WO2024158050A1 - ルシフェラーゼ変異体 - Google Patents

ルシフェラーゼ変異体 Download PDF

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WO2024158050A1
WO2024158050A1 PCT/JP2024/002442 JP2024002442W WO2024158050A1 WO 2024158050 A1 WO2024158050 A1 WO 2024158050A1 JP 2024002442 W JP2024002442 W JP 2024002442W WO 2024158050 A1 WO2024158050 A1 WO 2024158050A1
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amino acid
seq
position corresponding
acid residue
substituted
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French (fr)
Japanese (ja)
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千晶 稲本
敦 一柳
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Kikkoman Corp
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Kikkoman Corp
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Priority to JP2024573236A priority Critical patent/JPWO2024158050A1/ja
Priority to CN202480006843.4A priority patent/CN120457210A/zh
Priority to EP24747374.7A priority patent/EP4656723A1/en
Publication of WO2024158050A1 publication Critical patent/WO2024158050A1/ja
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y113/00—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13)
    • C12Y113/12—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13) with incorporation of one atom of oxygen (internal monooxygenases or internal mixed function oxidases)(1.13.12)
    • C12Y113/12007—Photinus-luciferin 4-monooxygenase (ATP-hydrolysing) (1.13.12.7), i.e. firefly-luciferase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase

Definitions

  • the present disclosure relates to a luciferase mutant having improved thermal stability, a polynucleotide encoding the luciferase mutant, a method for producing the luciferase mutant, a kit for detecting at least one of ATP, ADP, and AMP, which includes the luciferase mutant, and a method for detecting at least one of ATP, ADP, and AMP, which includes using the luciferase mutant.
  • Firefly luciferase is an enzyme that converts adenosine triphosphate (ATP), D-luciferin, and oxygen into adenosine monophosphate (AMP), oxyluciferin, and carbon dioxide in the presence of magnesium ions and oxygen, generating light.
  • ATP adenosine triphosphate
  • AMP adenosine monophosphate
  • Firefly luciferase is widely used, for example, to detect microorganisms in food and beverages using ATP as an indicator, to determine food residues and dirt on hands and utensils, and in highly sensitive measurement methods that utilize various antibody and gene amplification technologies.
  • beetle luciferases such as firefly luciferase generally have the disadvantage that they are unstable to heat and therefore easily inactivated when stored as a reagent. Therefore, attempts have been made to overcome this drawback and obtain luciferases with good thermal stability.
  • Non-Patent Document 1 reports that a North American firefly luciferase in which the amino acid at position 342 has been mutated to alanine has been obtained, and that the luminescence duration of this firefly luciferase has been improved.
  • Patent Document 1 also discloses that luciferase from Genji firefly or Heike firefly in which the amino acid at position 217 has been substituted with a hydrophobic amino acid has heat resistance.
  • Patent Document 2 discloses that heat stability has been improved in firefly luciferase having an amino acid sequence in which the amino acid corresponding to position 287 of Heike firefly luciferase has been mutated to alanine, or the amino acid corresponding to position 392 has been mutated to isoleucine.
  • Patent Document 3 reports a luciferase mutant in which the 393rd position of Heike firefly luciferase (SEQ ID NO: 1) is substituted.
  • the present disclosure aims to provide a firefly luciferase with improved thermal stability. Also, in certain embodiments, the present disclosure aims to provide a firefly luciferase with greatly improved thermal stability.
  • firefly luciferase mutants with specific amino acid substitutions exhibit improved thermal stability, and have completed the present invention, which includes this as one embodiment. Furthermore, the inventors have found that firefly luciferase mutants with multiple specific amino acid substitutions exhibit greatly improved thermal stability, and have completed the present invention, which includes this as one embodiment.
  • a luciferase mutant wherein the luciferase before the amino acid substitution has at least 70%, 80%, or 90% amino acid sequence identity with SEQ ID NO: 10, and the luciferase after the amino acid substitution has a position corresponding to position 252 of SEQ ID NO: 1 substituted with leucine, and the thermostability of the luciferase after the amino acid substitution is improved compared to that of the luciferase before the amino acid substitution.
  • the luciferase mutant according to embodiment 1 or 2 further comprising a substitution of an amino acid residue at a position corresponding to positions 90, 371, 41, 427, 53, 306, or 312 of SEQ ID NO:1.
  • amino acid residue at a position corresponding to position 90 of SEQ ID NO:1 is substituted, and the amino acid residue at the position corresponding to position 90 of SEQ ID NO:1 is an amino acid residue selected from the group consisting of tyrosine, leucine, isoleucine, cysteine, serine, threonine, aspartic acid, glutamic acid, proline, valine, tryptophan, methionine, alanine, glycine, glutamine, asparagine, lysine, histidine, and arginine; (b) an amino acid residue at a position corresponding to position 371 of SEQ ID NO:1 is substituted, and the amino acid residue at the position corresponding to position 371 of SEQ ID NO:1 is an amino acid residue selected from the group consisting of leucine, tyrosine, cysteine, threonine, aspartic acid, glutamic acid, isoleucine, proline, valine, tryp
  • amino acid residue at a position corresponding to position 221 of SEQ ID NO:1 has been substituted, and the amino acid residue at the position corresponding to position 221 of SEQ ID NO:1 is an amino acid residue selected from the group consisting of leucine, tyrosine, cysteine, serine, threonine, aspartic acid, glutamic acid, isoleucine, proline, valine, tryptophan, methionine, alanine, glycine, glutamine, asparagine, lysine, histidine, and arginine, or (ii) an amino acid residue at a position corresponding to position 17 of SEQ ID NO:1 is substituted, and the amino acid residue at the position corresponding to position 17 of SEQ ID NO:1 is an amino acid residue selected from the group consisting of aspartic acid, glutamic acid, lysine, histidine, arginine, tyrosine, cysteine, serine, threon
  • the luciferase before amino acid substitution has an amino acid sequence selected from the group consisting of the following (A) to (F): (A) an amino acid sequence having at least 70%, 80%, or 90% amino acid sequence identity to SEQ ID NO: 10; (B) an amino acid sequence in which a homologous region of SEQ ID NO: 1 in the amino acid sequence of (A) has a sequence identity of 90% or more with a homologous region of luciferase before the amino acid substitution; (C) an amino acid sequence of SEQ ID NO: 10 in which one or several amino acids are substituted, deleted or added at a position other than the position corresponding to position 252 of SEQ ID NO: 1; (D) an amino acid sequence in which one or several amino acids are substituted, deleted or added at positions other than positions corresponding to positions 90, 262, 371, 41, 427, 53, 306, 312, 294, 221, 17, 18, 75, 110, 223, 224, 257, 229 or 158 of SEQ ID NO: 1 in the amino acid sequence
  • the luciferase mutant comprising: [10]
  • the luciferase before amino acid substitution has an amino acid sequence selected from the group consisting of the following (a) to (d): (a) an amino acid sequence having 90% or more amino acid sequence identity with SEQ ID NO: 10; (b) an amino acid sequence in which one or more amino acids have been substituted, deleted or added at a position other than the position corresponding to position 252 of SEQ ID NO: 1; (c) an amino acid sequence in which, in the amino acid sequence of (b), one or several amino acids are substituted, deleted or added at positions other than those corresponding to positions 262, 371, 41, 427, 53, 306, 312, 252, 294, 221, 17, 18, 75, 110, 223, 224, 257, 229 or 158 of SEQ ID NO: 1; and (d) the amino acid sequence of SEQ ID NO: 10; 10.
  • the luciferase mutant of embodiment 9, comprising: [11] A polynucleotide encoding the luciferase mutant according to any one of embodiments 1 to 10. [12] A vector comprising the polynucleotide described in embodiment 11. [13] A host cell comprising the polynucleotide of embodiment 11 or the vector of embodiment 12. [14] A method for producing a luciferase mutant with improved thermostability, comprising the step of culturing the host cell according to embodiment 13. [15] A kit for detecting at least one of ATP, ADP, and AMP, comprising the luciferase mutant according to any one of embodiments 1 to 10.
  • a method for detecting at least one of ATP, ADP, and AMP comprising using a luciferase mutant according to any one of embodiments 1 to 10.
  • a luciferase mutant comprising an amino acid sequence in which one or more amino acid residues at positions corresponding to positions 252, 262, 294, 90, 371, 41, 427, 53, 306, 312, 221, 17, 18, 75, 110, 223, 224, 257, 229, and 158 of SEQ ID NO: 1 have been substituted, wherein the thermostability of the luciferase after amino acid substitution is improved compared to that of the luciferase before amino acid substitution.
