activity after 3 hours, whereas cysteine 59 → serine 59 substitution mutant Gaussia luciferase has a maximum activity (100%) at 25 hours. Compared to the above, it showed a regenerative activity of 80% or more within 7 hours.

  From the above, in order to produce Gaussia luciferase with luminescent catalytic activity, it is necessary to form an accurate intramolecular —S—S— bond involving cysteine residues other than 59th. In addition, by replacing the 59th cysteine residue with another amino acid such as a serine residue, an intramolecular —S—S— bond is formed more accurately and efficiently, and a highly active mutant Gaussia luciferase It was found that can be prepared.

  Similarly, cysteine 59 → serine 59 substitution mutant Gaussia luciferase (pCold-hGL-C59S) without ZZ domain was compared with natural luciferase (pCold-hGL) to confirm the efficiency and speed of refolding. I did it. Expression vectors pCold-hGL-C59S and pCold-hGL prepared in Example 2 (prepared by the method described in Inouye, S. & Sahara, Y. (2008) Biochem. Biophys. Res. Commun. 365, 96-101) Were introduced into Escherichia coli BL21 by a conventional method. Then, a Gaussia luciferase enzyme solution was obtained as a soluble fraction in the same manner as described above. The obtained soluble fraction was allowed to stand at 4 ° C. for 22.5 hours, and then the luminescent catalytic activity of Gaussia luciferase was measured in the same manner as described above. The results are shown in Table 2.

  Table 2 shows the measurement results of the luminescent catalytic activity of cysteine 59 → serine 59 substitution mutant Gaussia luciferase without ZZ domain and natural Gaussia luciferase without ZZ domain. The mutant Gaussia luciferase in the soluble fraction had a luminous catalytic activity that was about 19 times higher than that of the natural Gaussia luciferase. Further, the obtained soluble fraction was allowed to stand at 4 ° C. for 0, 0.5, 1, 2, 3, 5, 7, or 22.5 hours, and then the luminescence activity was measured in the same manner as described above. The results are shown in FIG. Natural Gaussia luciferase has lower activity than Cysteine 59 → Serine 59 mutation-substituted Gaussia luciferase, and its regenerative activity over time is slow. On the other hand, it was revealed that cysteine 59 → serine 59 mutation-substituted Gaussia luciferase is expressed as a soluble protein and efficiently regenerated.

Example 4 Measurement of Luminescence Pattern Expression vector pCold-ZZGL (prepared by the method described in Example 2 of JP-A-2008-99669), pCold-ZZGL-C59S of Example 1, pCold-hGL- of Example 2 C59S and pCold-hGL (prepared by the method described in Inouye, S. & Sahara, Y. (2008) Biochem. Biophys. Res. Commun. 365, 96-101) were introduced into Escherichia coli BL21 by conventional methods. did. In the same manner as in Example 3, a Gaussia luciferase enzyme solution was obtained as a soluble fraction.
The soluble fraction obtained above was allowed to stand at 4 ° C. for 24 hours, and then the luminescent catalytic activity of Gaussia luciferase was measured as follows. The substrate coelenterazine (0.5 μg / μl) dissolved in ethanol was dissolved in 50 ml Tris-HCl (pH 7.6) containing 0.1 ml 10 mM EDTA, and 1 μl of Gaussia luciferase was mixed to start the luminescence reaction, and the luminescence measurement Luminescence activity was measured for 30 seconds with an apparatus Luminescencer-PSN AB2200 (manufactured by Ato). A light emission pattern was drawn by plotting the relative activity (%) of the maximum light emission intensity (Imax) when the maximum light emission intensity (Imax) at the start of measurement was 100 (%). The results are shown in FIG. 5 for Gaussia luciferase having a ZZ domain and in FIG. 6 for Gaussia luciferase not having a ZZ domain.

