WO2020009215A1 - 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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WO2020009215A1
WO2020009215A1 PCT/JP2019/026784 JP2019026784W WO2020009215A1 WO 2020009215 A1 WO2020009215 A1 WO 2020009215A1 JP 2019026784 W JP2019026784 W JP 2019026784W WO 2020009215 A1 WO2020009215 A1 WO 2020009215A1
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amino acid
luciferase
seq
acid sequence
mutant
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French (fr)
Japanese (ja)
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華奈子 林
敦 一柳
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Kikkoman Corp
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Kikkoman Corp
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Priority to JP2020529058A priority patent/JP7472021B2/ja
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    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • 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
    • 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 invention provides a luciferase mutant having improved thermostability, a polynucleotide encoding the luciferase mutant, a method for producing the luciferase mutant, and at least one of ATP, ADP, and AMP containing the luciferase mutant. And a method for detecting at least one of ATP, ADP, and AMP, including using the luciferase mutant.
  • Firefly luciferase is an enzyme that converts adenosine triphosphate (ATP), D-luciferin and oxygen to adenosine monophosphate (AMP), oxyluciferin and carbon dioxide in the presence of magnesium ion and oxygen to produce light. It is. If the luminescence principle of firefly luciferase is applied, a very small amount of enzyme reaction substrate can be measured with extremely high sensitivity. For this reason, firefly luciferase, for example, detects microorganisms in food and drink using ATP as an index, determines food residues and stains attached to fingers and instruments, or high sensitivity using various antibody technologies and gene amplification technologies Widely used in measurement methods and the like.
  • ATP adenosine triphosphate
  • AMP adenosine monophosphate
  • beetle luciferases such as firefly luciferase have a drawback that they are easily deactivated when stored as reagents because they are unstable to heat. Therefore, attempts have been made to overcome such disadvantages and to obtain luciferase having good thermostability.
  • Non-Patent Document 1 reports that a firefly luciferase from North America in which the amino acid at position 342 has been mutated to alanine has been obtained, and that the firefly luciferase has improved luminescence persistence.
  • Patent Document 1 discloses that a Genji firefly or Heike firefly luciferase in which the amino acid at position 217 is substituted with a hydrophobic amino acid has heat resistance.
  • Patent Document 2 discloses that, in a 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, thermostability is improved. It is disclosed.
  • An object of the present invention is to provide a firefly luciferase having improved thermostability.
  • the present inventors have found that a mutation that substitutes cysteine at position 393 of Heike firefly for a non-acidic amino acid other than cysteine improves the thermal stability of firefly luciferase.
  • the present inventors have found that when the amino acid at the position corresponding to position 393 of SEQ ID NO: 1 is substituted, the thermostability of firefly luciferase can be improved, and completed the present invention.
  • the present invention includes the following aspects.
  • a firefly luciferase mutant comprising an amino acid sequence in which the amino acid residue at the position corresponding to position 393 of SEQ ID NO: 1 has been substituted, wherein the luciferase mutant has improved thermostability.
  • a mutant of wild-type firefly luciferase comprising an amino acid sequence in which the amino acid residue at the position corresponding to position 393 of SEQ ID NO: 1 is cysteine, wherein the amino acid at the position is a non-acidic amino acid other than cysteine
  • a luciferase variant having improved thermostability comprising the sequence.
  • a firefly luciferase mutant wherein the amino acid at the position corresponding to position 393 of SEQ ID NO: 1 is leucine, proline, valine, isoleucine, histidine, methionine, alanine, phenylalanine, glutamine, tryptophan, tyrosine, serine,
  • a luciferase variant having improved thermostability comprising an amino acid sequence selected from the group consisting of glycine, asparagine, lysine, threonine, and arginine.
  • a mutant of wild-type firefly luciferase comprising an amino acid sequence in which the amino acid residue at the position corresponding to position 393 of SEQ ID NO: 1 is tyrosine, wherein the amino acid at the position includes an amino acid sequence other than tyrosine and cysteine.
  • a luciferase mutant having improved thermostability.