  • the present disclosure provides a firefly luciferase with improved thermostability.
  • Figure 1 shows the alignment results of wild-type luciferases from Luciola lateralis, Luciola cruciata, Photinus pyralis, and Photuris pennsylvanica. In the figure, identical amino acid residues in the four amino acid sequences are boxed. This is a continuation of Figure 1.
  • each position of the sequence is defined with SEQ ID NO: 1 as the reference sequence.
  • SEQ ID NO: 1 is the amino acid sequence of luciferase derived from Heike firefly.
  • SEQ ID NO: 10 is a mutant created based on SEQ ID NO: 1. Compared with SEQ ID NO: 1, SEQ ID NO: 10 has amino acid substitutions A217L, E490K, C393L, G326S, V392I, and F467I.
  • the present disclosure relates to a firefly luciferase mutant comprising an amino acid sequence in which an amino acid residue at a position corresponding to positions 252, 262, 294, 90, 371, 41, 427, 53, 306, and/or 312 of SEQ ID NO: 1 has been substituted (these positions may be referred to as position 252, etc., in the present specification).
  • the present disclosure relates to a firefly luciferase mutant further comprising an amino acid sequence in which an amino acid residue at a position corresponding to positions 221, 17, 18, 75, 110, 223, 224, 257, 229, and/or 158 of SEQ ID NO: 1 has been substituted.
  • each of these positions may be substituted with any of 19 types of amino acids other than the amino acid residue in the wild-type sequence.
  • the present disclosure relates to firefly luciferase mutants having one or more amino acid substitutions corresponding to amino acid substitutions F252L, F262Y, F294L, F90Y, F371L, A41V, Y427F, Y53L, Y306F, F221L, F17(D,E,K), Y18L, V75K, I110(D,E,K,R), H223(V,I,L), A224I, and Y257F, Y229(F,L), and V158(D,E) with respect to SEQ ID NO: 1.
  • the luciferase mutants can be mutants with improved thermostability compared to the luciferase prior to the amino acid substitutions.
  • wild type refers to the trait that is most commonly present in nature within a homogeneous population.
  • Firefly luciferase can be derived from any firefly.
  • firefly luciferase derived from Luciola lateralis, Luciola cruciata, North American firefly (Photinus pyralis), Photuris pennsylvanica, European glow worm (Lampyris noctiluca), Pyrocoelia miyako, click beetle (Pyrophorus plagiophthalamus), or Luciola mingrelica, preferably Luciola lateralis, Genji firefly, North American firefly, or Photuris pennsylvanica, can be used.
  • chimeric proteins made from various firefly luciferase genes may be used.
  • the correspondence of amino acid positions can be easily identified by comparing the amino acid sequences of various firefly luciferases using, for example, existing amino acid homology analysis software, such as GENETYX (manufactured by GENETYX).
  • GENETYX existing amino acid homology analysis software
  • the amino acid position of a luciferase corresponding to position X of the amino acid sequence of SEQ ID NO: 1 can be identified by aligning the amino acid sequence of the luciferase with the amino acid sequence of SEQ ID NO: 1.
  • the "position corresponding to position 262 of SEQ ID NO: 1" is position 262 of the amino acid sequence of SEQ ID NO: 69, position 260 of SEQ ID NO: 71, and position 259 of SEQ ID NO: 73.
  • Figure 1 shows the alignment results of Heike firefly luciferase, Genji firefly luciferase, North American firefly luciferase, and Fotulis pennsylvanica luciferase. The corresponding positions in each amino acid sequence can be determined by referring to such alignment results.
  • the amino acid at the position corresponding to position 252 of SEQ ID NO: 1 in the luciferase mutant may be a non-acidic amino acid other than phenylalanine (e.g., leucine, tyrosine, isoleucine, cysteine, serine, threonine, aspartic acid, glutamic acid, tryptophan, methionine, valine, alanine, proline, glycine, glutamine, asparagine, lysine, histidine, or arginine).
  • the substitution (mutation) of phenylalanine at position 252 of SEQ ID NO: 1 to leucine may be referred to as F252L in this specification.
  • F252L is introduced in SEQ ID NO: 1, and it may be introduced into any luciferase at the position corresponding to position 252 of SEQ ID NO: 1.
  • the position corresponding to position 252 of SEQ ID NO: 1 before the substitution may not be F.
  • F252L is merely a description for convenience based on SEQ ID NO: 1, and if L is introduced at the position corresponding to position 252 of SEQ ID NO: 1, such an amino acid substitution corresponds to F252L.
  • a wild-type luciferase in which the position corresponding to position 252 of SEQ ID NO: 1 is originally L is excluded from the mutations of the present disclosure.
  • wild-type sequences and natural products are excluded from the present disclosure.
  • Other amino acid substitutions at other positions may also be described as (amino acid before substitution-position-amino acid after substitution).
  • the luciferase mutant may be a mutant containing an amino acid sequence having high sequence identity to the amino acid sequence of SEQ ID NO: 10, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity.
  • the luciferase before amino acid substitution may be Heike firefly-derived luciferase or Genji firefly-derived luciferase.
  • the mutation at a position corresponding to position 252, etc., of SEQ ID NO:1 is artificially introduced. This can be achieved by artificially introducing a mutation into the sequence of a gene encoding luciferase.
  • the firefly luciferase mutant may further include a mutation other than the mutation at a position corresponding to position 252 of SEQ ID NO:1.
  • the further mutation may be artificially introduced with the intention of achieving some specific effect, or may be randomly or non-artificially introduced.
  • Examples of mutations introduced with the intention of achieving a specific effect include addition, deletion, or modification of a sequence for enhancing the expression level of firefly luciferase, addition, deletion, or modification of a sequence for improving the purification efficiency of firefly luciferase, as well as various mutations that impart practically preferable properties to firefly luciferase.
  • Such known mutations include a mutation that enhances the luminescence persistence as described in JP-A-2000-197484, a mutation that changes the luminescence wavelength as described in JP-A-3-285683 or JP-T-2003-512071, a mutation that enhances surfactant resistance as described in JP-A-11-239493, a mutation that enhances the surfactant resistance as described in WO-99/02697, JP-T-2003-512750, or JP-T-2003-512071, and a mutation that enhances the surfactant resistance as described in WO-A-2003-239493.
  • mutations include mutations that change substrate affinity as described in JP-A-1-518799, mutations that increase stability as described in JP-A-5-244942, JP-A-2011-120559, JP-A-2000-197487, JP-T-9-510610, or JP-T-2003-518912, and mutations that improve luminescence duration, stability, and luminescence amount as described in JP-A-2011-188787.
  • JP-A-2011-120559 discloses that the thermal stability is improved in firefly luciferase having an amino acid sequence in which the amino acid corresponding to position 287 of Heike firefly luciferase is mutated to alanine, or the amino acid corresponding to position 392 is mutated to isoleucine.
  • JP 2011-120559 A describes that by combining these mutations with a mutation in which the amino acid at position 326 is replaced with serine and/or a mutation in which the amino acid at position 467 is replaced with isoleucine, it is possible to obtain a firefly luciferase with improved stability.
  • WO 2020/009215 A describes a thermostable luciferase mutant in which position 393 of SEQ ID NO:1 is replaced.
  • the luciferase mutant of the present disclosure comprises: (a) The amino acid residue at the position corresponding to 252 in SEQ ID NO:1 is an amino acid residue selected from the group consisting of leucine, tyrosine, cysteine, serine, threonine, aspartic acid, glutamic acid, isoleucine, proline, valine, tryptophan, alanine, glycine, glutamine, asparagine, lysine, histidine, arginine, and methionine, e.g., leucine.
  • the luciferase mutant of the present disclosure comprises: (b) the amino acid residue at the position corresponding to position 262 of SEQ ID NO:1 has been substituted, and the amino acid residue at the position corresponding to position 262 of SEQ ID NO:1 is an amino acid residue selected from the group consisting of tyrosine, leucine, isoleucine, cysteine, serine, threonine, aspartic acid, glutamic acid, tryptophan, methionine, valine, alanine, proline, glycine, glutamine, asparagine, lysine, histidine, and arginine, for example, tyrosine.
  • the luciferase mutant of the present disclosure comprises: (c) the amino acid residue at the position corresponding to position 294 of SEQ ID NO:1 is an amino acid residue selected from the group consisting of leucine, tyrosine, cysteine, serine, threonine, aspartic acid, glutamic acid, isoleucine, proline, valine, tryptophan, methionine, alanine, glycine, glutamine, asparagine, lysine, histidine, and arginine, e.g., leucine.