  Natural Gaussia luciferase rapidly attenuates the luminescence intensity immediately after the addition of the substrate, and becomes less than 20% in relative activity after 30 seconds. On the other hand, cysteine 59 → serine 59 substitution mutant Gaussia luciferase showed luminescence intensity of 70% or more in relative activity even after 30 seconds, and it was revealed that the decay time was significantly longer than that of natural Gaussia luciferase. Moreover, regardless of the presence or absence of the ZZ domain, the mutant Gaussia luciferase substituted with cysteine 59 → serine 59 has a significantly long decay time, and although not shown in FIGS. 5 and 6, the half-life of luminescence is 3 minutes or more. It became clear.

[SEQ ID NO: 1] This shows a DNA sequence encoding ZZ-gaussia luciferase-C59S protein inserted into expression vector pCold-ZZGL-C59S prepared in Example 1.
[SEQ ID NO: 2] This shows the amino acid sequence of ZZ-Gaussia luciferase-C59S inserted into expression vector pCold-ZZGL-C59S prepared in Example 1.
[SEQ ID NO: 3] This shows the DNA sequence encoding Gaussia luciferase-C59S protein inserted into expression vector pCold-hGL-C59S prepared in Example 2.
[SEQ ID NO: 4] This shows the amino acid sequence of Gaussia luciferase-C59S inserted in expression vector pCold-hGL-C59S prepared in Example 2.
[SEQ ID NO: 5] This shows the base sequence of the primer of the promoter region of pCold vector.
[SEQ ID NO: 6] This shows the base sequence of the primer in the terminator region of pCold vector.
[SEQ ID NO: 7] This shows the base sequence of the amino terminal primer of Gaussia luciferase.
[SEQ ID NO: 8] This shows the base sequence of the carboxyl terminal primer of Gaussia luciferase.
[SEQ ID NO: 9] This shows the base sequence of the primer used for the introduction of C52S mutation in Gaussia luciferase.
[SEQ ID NO: 10] This shows the base sequence of the primer used for the C52S mutation introduction of Gaussia luciferase.
[SEQ ID NO: 11] This shows the base sequence of the primer used for the introduction of C56S mutation in Gaussia luciferase.
[SEQ ID NO: 12] This shows the base sequence of the primer used for the introduction of C56S mutation in Gaussia luciferase.
[SEQ ID NO: 13] This shows the base sequence of the primer used for the introduction of C59S mutation in Gaussia luciferase.
[SEQ ID NO: 14] This shows the base sequence of the primer used for the introduction of C59S mutation in Gaussia luciferase.
[SEQ ID NO: 15] This shows the base sequence of the primer used for the introduction of C65S mutation in Gaussia luciferase.
[SEQ ID NO: 16] This shows the base sequence of the primer used for introducing C65S mutation in Gaussia luciferase.
[SEQ ID NO: 17] This shows the base sequence of the primer used for the introduction of C77S mutation in Gaussia luciferase.
[SEQ ID NO: 18] This shows the base sequence of the primer used for the mutation introduction of Gaussia luciferase C77S.
[SEQ ID NO: 19] This shows the base sequence of the primer used for the introduction of C120S mutation in Gaussia luciferase.
[SEQ ID NO: 20] This shows the base sequence of the primer used for the introduction of C120S mutation in Gaussia luciferase.
[SEQ ID NO: 21] This shows the base sequence of the primer used for the introduction of C123S mutation in Gaussia luciferase.
[SEQ ID NO: 22] This shows the base sequence of the primer used for the introduction of C123S mutation in Gaussia luciferase.
[SEQ ID NO: 23] This shows the base sequence of the primer used for the introduction of C127S mutation in Gaussia luciferase.
[SEQ ID NO: 24] This shows the base sequence of the primer used for the introduction of C127S mutation in Gaussia luciferase.
[SEQ ID NO: 25] This shows the base sequence of the primer used for the introduction of C136S mutation in Gaussia luciferase.
[SEQ ID NO: 26] This shows the base sequence of the primer used for the introduction of C136S mutation in Gaussia luciferase.
[SEQ ID NO: 27] This shows the base sequence of the primer used for the introduction of C148S mutation in Gaussia luciferase.
[SEQ ID NO: 28] This shows the base sequence of the primer used for the introduction of C148S mutation in Gaussia luciferase.
[SEQ ID NO: 29] This represents the nucleotide sequence of a polynucleotide encoding the catalytic portion of Gaussia luciferase.
[SEQ ID NO: 30] This represents the amino acid sequence of the catalytic portion of Gaussia luciferase.
[SEQ ID NO: 31] This shows the base sequence of the polynucleotide encoding the protein in which the 59th cysteine residue in the amino acid sequence of the catalytic portion of Gaussia luciferase is substituted with serine.
[SEQ ID NO: 32] This represents a protein in which the 59th cysteine residue in the amino acid sequence of the catalytic portion of Gaussia luciferase is substituted with serine.
[SEQ ID NO: 33] This shows the base sequence of DNA encoding the amino acid sequence of the polypeptide represented by formula Z.
[SEQ ID NO: 34] This shows the amino acid sequence of the polypeptide represented by formula Z.
[SEQ ID NO: 35] This shows the base sequence of DNA encoding the amino acid sequence of the polypeptide represented by formula (Z) ₂ .
[SEQ ID NO: 36] This shows the amino acid sequence of the polypeptide represented by formula (Z) ₂ .