  • an amino acid sequence in which the luciferase variant is selected from the group consisting of the following (i) to (iii): (I) an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, and 7; (Ii) an amino acid sequence in which one or several amino acids are substituted, deleted or added at a position other than the position corresponding to position 393 of SEQ ID NO: 1 in any one of the amino acid sequences of the above (i); and (iii) A) an amino acid sequence having a sequence identity of 70% or more in total length to any of the amino acid sequences of (i), and the following positions of SEQ ID NO: 1: positions 4 to 5, 9 to 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
  • the luciferase mutant according to (8) comprising: (10) A polynucleotide encoding the luciferase mutant according to any one of (1) to (9). (11) A vector comprising the polynucleotide according to (10).
  • a host cell comprising the polynucleotide according to (10) or the vector according to (11).
  • a method for producing a luciferase mutant having improved thermostability comprising a step of culturing the host cell according to (12).
  • a kit for detecting at least one of ATP, ADP, and AMP comprising the luciferase mutant according to any one of (1) to (9).
  • a method for detecting at least one of ATP, ADP, and AMP comprising using the luciferase mutant according to any one of (1) to (9).
  • thermostability a firefly luciferase having improved thermostability is provided.
  • Fig. 1-1 shows the wild type luciferase of wild-type luciferase of Heike firefly (Luciola @ latalis), Genji firefly (Luciola @ cruciata), North American firefly (Photinus @ pyralis), and Foturis pennsylvanica (Photuris @ pennsylvanica).
  • identical amino acid residues in four amino acid sequences are boxed. It is a continuation of FIG.
  • the present invention relates to a firefly luciferase comprising an amino acid sequence in which the amino acid residue corresponding to position 393 of SEQ ID NO: 1 has been substituted, for example, a mutant of wild-type firefly luciferase.
  • the present invention relates to a firefly luciferase comprising an amino acid sequence wherein the amino acid residue at the position corresponding to position 393 of SEQ ID NO: 1 is cysteine, for example, a mutant of wild-type firefly luciferase.
  • the luciferase variants of the present invention have improved thermostability.
  • wild type refers to a trait that is most frequently found in nature in a population of the same species.
  • any firefly-derived firefly can be used.
  • Heike firefly (Luciola lateralis), Genji firefly (Luciola cruciata), North American firefly (Photinus pyralis), Fotsurisu-Penshirubanika (Photuris pennsylvanica), Europe glow worm (Lampyris noctiluca), Miyako Mado firefly (Pyrocoelia miyako), click beetle (Pyrophorus plagiophthalamus)
  • Luciola mingrelica preferably firefly, Genji firefly, North American firefly, or firefly luciferase derived from Foturis pennsylvanica.
  • chimeric proteins prepared based on various firefly-derived luciferase genes may be used.
  • correspondence between amino acid positions can be easily determined by comparing the amino acid sequences of various firefly luciferases using, for example, existing amino acid homology analysis software, for example, GENETYX (manufactured by GENETYX).
  • GENETYX manufactured by GENETYX
  • the amino acid position of luciferase corresponding to position X of the amino acid sequence of SEQ ID NO: 1 can be specified by aligning the amino acid sequence of the luciferase with the amino acid sequence of SEQ ID NO: 1.
  • “the position corresponding to position 393 of SEQ ID NO: 1” may be position 393 of the amino acid sequence of SEQ ID NO: 3, position 391 of SEQ ID NO: 5, and position 390 of SEQ ID NO: 7.
  • the amino acid at a position corresponding to position 393 of SEQ ID NO: 1 in the luciferase variant is a non-acidic amino acid other than cysteine (ie, leucine, proline, valine, isoleucine, histidine, methionine, alanine, phenylalanine, glutamine, Tryptophan, tyrosine, serine, glycine, asparagine, lysine, threonine, or arginine).
  • cysteine ie, leucine, proline, valine, isoleucine, histidine, methionine, alanine, phenylalanine, glutamine, Tryptophan, tyrosine, serine, glycine, asparagine, lysine, threonine, or arginine.
  • the luciferase variant has high sequence identity with the luciferase variant from Heike firefly, or the amino acid sequence of SEQ ID NO: 1, such as 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.
  • Variants containing an amino acid sequence having the sequence identity of The amino acid at a position corresponding to position 393 of SEQ ID NO: 1 is preferably selected from the group consisting of leucine, proline, valine, isoleucine, histidine, methionine, alanine, phenylalanine, glutamine, tryptophan, tyrosine, serine, and glycine; Preferably it is selected from the group consisting of leucine, proline, valine, isoleucine, histidine, methionine and alanine, more preferably leucine, proline or valine.