  • the luciferase mutant of the present disclosure further comprises: (a) an amino acid residue at a position corresponding to position 90 of SEQ ID NO:1 is substituted, and the amino acid residue at the position corresponding to position 90 of SEQ ID NO:1 is an amino acid residue selected from the group consisting of tyrosine, leucine, isoleucine, cysteine, serine, threonine, aspartic acid, glutamic acid, proline, valine, tryptophan, methionine, alanine, glycine, glutamine, asparagine, lysine, histidine, and arginine, e.g., tyrosine; (b) the amino acid residue at the position corresponding to position 371 of SEQ ID NO:1 is substituted, and the amino acid residue at the position corresponding to position 371 of SEQ ID NO:1 is an amino acid residue selected from the group consisting of leucine, tyrosine, cysteine, th
  • the luciferase mutants of the present disclosure further include substitutions of amino acid residues at positions corresponding to 221, 17, 18, 75, 110, 223, 224, 257, 229, and/or 158 of SEQ ID NO:1, and the thermal stability of the luciferase after amino acid substitution may be improved compared to the luciferase before amino acid substitution.
  • luciferase variants with additional amino acid substitutions may have one or more of the following amino acid substitutions: (i) the amino acid residue at the position corresponding to position 221 of SEQ ID NO:1 is substituted, and the amino acid residue at the position corresponding to position 221 of SEQ ID NO:1 is an amino acid residue selected from the group consisting of leucine, tyrosine, cysteine, serine, threonine, aspartic acid, glutamic acid, isoleucine, proline, valine, tryptophan, methionine, alanine, glycine, glutamine, asparagine, lysine, histidine, and arginine, e.g., leucine; (ii) the amino acid residue at the position corresponding to position 17 of SEQ ID NO:1 is substituted, and the amino acid residue at the position corresponding to position 17 of SEQ ID NO:1 is an amino acid residue selected from the group consisting of aspartic acid, glut
  • the luciferase mutant of the present disclosure may have a double mutation selected from the group consisting of F252L/F262Y, F262Y/F294L, and F252L/F294L, based on SEQ ID NO:1.
  • the luciferase mutant of the present disclosure may have a triple mutation consisting of F252L/F262Y/F294L based on SEQ ID NO: 1.
  • One or more amino acid substitutions of the present disclosure may be further introduced into this triple mutant.
  • the triple mutant may have an amino acid sequence identity of 90% or more with SEQ ID NO: 1, 2, or 10.
  • the triple mutant has an improved residual activity rate after heat treatment at 60° C. for 5 minutes, compared to the luciferase before the introduction of the three amino acid substitutions, by 2-fold, 3-fold, 4-fold, or 5-fold or more.
  • the triple mutant has an improved residual activity rate after heat treatment at 60° C.
  • a firefly luciferase with significantly improved thermal stability refers to a firefly luciferase whose residual activity after heat treatment at 60°C for 5 minutes is improved by 100% or more, 200% or more, 300% or more, or 400% or more, compared to a comparable unmutated firefly luciferase.
  • the luciferase prior to amino acid substitution may have an amino acid sequence selected from the group consisting of (A) to (F) below: (A) an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to any of SEQ ID NOs: 1, 2, or 10; (B) an amino acid sequence in which the homologous region of SEQ ID NO: 1 in the amino acid sequence of (A) has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the homologous region of luciferase before the amino acid substitution; (C) an amino acid sequence of SEQ ID NO: 1 or 10 in which one or several amino acids have been substituted, deleted or added at a position other than the position corresponding to position 252 of SEQ ID NO: 1; (D)
  • the homologous regions can be identified as regions where identical amino acid residues are conserved in the four firefly luciferases shown in Figure 1 (Lucifera cruciata, L. genji, North American firefly, and Photuris pennsylvanica).
  • the homologous regions of SEQ ID NO: 1 are the following positions of SEQ ID NO: 1: positions 4-5, 9-10, 13-14, 16-17, 19, 23, 25-26, 28, 35-37, 40, 42-43, 45, 47, 55, 57, 62, 65, 72-74, 80, 83-86, 90-91, 93, 98, 101, 105-106, 111, 114-116, 119, 122, 125, 129, 131-132, 133-134, 135-136, 137-138, 139-140, 141-142, 143-144, 145-146, 147-148, 149-250, 151-152, 153-154, 155-156, 157-158, 159-259, 160-161, 161-162, 162-163, 163-164, 164-165, 165-166, 167-168, 168-169, 170-171, 172-173, 173-174, 175-176,
  • the homologous region of the firefly luciferase of interest in this specification is a region corresponding to the homologous region of SEQ ID NO: 1.
  • the respective positions correspond to each other.
  • the position in sequence A corresponding to position 4 of SEQ ID NO: 1 and the position in sequence B corresponding to position 4 of SEQ ID NO: 1 are compared as corresponding positions.
  • the homologous region is defined with respect to the sequence before amino acid substitution, and does not prevent a mutation in luciferase from being introduced into the homologous region.
  • position 262 of SEQ ID NO: 1 is included in the homologous region of SEQ ID NO: 1, but a mutation may be introduced into this position. The same applies to other mutation positions described in this specification.
  • the identity of amino acid sequences and gene sequences can be calculated using programs such as maximum matching and search homology in GENETYX (GENETYX), multiple alignment in CLUSTAL W, pairwise alignment by BLAST, and other programs.
  • GENETYX GENETYX
  • CLUSTAL W CLUSTAL W
  • pairwise alignment by BLAST pairwise alignment by BLAST
  • other programs such as maximum matching and search homology in GENETYX (GENETYX), multiple alignment in CLUSTAL W, pairwise alignment by BLAST, and other programs.
  • the positions of amino acids that are identical in two or more luciferases can be examined when the two or more luciferases are aligned. Based on this information, identical regions in the amino acid sequences can be determined.
  • the percent identity refers to the percentage calculated by aligning two or more amino acid sequences using BLAST (BLASTP) or the like targeting amino acid sequences, with the total number of amino acids in the aligned regions as the denominator and the number of positions occupied by identical amino acids as the numerator. Therefore, normally, when two or more amino acid sequences have a region where no identity is observed, for example when one of the amino acid sequences has an additional sequence at the C-terminus where no identity is observed, the region where no identity is observed cannot be aligned and is therefore not used in calculating the percent identity.
  • BLASTP BLAST
  • amino acids that are similar in two or more luciferases can be aligned using CLUSTALW, in which case the algorithm Blosum62 is used, and amino acids that are determined to be similar when multiple amino acid sequences are aligned are sometimes called similar amino acids.
  • amino acid substitutions may be due to substitutions between such similar amino acids.
  • alignments can be used to examine regions of identical amino acid sequences and positions occupied by similar amino acids for multiple amino acid sequences. Based on this information, regions of homology (also called highly conserved regions) in the amino acid sequences can be determined.
  • Luciferases can also be subjected to high-throughput screening to obtain functional luciferase mutants.
  • a library of transformed or transduced strains carrying mutated luciferase genes can be prepared and subjected to microtiter plate-based high-throughput screening or droplet microfluidic-based ultra-high-throughput screening. Examples include constructing a combinatorial library of mutant genes encoding variants and then screening a large population of mutant luciferases using phage display (e.g., Chem. Rev. 105 (11): 4056-72, 2005), yeast display (e.g., Comb Chem High Throughput Screen.
  • Libraries may be transformed into suitable cells, such as electrocompetent EBY-100 cells, to obtain approximately 10 to the power of 7 mutants (10 million).
  • Yeast cells transformed with the libraries may then be subjected to cell sorting.
  • Polydimethoxylsiloxane (PDMS) microfluidic devices fabricated using standard soft lithography techniques may also be used.
  • Flow focus devices may be used to form monodisperse droplets.
  • the droplets formed containing the individual mutants may be subjected to an appropriate sorting device.
  • the presence or absence of luciferase activity may be utilized to select cells.
  • a reaction solution having a composition that emits light when the above-mentioned luciferase acts may be used.
  • luminescence may be measured using a 96-well plate, 192-well plate, 384-well plate, 9600-well plate, etc., and a plate reader.
  • Mutation introduction and selection may be repeated multiple times. Mutations as used herein include substitutions, insertions, deletions, and/or additions of amino acids.
  • 1 to 10 mutations may be introduced into luciferase, and luciferase activity may be confirmed.
  • 1 to 10 further mutations may be introduced, and activity may be confirmed.
  • a series of high-throughput screening (for example, the above-mentioned method of obtaining and screening approximately 10 to the power of 7 mutants) may be repeated for 2 or more rounds, 5 or more rounds, 10 or more rounds, 15 or more rounds, for example, 20 or more rounds.