Claims (16)

The protein according to any one of the following (a) to (d):
(A) a protein comprising an amino acid sequence in which the 59th cysteine is replaced with another amino acid in the amino acid sequence of SEQ ID NO: 30;
(B) In the amino acid sequence of SEQ ID NO: 30, the 59th cysteine is replaced with another amino acid, and the 52nd, 56th, 65th, 77th, 120th, 123rd, 127th, 136th and 148th positions A protein having an amino acid sequence in which one to a plurality of amino acids are substituted with other amino acids other than the position, and having a luminescence catalytic activity using luciferin as a substrate;
(C) a protein having an amino acid sequence in which the 59th cysteine in the amino acid sequence of SEQ ID NO: 30 is substituted with another amino acid and having a luminescence catalytic activity using luciferin as a substrate; and (d) of SEQ ID NO: 30 In the amino acid sequence, the 59th cysteine is substituted with another amino acid, and 1 to a plurality of amino acids other than positions 52, 56, 65, 77, 120, 123, 127, 136 and 148 A protein having an amino acid sequence in which an amino acid is substituted with another amino acid and having a luminescence catalytic activity using luciferin as a substrate. 2. The protein according to claim 1, which is any of the following (a) to (d):
(A) a protein comprising an amino acid sequence in which the 59th cysteine is replaced with another amino acid in the amino acid sequence of SEQ ID NO: 30;
(B) In the amino acid sequence of SEQ ID NO: 30, the 59th cysteine is replaced with another amino acid, and the 52nd, 56th, 65th, 77th, 120th, 123rd, 127th, 136th and 148th positions A protein having a photocatalytic activity comprising an amino acid sequence in which 1 to 25 amino acids other than the position are substituted with other amino acids, and luciferin as a substrate;
(C) a protein having an amino acid sequence in which the 59th cysteine in the amino acid sequence of SEQ ID NO: 30 is substituted with another amino acid and having a luminescence catalytic activity using luciferin as a substrate; and (d) of SEQ ID NO: 30 In the amino acid sequence, the 59th cysteine is substituted with another amino acid, and 1 to 25 other than positions 52, 56, 65, 77, 120, 123, 127, 136 and 148 A protein having an amino acid sequence in which an amino acid is substituted with another amino acid and having a luminescence catalytic activity using luciferin as a substrate. The protein according to claim 1, which is the following (a) or (b):
(A) a protein comprising an amino acid sequence in which the 59th cysteine is replaced with another amino acid in the amino acid sequence of SEQ ID NO: 30; and (b) the 59th cysteine is replaced with another amino acid in the amino acid sequence of SEQ ID NO: 30. A protein having a light-emitting catalytic activity comprising the amino acid sequence and luciferin as a substrate.   The protein according to any one of claims 1 to 3, wherein the other amino acid substituted for the 59th cysteine in the amino acid sequence of SEQ ID NO: 30 is serine. The protein according to claim 4, which is the following (a) or (b):