  • mutants having 10 or more mutations, 50 or more mutations, for example 100 or more mutations, and active mutants may be rapidly obtained.
  • mutants having 20 or more mutations, 100 or more mutations, for example 200 or more mutations, and active mutants may be rapidly obtained.
  • Such operations may be performed by an automatic device or by repeating a routine process.
  • Mutations may be introduced at one or more positions from the first amino acid to the last amino acid in the full-length amino acid sequence of luciferase.
  • regions important for the function of the enzyme such as the active center, substrate recognition site, ATP recognition site, and their vicinity, are excluded.
  • Luciferase is widely used in industry, and those skilled in the art are familiar with the regions important for the function of the enzyme, including the active center, substrate recognition site, and ATP recognition site.
  • one or more mutations may be introduced first at positions 1 to 10 of the full-length sequence of luciferase. Next, starting from a luciferase mutant confirmed to have activity, one or more mutations may be further introduced at positions 11 to 20, and the activity may be confirmed.
  • This may be repeated n times (n ⁇ 55). For example, one or more mutations may be introduced at positions 541 to 548 in the 55th round. Regions important for the function of the enzyme or regions not intended to be modified may be skipped as appropriate along the way. For example, position 208 of SEQ ID NO: 1 may be skipped.
  • any mutation to be introduced at any position in the full-length sequence, except for regions important for the function of the enzyme, and allows for rapid acquisition of active luciferase mutants having, for example, 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 110 or more, 120 or more, 130 or more, 140 or more, 150 or more, 160 or more, 170 or more, 180 or more, 190 or more, for example, 200 or more mutations.
  • no mutations that eliminate the activity of luciferase are introduced.
  • no mutations are introduced at positions 202, 208, 346, 424, 439, 448, and 457 of SEQ ID NO: 1.
  • Mutations may be introduced randomly or by rational design.
  • the mutations introduced by rational design or randomly may be conservative amino acid substitutions.
  • Conservative amino acid substitutions include amino acid substitutions in which the amino acid before and after the substitution have similar chemical properties (e.g., Stryer et al., Biochemistry, 5th ed., 2002, pp. 44-49).
  • conservative amino acid substitutions may be selected from the group consisting of (i) a basic amino acid with a different kind of basic amino acid; (ii) an acidic amino acid with a different kind of acidic amino acid; (iii) an aromatic amino acid with a different kind of aromatic amino acid; (iv) a non-polar aliphatic amino acid with a different kind of non-polar aliphatic amino acid; and (v) a polar uncharged amino acid with a different kind of polar uncharged amino acid.
  • the basic amino acid may be selected from, for example, arginine, histidine, and lysine.
  • the acidic amino acid may be, for example, aspartic acid or glutamic acid.
  • Aromatic amino acids may be selected from, for example, phenylalanine, tyrosine, and tryptophan.
  • Nonpolar aliphatic amino acids may be selected from, for example, glycine, alanine, valine, leucine, methionine, proline, and isoleucine.
  • Polar uncharged amino acids may be selected from, for example, serine, threonine, cysteine, asparagine, and glutamine.
  • Conservative amino acid substitutions are highly likely to maintain the tertiary structure and have activity because the chemical properties of the amino acid residue before and after substitution are similar, and the position at which the conservative amino acid substitution is made is the same position in the tertiary structure of the protein.
  • the mutations introduced by rational design or randomly include substitutions with functionally similar amino acids.
  • Tables of functionally similar amino acids are widely known in the art.
  • the substitutions with functionally similar amino acids may be of any of the following amino acid classes: 1) Glycine (G), Alanine (A); 2) Aspartic acid (D), glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) arginine (N), lysine (K), histidine (H); 5) Isoleucine (I), Leucine (L), Valine (V), Proline (P); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) Cysteine (C), Methionine (M).
  • non-conservative amino acid substitutions are substitutions of an amino acid with any amino acid that does not fall within the conservative substitutions (i) to (v) outlined above.
  • the amino acid substitution may be a non-conservative amino acid substitution. In this case, for example, it may be confirmed whether dehydrogenase activity is maintained before and after the non-conservative amino acid substitution is introduced. If activity is confirmed, the non-conservative amino acid substitution may be adopted.
  • the amino acid substitution may be a substitution with a similar amino acid (similarity substitution).
  • a substitution with a similar amino acid refers to a substitution with an amino acid that is evaluated as a positive value or a neutral value (zero) in the amino acid substitution matrix used in the ClustalW software and the Blosum62 algorithm (see, e.g., S. Heinkoff and J. G. Henikoff, Proc. Natl. Acad. Sci. USA, Vol. 89, pp. 10915-10919, 1992, especially FIG. 2 therein, and Thompson, Nucleic Acid Research, 1994, Vol. 22, No. 22, pp. 4673-4680).
  • the matrix table was generated from aligned sequence segments of approximately 2000 blocks of over 500 related proteins. Moreover, whether starting from a single matrix or using a subset of proteins, repeated application leads to approximately the same set of scores. Therefore, the substitution matrix is considered versatile. This is the most widely used approach, taking advantage of the evolutionary relationship of homologous sequences. Therefore, variants with similar substitutions introduced into a luciferase are likely to be active.
  • a serine-to-threonine substitution is evaluated as a positive value of "1”, so that, for example, a serine-to-threonine substitution at position 24 of SEQ ID NO: 1 corresponds to a similar amino acid substitution. This position is threonine in SEQ ID NO: 71, and both enzymes are active, so the S24T mutant of SEQ ID NO: 1 is likely to be active. The same is true for other similar substitution variants.
  • the conservative amino acid substitution or the substitution with a functionally similar amino acid is not present in an area important to the function of the enzyme, such as the active center, substrate recognition site, coenzyme recognition motif, or the vicinity thereof, and therefore does not significantly affect the activity of the enzyme.
  • the conservative amino acid substitution or the substitution with a functionally similar amino acid is present in the active center, substrate recognition site, coenzyme recognition motif, or the vicinity thereof, but does not substantially affect the activity of the enzyme.
  • amino acid substitutions, deletions, or additions can mean 1 to 10, for example 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, or 1 to 3, for example 1 or 2.
  • Luciferase variants may contain amino acid deletions compared to the sequence prior to the amino acid substitution.
  • the amino acid deletions are not in regions critical to the function of the enzyme and therefore do not significantly affect the activity of the enzyme.
  • the deletions may be short deletions of 1-2 amino acids.
  • the amino acid sequence of one luciferase may be compared to that of another luciferase, and if an amino acid is deleted in one sequence, the deletion may be introduced into the other luciferase. As both luciferases exhibit activity, such deletions are unlikely to significantly affect the activity of the enzyme.
  • Luciferase mutants may also include those in which additional amino acids have been inserted compared to the pre-mutation sequence.
  • the amino acid insertion is not in an area critical to the function of the enzyme, such as the active center, substrate recognition site, coenzyme recognition motif, or adjacent thereto, and therefore does not significantly affect the activity of the enzyme.
  • the insertion may be 1-4 amino acids.
  • the amino acid sequence of one luciferase is compared to that of another luciferase, and if an amino acid has been inserted in one sequence, the inserted amino acid may be introduced into the other luciferase. Since both luciferases exhibit activity, such an insertion is unlikely to significantly affect the activity of the enzyme.
  • an inserted amino acid is given below.
  • SEQ ID NO:1 and SEQ ID NO:73 are aligned, a proline has been inserted between positions 39 and 40 of SEQ ID NO:1 at a position corresponding to position 37 of SEQ ID NO:1.
  • an amino acid e.g., proline
  • Other insertion positions may be identified from FIG. 1.
  • the inserted amino acid may be an amino acid inserted in another sequence, or it may be an amino acid that is a conservative amino acid substitution for the amino acid in question.
  • Luciferase mutants may also include those in which additional amino acids have been added compared to the pre-mutation sequence.
  • the amino acid additions are made to the N-terminus or C-terminus of the luciferase and do not significantly affect the activity of the enzyme.
  • the additions may be 1-6 amino acids, 1-5 amino acids, e.g., 1-4 amino acids.
  • Examples of additions include, but are not limited to, short stretches of histidine residues (e.g., 2-6 histidine residues) to aid in purification of the luciferase.
  • additions also include, but are not limited to, the addition of a signal peptide to aid in expression of the luciferase. Signal peptides include known signal sequences or functional equivalents thereof.
  • SEQ ID NO:1 When SEQ ID NO:1 and SEQ ID NO:71 are aligned, SEQ ID NO:1 has three amino acids added upstream of its N-terminus when viewed from SEQ ID NO:71. As both luciferases exhibit activity, it is considered highly likely that such an addition will not significantly affect the activity of the enzyme. Therefore, one, two or three amino acids may be added to the N-terminus of SEQ ID NO:71.
  • the added amino acids may be corresponding amino acids in another sequence, or may be amino acids that are conservative amino acid substitutions for the corresponding amino acids.
  • Mutations can be introduced into luciferase in a manner that does not disrupt secondary structures or structural motifs, such as alpha helices or beta sheets. Regions of secondary structure can be identified, for example, by secondary structure prediction algorithms. Examples of prediction algorithms include, but are not limited to, NetSurfP-2.0. The same applies to other structural motifs, such as nests and niches.
  • amino acid residues or amino acid sequence motifs essential for luciferase activity are not substituted.
  • conservative amino acid substitutions or similar substitutions can be made at these positions, but the activity of the variants after the substitutions is confirmed.
  • no amino acid deletions or insertions are made before or after the amino acid residues essential for luciferase activity.
  • the positions before or after the amino acid residues essential for luciferase activity refer to positions one or two positions N-terminal or one or two positions C-terminal to the amino acid residue essential for the activity.
  • amino acid deletions or insertions can be made before or after the amino acid residues essential for luciferase activity, but the activity of the variants after the substitutions is confirmed. Whether or not a variant has activity can be routinely confirmed, for example, by high-throughput screening.
  • the luciferase mutant of the present disclosure has luciferase activity.
  • the presence or absence of luciferase activity can be measured using a Lumitester C-110 (Kikkoman Biochemifa Corporation), for example, according to the method described in the Examples.
  • thermal stability can be evaluated, for example, by using the remaining activity when firefly luciferase is heat-treated at a predetermined temperature for a predetermined time as an indicator.
  • the thermal stability of firefly luciferase can be evaluated by comparing the remaining activity rate after heat treatment of firefly luciferase under high temperature conditions, for example, at a reaction temperature of usually 30 to 50°C, for example 35 to 45°C or 35 to 40°C, for a certain period of time, usually 5 to 180 minutes or 10 to 180 minutes, for example 60 to 180 minutes or about 90 minutes.
  • the residual activity of firefly luciferase is the activity after heat treatment when the activity of firefly luciferase before acting under the above-mentioned high temperature conditions is set to 1. For example, if the residual activity after heat treatment is halved, the residual activity after heat treatment will be 0.5 relative to the activity before heat treatment of 1.
  • the residual activity rate of firefly luciferase is calculated as the ratio of the activity after heat treatment to the firefly luciferase activity before acting under the above-mentioned high temperature conditions.
  • improved thermal stability refers to a case where the residual activity rate when the firefly luciferase mutant is acted under the above conditions shows an improvement of 1.01 times or more, 1.02 times or more, 1.1 times or more, 1.2 times or more, 1.3 times or more, 1.4 times or more, 1.5 times or more, 1.6 times or more, 1.7 times or more, 1.8 times or more, 1.9 times or more, or 2 times or more compared to the luciferase before introducing the mutation of the present disclosure (a mutation at a position corresponding to position 252 of SEQ ID NO: 1, etc.).
  • improved thermostability refers to a case in which the residual activity of the firefly luciferase mutant when acted under the above conditions shows an improvement of 1% or more, 2% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% or more compared to the residual activity of the luciferase before the introduction of the mutation of the present disclosure.
  • polynucleotides in one embodiment, provides a polynucleotide encoding a luciferase mutant (hereinafter also referred to as a "luciferase gene").
  • the sequence of the polynucleotide can be easily determined based on the amino acid sequence of the firefly luciferase mutant.
  • polynucleotides encoding the amino acid sequences of SEQ ID NO: 2 and 10 include polynucleotides of SEQ ID NO: 3 and 11, respectively.
  • a polynucleotide encoding a luciferase before amino acid substitution can be, for example, (i) a nucleotide sequence having a sequence identity over its entire length of 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, more preferably 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, or 99% or more to any of the nucleotide sequences of SEQ ID NOs: 3 and 11, and encoding an active luciferase; (ii) a nucleotide sequence encoding an active luciferase, in which one or several nucleotides are substituted, deleted or added in any of the nucleotide sequences of SEQ ID NOs: 3 and 11; or (iii) a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3 and 11
  • chromosomal DNA or mRNA can be extracted from firefly tissues or cells capable of producing luciferase by a conventional method, for example, the method described in Current Protocols in Molecular Biology (WILEY Interscience, 1989).
  • cDNA can be synthesized using the mRNA as a template.
  • a chromosomal DNA or cDNA library can be prepared using the chromosomal DNA or cDNA obtained in this way.
  • an appropriate probe DNA is synthesized based on the amino acid sequence of the luciferase, and a polynucleotide encoding the luciferase is selected from a chromosomal DNA or cDNA library using the probe DNA, or a method is used in which an appropriate primer DNA is prepared based on the amino acid sequence, and a DNA containing a desired nucleotide fragment encoding the luciferase is amplified by an appropriate polymerase chain reaction (PCR) such as the 5'RACE method or the 3'RACE method, and these DNA fragments are linked to obtain a DNA containing the full length of the nucleotide sequence encoding the desired luciferase.
  • PCR polymerase chain reaction
  • the nucleotide sequence encoding luciferase may be artificially synthesized.
  • Such an artificial gene synthesis service is provided, for example, by Integrated DNA Technologies.
  • Mutation of the luciferase gene can be performed by any known method according to the intended form of mutation, including a method of contacting the luciferase gene or a recombinant DNA incorporating the gene with a mutagenic agent, an ultraviolet irradiation method, a genetic engineering method, or a method making full use of a protein engineering method.
  • mutagenic agents used in the above mutation treatment include hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine, nitrous acid, sulfurous acid, hydrazine, formic acid, and 5-bromouracil.
  • the conditions for this contact and action can be adjusted according to the type of drug used, and are not particularly limited as long as the desired mutation can be actually induced in the luciferase gene.
  • the desired mutation can be induced by contacting and acting the drug at a concentration of preferably 0.5 to 12 M at a reaction temperature of 20 to 80°C for 10 minutes or more, preferably 10 to 180 minutes.
  • ultraviolet irradiation it can also be performed according to the usual method as described above (Modern Chemistry, vol. 24-30, June 1989 issue).
  • Methods that make full use of protein engineering techniques include the method generally known as Site-Specific Mutagenesis. Examples include the Kramer method (Nucleic Acids Res., 12, 9441-9456 (1984)), the Eckstein method (Nucleic Acids Res., 13, 8749-8764 (1985): Nucleic Acids Res., 13, 8765 (1985): Nucleic Acids Res., 14, 9679 (1986)), and the Kunkel method (Proc. Natl. Acids Sci. U.S.A., 82, 488-492 (1985)).
  • the desired modified luciferase gene can also be directly synthesized by organic synthesis or enzymatic synthesis.
  • the base sequence of the luciferase gene can be confirmed, for example, using a multi-capillary DNA analysis system such as the Applied Biosystems 3730xl DNA Analyzer (Thermo Fisher Scientific).
  • the present disclosure relates to a vector comprising the polynucleotide.
  • these luciferase genes are linked to various vectors according to standard methods.
  • examples of vectors include plasmids, but any other vectors known to those skilled in the art, such as bacteriophages and cosmids, can also be used.
  • the type of vector can be selected depending on the host cell, and specifically, for example, pET16-b or pKK223-3, etc. are preferred.
  • the present disclosure relates to a host cell comprising the polynucleotide or vector described above.
  • the host cell may be, but is not limited to, a bacterium such as E. coli or Bacillus subtilis, a yeast cell, an insect cell, an animal cell (e.g., a mammalian cell), or a plant cell, preferably a bacterial cell such as E. coli.
  • the luciferase gene obtained as described above can be incorporated into a vector such as a bacteriophage, cosmid, or a plasmid used for transformation of prokaryotic or eukaryotic cells by a conventional method, and a host corresponding to each vector can be transformed or transduced by a conventional method.
  • a microorganism belonging to the genus Escherichia for example, the obtained recombinant DNA can be used as a host to transform or transduce, for example, E. coli K-12 strain, or E. coli B strain, preferably E. coli JM109 strain, E. coli DH5 ⁇ strain, E. coli BL21 strain, E.
  • coli BL21(DE3) strain both manufactured by Takara Bio Inc.
  • a method for transferring a recombinant vector into such a host cell for example, when the host cell is a microorganism belonging to Escherichia coli, a method of transferring recombinant DNA in the presence of calcium ions can be adopted, and further, electroporation method can be used.
  • commercially available competent cells for example, ECOS Competent Escherichia coli BL21(DE3); Nippon Gene
  • the luciferase gene may be codon-optimized depending on the expression host.