(A) a protein comprising the amino acid sequence of SEQ ID NO: 2, 4 or 32; and (b) a protein comprising the amino acid sequence of SEQ ID NO: 2, 4 or 32 and having a luminescence catalytic activity using luciferin as a substrate.   A polynucleotide comprising a polynucleotide encoding the protein according to any one of claims 1 to 5.   A recombinant vector containing the polynucleotide according to claim 6.   A transformant into which the recombinant vector according to claim 7 has been introduced.   The method for producing a protein according to any one of claims 1 to 5, comprising a step of culturing the transformant according to claim 8 to produce the protein according to any one of claims 1 to 5.   A kit comprising the protein according to any one of claims 1 to 5.   A kit comprising the polynucleotide according to claim 6, the recombinant vector according to claim 7, or the transformant according to claim 8.   The kit according to claim 10 or 11, further comprising luciferin.   13. The kit according to claim 12, wherein the luciferin is coelenterazines.   14. The kit according to claim 13, wherein the coelenterazine is coelenterazine. The protein according to any one of the following (a) to (d):
(A) a protein comprising an amino acid sequence in which the 59th cysteine is replaced with another amino acid in the amino acid sequence of SEQ ID NO: 30;
(B) In the amino acid sequence of SEQ ID NO: 30, the 59th cysteine is replaced with another amino acid, and the 52nd, 56th, 65th, 77th, 120th, 123rd, 127th, 136th and 148th positions A protein having an amino acid sequence in which one to a plurality of amino acids are substituted with other amino acids other than the position, and having a luminescence catalytic activity using luciferin as a substrate;
(C) a protein having an amino acid sequence in which the 59th cysteine in the amino acid sequence of SEQ ID NO: 30 is substituted with another amino acid and having a luminescence catalytic activity using luciferin as a substrate; and (d) of SEQ ID NO: 30 In the amino acid sequence, the 59th cysteine is substituted with another amino acid, and 1 to a plurality of amino acids other than positions 52, 56, 65, 77, 120, 123, 127, 136 and 148 A method for performing a luminescent reaction, comprising contacting a luciferin with a protein having an amino acid sequence in which an amino acid is substituted with another amino acid and having a luminescence catalytic activity using luciferin as a substrate. The protein according to any one of the following (a) to (d):
(A) a protein comprising an amino acid sequence in which the 59th cysteine is replaced with another amino acid in the amino acid sequence of SEQ ID NO: 30;
(B) In the amino acid sequence of SEQ ID NO: 30, the 59th cysteine is replaced with another amino acid, and the 52nd, 56th, 65th, 77th, 120th, 123rd, 127th, 136th and 148th positions A protein having an amino acid sequence in which one to a plurality of amino acids are substituted with other amino acids other than the position, and having a luminescence catalytic activity using luciferin as a substrate;
(C) a protein having an amino acid sequence in which the 59th cysteine in the amino acid sequence of SEQ ID NO: 30 is substituted with another amino acid and having a luminescence catalytic activity using luciferin as a substrate; and (d) of SEQ ID NO: 30 In the amino acid sequence, the 59th cysteine is substituted with another amino acid, and 1 to a plurality of amino acids other than positions 52, 56, 65, 77, 120, 123, 127, 136 and 148 A bioluminescence resonance energy transfer (BRET) method is performed using a protein having an amino acid sequence substituted with another amino acid and having a luminescence catalytic activity using luciferin as a substrate as a donor protein. Physiological function analysis or enzyme activity measurement method.