  • the present disclosure relates to a method for producing a luciferase mutant having improved thermostability, comprising the step of culturing the host cell.
  • the culture can be carried out by any of a variety of known methods, including a solid culture method, but is preferably carried out by a liquid culture method.
  • the production method may include culturing the host cells under conditions that allow expression of the luciferase protein, and optionally isolating the luciferase from the culture or culture medium.
  • the conditions that allow expression of the luciferase protein refer to conditions in which the luciferase gene is transcribed and translated to produce a polypeptide encoded by the gene.
  • the production method includes artificially introducing a mutation into the luciferase protein at a position corresponding to, for example, position 252 of SEQ ID NO:1, prior to the culturing step. This can be done by artificially introducing a mutation into the sequence of the gene encoding luciferase.
  • the medium for culturing the host cells may be, for example, one or more nitrogen sources such as yeast extract, tryptone, peptone, meat extract, corn steep liquor, or soybean or wheat bran infusion, to which one or more inorganic salts such as sodium chloride, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, magnesium chloride, ferric chloride, ferric sulfate, or manganese sulfate have been added, and further, if necessary, carbohydrate raw materials, vitamins, etc. may be appropriately added.
  • nitrogen sources such as yeast extract, tryptone, peptone, meat extract, corn steep liquor, or soybean or wheat bran infusion
  • inorganic salts such as sodium chloride, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, magnesium chloride, ferric chloride, ferric sulfate, or manganese sulfate have been added, and further, if necessary, carbohydrate raw materials,
  • the initial pH of the medium should be adjusted to pH 7-9.
  • Cultivation is preferably carried out at a culture temperature of 20-42°C, preferably around 25-37°C for 4-24 hours, more preferably around 25-37°C for 8-16 hours, by aeration and agitation submerged culture, shaking culture, static culture, etc.
  • luciferase can be collected from the culture by using a conventional enzyme collection method.
  • the cells can be subjected to ultrasonic destruction, grinding, etc., or the enzyme can be extracted using a lytic enzyme such as lysozyme, or the cells can be lysed by shaking or standing in the presence of toluene, etc., to excrete the enzyme from the cells.
  • the solution can then be filtered, centrifuged, etc. to remove solids, and nucleic acids can be removed as necessary using streptomycin sulfate, protamine sulfate, manganese sulfate, etc., after which ammonium sulfate, alcohol, acetone, etc. can be added to fractionate, the precipitate can be collected, and the crude luciferase enzyme can be obtained.
  • a purified luciferase enzyme from the above-mentioned crude luciferase enzyme, for example, a gel filtration method using Sephadex, Superdex, Ultrogel, etc., an adsorption elution method using an ion exchange carrier, a hydrophobic carrier, or hydroxyapatite, an electrophoresis method using polyacrylamide gel, etc., a sedimentation method such as sucrose density gradient centrifugation, an affinity chromatography method, a fractionation method using a molecular sieve membrane or a hollow fiber membrane, etc., can be appropriately selected or performed in combination to obtain a purified luciferase enzyme. In this way, the desired luciferase can be obtained.
  • a gel filtration method using Sephadex, Superdex, Ultrogel, etc. an adsorption elution method using an ion exchange carrier, a hydrophobic carrier, or hydroxyapatite
  • the luciferase produced can be used in the kits described herein or in methods for detecting at least one of ATP, ADP, and AMP.
  • the present disclosure relates to a kit for detecting at least one of ATP, ADP, and AMP, comprising the luciferase mutant described herein.
  • the kit may include luciferin in addition to the luciferase mutant.
  • metal ions such as magnesium, manganese, calcium, etc. may also be included in the kit.
  • Luciferase converts ATP, O2 , and luciferin to AMP, pyrophosphate, CO2 , and oxyluciferin, which then produces luminescence.
  • the reaction that occurs at this time is represented as follows:
  • the kit further comprises an enzyme that catalyzes a reaction to generate ATP from ADP.
  • the enzyme that catalyzes a reaction to generate ATP from ADP may be selected from the group consisting of pyruvate kinase (PK), acetate kinase (AK), creatine kinase (CK), polyphosphate kinase (PPK), hexokinase, glucokinase, glycerol kinase, fructokinase, phosphofructokinase, riboflavin kinase, and fructose bisphosphatase.
  • the kit further comprises pyruvate orthophosphate dikinase (PPDK), adenylate kinase (ADK), or pyruvate water dikinase (PWDK).
  • the sample contains ATP, it is converted to AMP by luciferase and luminescence occurs. If the sample contains ADP in a system in which an enzyme that catalyzes the reaction of generating ATP from ADP is present, the enzyme converts ADP to ATP, and then the ATP is subjected to a luminescence reaction. This allows the total amount of ATP and ADP present in the system to be measured. Furthermore, if the sample contains AMP in a system in which PPDK is present, it is converted to ATP by PPDK, PEP, and PPi. Alternatively, if the sample contains AMP in a system in which PWDK is present, it is converted to ATP by PWDK, PEP, and phosphate.
  • the generated ATP again emits light by luciferase.
  • the luminescence is maintained stably, and the amount of luminescence correlates with the total amount of ATP and AMP present in the system, making it possible to quantify ATP and AMP. If an enzyme that catalyzes the reaction of generating ATP from ADP and PPDK, ADK, or PWDK are present, the total amount of ATP, ADP, and AMP can be measured.
  • luciferin Any luciferin may be used as long as it is recognized as a substrate by the luciferase used, and may be natural or chemically synthesized. Any known luciferin derivative may also be used.
  • the basic structure of luciferin is imidazopyrazinones, and there are many tautomers.
  • An example of luciferin is firefly luciferin. Firefly luciferin is a substrate for firefly luciferase (EC 1.13.12.7). Luciferin derivatives may be those described in JP 2007-91695 A, JP 2010-523149 A (International Publication WO 2008/127677), etc.
  • the kit may also include at least one of a stabilizer, a buffer, and instructions.
  • the present disclosure relates to a method for detecting at least one of ATP, ADP, and AMP, comprising using a luciferase variant described herein, which may include catalyzing an oxidation reaction of luciferin with a luciferase variant described herein, and measuring the luminescence produced by the oxidation reaction.
  • Catalysis of the oxidation reaction of luciferin by luciferase mutants is as described in "Kit for detecting at least one of ATP, ADP, and AMP". This can be done by reacting a sample with the luciferase mutants and luciferin described in this specification. If the sample contains ATP, it is converted to AMP by luciferase and luminescence is generated, so that ATP can be measured. In a system in which an enzyme that catalyzes the reaction to generate ATP from ADP is present, the total amount of ATP and ADP present in the system can be measured. In addition, in a system in which PPDK or PWDK is present, ATP and AMP can be quantified. When an enzyme that catalyzes the reaction to generate ATP from ADP and PPDK, ADK, or PWDK are present, the total amount of ATP, ADP, and AMP can be measured.
  • the amount of luminescence produced by luciferase can be measured by known methods, for example, using a suitable luminescence measuring device, such as a luminometer (CentroLB960 or Lumat3 LB9508 manufactured by Berthold, Lumitester C-110, Lumitester C-100, Lumitester PD-20, Lumitester PD-30, etc. manufactured by Kikkoman Biochemifa), and the relative luminescence intensity (RLU) obtained can be used as an indicator.
  • a suitable luminescence measuring device such as a luminometer (CentroLB960 or Lumat3 LB9508 manufactured by Berthold, Lumitester C-110, Lumitester C-100, Lumitester PD-20, Lumitester PD-30, etc. manufactured by Kikkoman Biochemifa)
  • RLU relative luminescence intensity
  • a device equipped with a photomultiplier tube manufactured by 3M, etc.
  • a device equipped with a photodiode manufactured by Hygiena, Neogen, etc.
  • a luciferase variant of the present disclosure e.g., a luciferase variant having at least 70%, 75%, 80%, 85%, 90% or 95% amino acid sequence identity to SEQ ID NO:10 and having a leucine at the position corresponding to position 252 of SEQ ID NO:1 has an isoleucine at the position corresponding to position 393 of SEQ ID NO:1, a serine at the position corresponding to position 326 of SEQ ID NO:1, an isoleucine at the position corresponding to position 392 of SEQ ID NO:1, and an isoleucine at the position corresponding to position 467 of SEQ ID NO:1.
  • the variant has one or more amino acid substitutions corresponding to 252L, 262Y, 294L, 90Y, 371L, 41V, 427F, 53L, 306F, 221L, 17D, 17E, 17K, 18L, 75K, 110D, 110E, 110K, 110R, 223V, 223L, 223I, 224I, 257F, 229F, 229L, 158D, or 158E, based on SEQ ID NO:1.
  • the luciferase mutants disclosed herein do not include the wild-type sequence itself. In addition, the luciferase mutants disclosed herein do not include natural products.
  • the peroxisomal targeting signal 1 of luciferase may be deleted.
  • PTS1 is located at the 3 amino acids from the C-terminus, and its consensus sequence is "-(S/A/C)-(K/R/H)-(L/M)-COOH".
  • 3, 4, or 5 amino acids may be deleted from the C-terminus of luciferase, but this disclosure is not limited thereto.
  • 6, 7, 8, 9, 10, 11, or 12 amino acids may be deleted from the C-terminus of luciferase, but this disclosure is not limited thereto.
  • the presence or absence of activity of the deletion mutant may be easily confirmed by routine procedures.
  • mutant luciferases of the present disclosure are further illustrated by the following examples. However, these are for illustrative purposes only, and the present disclosure is not limited thereto.
  • pET-16b-LlLuc-1M is a plasmid for expressing luciferase, and a gene (sequence number 3) encoding mutant Heike firefly luciferase (sequence number 2) has been inserted into the multicloning site of the pET-16b vector (see WO2020/009215).
  • a plasmid for expressing LlLuc-2M was constructed by performing PCR using primers of SEQ ID NO: 4 and SEQ ID NO: 5 with pET-16b-LlLuc-1M as a template.
  • the names of the mutants prepared, the amino acid substitutions introduced into LlLuc-1M, and the template plasmid and primers used in PCR are shown in Table 1.
  • LlLuc-4M Mutant A mutant of LlLuc-4M (SEQ ID NO: 10) was prepared according to the method described in "1. Preparation of Heike firefly-derived luciferase (LlLuc) mutant". The nucleotide sequence of the gene encoding LlLuc-4M is shown in SEQ ID NO: 11. An expression plasmid for multiple mutant LlLuc-4M was constructed by repeated single mutation introduction. For example, an expression plasmid for LlLuc-6M was constructed by performing PCR using pET-16b-LlLuc-5M as a template and primers of SEQ ID NO: 16 and SEQ ID NO: 17. The names of the mutants prepared, the amino acid substitutions introduced into LlLuc-4M, and the template plasmid and primers used in PCR are shown in Table 2.
  • the culture medium was centrifuged at 15,000 rpm for 2 minutes, and the pellet was resuspended in 1 mL of 230 mM Tricine buffer (pH 7.85).
  • the bacterial suspension was ultrasonically disrupted, and then centrifuged at 15,000 rpm for 15 minutes to obtain the supernatant, which was then used as a crude enzyme solution of LlLuc-4M or its mutant enzyme.
  • the residual activity of the LlLuc-4M mutants shown in Table 3 increased by 0.19 to 0.62 compared to LlLuc-4M, in other words, improved by 58% to 187%, and it can be said that all LlLuc-4M mutants have improved thermal stability.
  • E. coli BL21 (DE3) was transformed to obtain strains producing LlLuc-4M or its mutant enzymes. These producing strains were inoculated into 2.5 mL of LB medium containing 100 ⁇ g/mL ampicillin and pre-cultured at 30° C. and 160 rpm for 12 hours. 1 mL of the pre-culture solution was inoculated into 100 mL of 2 ⁇ LB medium containing 50 ⁇ g/mL ampicillin and cultured at 30° C. and 120 rpm for 21 hours, after which 1 mL of 10 mM IPTG was added and cultured at 28° C. and 110 rpm for 17 hours.
  • the culture medium was centrifuged at 10,000 rpm for 10 minutes to obtain a pellet, which was then resuspended in 40 mL of A buffer (5% glycerol, 1 mM EDTA-2Na, 2 mM 3-mercapto-1,2-propanediol, phosphoric acid, pH 7.1).
  • a buffer 5% glycerol, 1 mM EDTA-2Na, 2 mM 3-mercapto-1,2-propanediol, phosphoric acid, pH 7.1.
  • the bacterial suspension was then ultrasonically disrupted, and centrifuged at 10,000 rpm for 50 minutes to obtain a supernatant.
  • the pH of the supernatant was adjusted to 7.1 with phosphoric acid, and the crude enzyme solutions were then filtered through a 0.2 ⁇ m filter to recover the respective solutions.
  • the crude enzyme solution was applied to a cation exchange chromatography column (Cytiva HiScreen SP FF) equilibrated with the above-mentioned buffer A, and gradient elution was performed with buffer B (200 mM potassium phosphate, 5% glycerol, 1 mM EDTA-2Na, 2 mM 3-mercapto-1,2-propanediol, pH 7.1) to recover the active fraction.
  • the recovered fraction was concentrated approximately 30-fold using an Amicon Ultra-3K (Merck) to obtain a purified enzyme solution.
  • LlLuc-7Md is a mutant of LlLuc-7M lacking the five C-terminal amino acids (P544, V545, A546, K547, and M548) that contain peroxisome targeting signal 1 (PTS1).
  • PTS1 is located at the three C-terminal amino acids, and its consensus sequence is "-(S/A/C)-(K/R/H)-(L/M)-COOH" (see, for example, FEBS J., 272, 2362 (2005), Plant Cell Physiol., 38, 759 (1997), Eur. J. Cell Biol., 71, 248 (1996)).
  • mutants of LlLuc-7Md were created according to the method described in "1. Creation of Heike firefly-derived luciferase (LlLuc) mutants".
  • the expression plasmid for multiple mutant LlLuc-7Md was constructed by repeatedly introducing single mutations.
  • an expression plasmid for LlLuc-9Md was constructed by performing PCR using pET-16b-LlLuc-8Md as a template and primers of SEQ ID NO:66 and SEQ ID NO:67.
  • the names of the mutants created, the amino acid substitutions introduced into LlLuc-7Md, and the template plasmid and primers used in PCR are shown in Table 5.
  • the residual activity of the LlLuc-7M mutants shown in Table 6 is increased by 0.04 to 0.50 compared to LlLuc-7M, in other words, improved by 13% to 161%, and it can be said that all LlLuc-7M mutants have improved thermal stability. It is highly likely that these mutations will similarly improve thermal stability when introduced into luciferases of other origins.
  • the residual activity of the LlLuc-8Md to 16Md and 15Md-2 mutants shown in Table 7 is increased by 0.02 to 1.01 compared to LlLuc-7Md, in other words, improved by 200% to 10100%. It can be said that the combination of the amino acid substitutions shown in Table 6 has dramatically improved the thermal stability of the LlLuc-7Md mutant.
  • the present disclosure provides luciferase with improved thermal stability.
  • the present disclosure also provides luciferase with greatly improved thermal stability.
  • the present disclosure also provides luciferase with improved affinity for substrates. This can be used for ATP measurement, ATP detection, luminescence measurement, etc.
  • SEQ ID NO: 1 Luciola lateralis-derived luciferase
  • SEQ ID NO: 2 Mutant L. lateralis-derived luciferase (also called HLK-C393L; see WO2020/009215)
  • SEQ ID NO: 3 Nucleotide sequence of the gene encoding SEQ ID NO: 2
  • SEQ ID NOs: 4 to 9 Primer sequences for introducing mutations
  • SEQ ID NO: 10 Mutant L.
  • LlLuc-4M lateralis-derived luciferase
  • SEQ ID NO: 11 Nucleotide sequence of the gene encoding SEQ ID NO: 10
  • SEQ ID NOs: 12 to 26 Primer sequences for introducing mutations
  • SEQ ID NO: 27 Mutant L.
  • lateralis-derived luciferase LlLuc-7Md
  • SEQ ID NO:28 Nucleotide sequence of the gene encoding SEQ ID NO:27
  • SEQ ID NOs:29 to 68 Primer sequences for mutagenesis
  • Amino acid sequence of Genji firefly luciferase SEQ ID NO:70
  • Nucleotide sequence of Genji firefly luciferase SEQ ID NO:71
  • Amino acid sequence of North American firefly luciferase SEQ ID NO:72
  • Nucleotide sequence of North American firefly luciferase Nucleotide sequence of North American firefly luciferase
  • Amino acid sequence of Fotulis pennsylvanic luciferase SEQ ID NO:74
  • Nucleotide sequence of Fotulis pennsylvanic luciferase Nucleotide sequence of Fotulis pennsylvanic lucifera
  • SEQ ID NO: 1 Amino acid sequence of luciferase derived from Luciola lateralis (Heike firefly) MENMENDENIVYGPEPFYPIEEGSAGAQLRKYMDRYAKLGAIAFTNALTG VDYTYAEYLEKSCCLGEALKNYGLVVDGRIALCSENCEEFFIPVLAGLFI GVGVAPTNEIYTLRELVHSLGISKPTIVFSSKKGLDKVITVQKTVTAIKT IVILDSKVDYRGYQSMDNFIKKNTPQGFKGSSFKTVEVNRKEQVALIMNS SGSTGLPKGVQLTHENAVTRFSHARDPIYGNQVSPGTAILTVVPFHHGFG MFTTLGYLTCGRIVMLTKFDEETFLKTLQDYKCSSVILVPTLFAILNRS ELLDKYDLSNLVEIASGGAPLSKEIGEAVARRFNLPGVRQGYGLTETTSA IIITPEGDDKPGASGKVVPLFKAKVIDLDTKKTLGPNR
  • SEQ ID NO: 2 Amino acid sequence of mutant Luciola lateralis luciferase (also called HLK-C393L) MENMENDENIVYGPEPFYPIEEGSAGAQLRKYMDRYAKLGAIAFTNALTG VDYTYAEYLEKSCCLGEALKNYGLVVDGRIALCSENCEEFFIPVLAGLFI GVGVAPTNEIYTLRELVHSLGISKPTIVFSSKKGLDKVITVQKTVTAIKT IVILDSKVDYRGYQSMDNFIKKNTPQGFKGSSFKTVEVNRKEQVALIMNS SGSTGLPKGVQLTHENLVTRFSHARDPIYGNQVSPGTAILTVVPFHHGFG MFTTLGYLTCGRIVMLTKFDEETFLKTLQDYKCSSVILVPTLFAILNRS ELLDKYDLSNLVEIASGGAPLSKEIGEAVARRFNLPGVRQGYGLTETTSA IIITPEGDDKPGASGKVVPLFKAKVIDLDTKKTLGPNRRG
  • SEQ ID NO: 3 Nucleotide sequence of mutant Luciola lateralis luciferase gene (also called HLK-C393L) atggaaaacatggagaacgatgaaaatattgtgtatggtcctgaaccattttaccctatt gaagagggatctgctggagcacaattgcgcaagtatatggatcgatatgcaaaacttgga gcaattgcttttactaacgcacttaccggtgtcgattatacgtacgccgaatacttagaa aaatcatgctgtctaggagaggcttttaaagaattatggtttggttgttgatggaagaatt gcgttatgcagtgaaaactgtgaagagttctttattcctgtattagcggttttatttata ggtgtcggtg
  • SEQ ID NO: 4 Primer for introducing G326S aatttctttagataaaggtgctccgccaga
  • SEQ ID NO: 5 Primer for introducing G326S tctaaagaaattagcgaagctgttgctaga
  • SEQ ID NO: 11 Nucleotide sequence of mutant Luciola lateralis-derived luciferase (LlLuc-4M) gene atggaaaacatggagaacgatgaaaatattgtgtatggtcctgaaccattttaccctatt gaagagggatctgctggagcacaattgcgcaagtatatggatcgatatgcaaaacttgga gcaattgcttttactaacgcacttaccggtgtcgattatacgtacgccgaatacttagaa aaatcatgctgtctaggagaggcttttaaagaattatggtttggttgttgatggaagaatt gcgttatgcagtgaaaactgtgtctttattcctgtattagcggttttatttata ggtgtcggtggtggtggtt
  • SEQ ID NO: 14 Primer for introducing F221L acgcgtgaccaaattctcgtgagtaagttg
  • SEQ ID NO: 15 Primer for introducing F221L ttggtcacgcgtttatctcacgccagagat
  • SEQ ID NO: 24 Primer for introducing F252/F262Y accacaagttagatagcctaaagtagttaa
  • SEQ ID NO: 28 Nucleotide sequence of mutant Luciola lateralis-derived luciferase (LlLuc-7Md) gene atggaaaacatggagaacgatgaaaatattgtgtatggtcctgaaccattttaccctatt gaagagggatctgctggagcacaattgcgcaagtatatggatcgatatgcaaaacttgga gcaattggtttactaacgcacttaccggtgtcgattatacgtacgccgaatacttagaa aaatcatgctgtctaggagaggcttttaaagaattatggtttggttgttgatggaagaatt gcgttatgcagtgaaaactgtgtctttattcctgtattagcggttttatttata ggtgtcggtgtggtggtgga
  • SEQ ID NO: 33 Primer for introducing Y18L aaatggttcaggaccatacacaatattttc
  • SEQ ID NO: 34 Primer for introducing Y18L cctgaaccatttttacctattgaagaggga
  • SEQ ID NO: 56 Primer for introducing Y257F gcctaaagtagttaacatacccaaaaccatg SEQ ID NO: 57 Primer for introducing Y257F actactttaggctttctaacttgtggttat
  • SEQ ID NO: 68 Primer for introducing L229Y (Y229L back mutation) agagatccaatttatggaaaccaagtttca
  • SEQ ID NO: 69 Amino acid sequence of Genji firefly luciferase Met Glu Asn Met Glu Asn Asp Glu Asn Ile Val Val Gly Pro Lys Pro Phe Tyr Pro Ile Glu Glu Gly Ser Ala Gly Thr Gln Leu Arg Lys Tyr Met Glu Arg Tyr Ala Lys Leu Gly Ala Ile Ala Phe Thr Asn Ala Val Thr Gly Val Asp Tyr Ser Tyr Ala Glu Tyr Leu Glu Lys Ser Cys Cys Leu Gly Lys Ala Leu Gln Asn Tyr Gly Leu Val Val Asp Gly Arg Ile Ala Leu Cys Ser Glu Asn Cys Glu Glu Phe Phe Ile Pro Val Ile Ala Gly Leu Phe Ile Gly Val Gly Val Ala Pro Thr Asn Glu Ile Tyr Thr Leu Arg Glu Leu Val His Ser Leu Gly Ile Ser Lys Pro Thr Ile Val Phe Ser Ser Lys Ly
  • SEQ ID NO: 70 Base sequence of Genji firefly luciferase atggaaaaca tggaaaacga tgaaatatt gtagttggac ctaaaccgtt ttaccctatc gaagagggat ctgctggaac acaattacgc aaatacatgg agcgatatgc aaaacttggc gcaattgctt tttacaaatgc agttactggt gttgattatt cttacgccga atacttggag aaatcatgttt gtctaggaaaa agctttgcaa aattatggtt tggttgttga tggcagaatt gcgttatgca gtgaaaactg ttttatgca gtgaaaactg
  • SEQ ID NO: 71 Amino acid sequence of North American firefly luciferase Met Glu Asp Ala Lys Asn Ile Lys Lys Gly Pro Ala Pro Phe Tyr Pro Leu Glu Asp Gly Thr Ala Gly Glu Gln Leu His Lys Ala Met Lys Arg Tyr Ala Leu Val Pro Gly Thr Ile Ala Phe Thr Asp Ala His Ile Glu Val Asn Ile Thr Tyr Ala Glu Tyr Phe Glu Met Ser Val Arg Leu Ala Glu Ala Met Lys Arg Tyr Gly Leu Asn Thr Asn His Arg Ile Val Val Cys Ser Glu Asn Ser Leu Gln Phe Phe Met Pro Val Leu Gly Ala Leu Phe Ile Gly Val Ala Val Ala Pro Ala Asn Asp Ile Tyr Asn Glu Arg Glu Leu Leu Asn Ser Met Asn Ile Ser Gln Pro Thr Val Val Phe Val Ser Lys Lys Gly Leu
  • SEQ ID NO: 72 Base sequence of North American firefly luciferase atggaagacg ccaaaacat aaagaaaggc ccggcgccat tctatccgct agaggatgga accgctggag agcaactgca taaggctatg aagagatacg ccctggttcc tggaacaatt gcttttacag atgcacatat cgaggtgaac atcacgtacg cggaatactt cgaaatgtcc gttcggttgg cagaagctat gaaacgatat gggctgaata caaatcacag aatcgtcgta tgaaa actctcttca attctttatg ccggtgtgggg gtgg gttgg gaa
  • SEQ ID NO: 74 Nucleotide sequence of Fotulis pennsylvanica luciferase atggaagata aaaatatttt atatggacct gaaccatttc atcccttggc tgatgggacg gctggagaac agatgtttta cgcattatct cggtatgcag atatttcagg atgcattgca ttgacaaatg ctcatacaa agaaaatgttt ttatatgaag aatttttaaa attgtcgtgt cgtttagcgg aaagtttttaa aaagtatgga ttaaacaaaacgacacaat agcggtgtgt agtgaaaaaaacacaat agcggtgtgt agtgtg

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