  • the amino acid at the position corresponding to position 393 of SEQ ID NO: 1 (eg, the amino acid at position 393 of SEQ ID NO: 3) in the luciferase variant is an amino acid other than cysteine.
  • the luciferase variant has a high sequence identity with the luciferase variant from Genji firefly or the amino acid sequence of SEQ ID NO: 3, for example, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.
  • Variants containing an amino acid sequence having the sequence identity of The amino acid at the position corresponding to position 393 of SEQ ID NO: 1 may be asparagine, alanine, serine, arginine, leucine, threonine, histidine, valine, phenylalanine, glycine, tryptophan, tyrosine, isoleucine, proline, methionine, or glutamine.
  • the amino acid at the position corresponding to position 393 of SEQ ID NO: 1 (eg, the amino acid at position 391 of SEQ ID NO: 5) in the luciferase variant is tryptophan.
  • the luciferase variant has a high sequence identity with the luciferase variant from North American firefly or the amino acid sequence of SEQ ID NO: 5, for example, 70% or more, 80% or more, 90% or more, 95% or more, 96%.
  • a mutant containing an amino acid sequence having 97% or more, 98% or more, or 99% or more sequence identity may be used.
  • the present invention relates to any of the above, wherein the amino acid at the position corresponding to position 393 of SEQ ID NO: 1 is leucine, proline, valine, isoleucine, histidine, methionine, alanine, phenylalanine, glutamine, tryptophan, tyrosine, serine, glycine, asparagine
  • a luciferase variant having improved thermostability comprising an amino acid sequence selected from the group consisting of lysine, threonine, and arginine.
  • the amino acid at the position corresponding to position 393 of SEQ ID NO: 1 in the firefly luciferase before mutagenesis is not limited as long as it is not any of the above amino acids, but may be, for example, tyrosine. (In this case, provided that the amino acid after substitution is not tyrosine).
  • the amino acid at position corresponding to position 393 of SEQ ID NO: 1 in the luciferase variant is not tryptophan.
  • the amino acid at a position corresponding to position 393 of SEQ ID NO: 1 in the luciferase variant is preferably selected from the group consisting of leucine, proline, valine, isoleucine, histidine, methionine, and alanine, more preferably leucine , Proline, or valine.
  • the present invention relates to a firefly luciferase mutant comprising an amino acid sequence in which the amino acid residue at the position corresponding to position 393 of SEQ ID NO: 1 (for example, position 390 of SEQ ID NO: 7) is tyrosine,
  • a luciferase variant having improved thermostability wherein the amino acid at the position comprises an amino acid sequence other than tyrosine and cysteine (eg, an amino acid sequence other than tyrosine, cysteine and tryptophan).
  • the amino acid residue at a position corresponding to position 393 of SEQ ID NO: 1 is proline, asparagine, arginine, glycine, serine, lysine, phenylalanine, aspartic acid, glutamine, threonine, glutamic acid, isoleucine, or alanine.
  • it can be, for example, proline, asparagine, arginine, glycine, serine, lysine, phenylalanine, aspartic acid, glutamine, or threonine.
  • the mutation at a position corresponding to position 393 of SEQ ID NO: 1 has been introduced artificially. This can be done by artificially introducing a mutation into the sequence of the gene encoding luciferase.
  • the firefly luciferase mutant of the present invention may further include a mutation other than the mutation at the position 393 or the position corresponding to the position 393.
  • the mutation may be artificially introduced for the purpose of any particular effect, or may be randomly or non-artificially introduced.
  • Examples of the mutation introduced with the intention of a specific effect include, for example, addition and modification of a sequence for enhancing the expression level of firefly luciferase, modification for improving the purification efficiency of firefly luciferase, and the like, as well as firefly luciferase.
  • Various mutations that impart practically favorable properties may also be included.
  • JP-A-2011-120559 discloses a 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. It is disclosed that thermal stability is improved.
  • 2011-120559 discloses that the stability is further improved by combining these mutations, a mutation in which the amino acid at position 326 is substituted with serine, and / or a mutation in which the amino acid at position 467 is substituted with isoleucine. It is described that improved firefly luciferase can be obtained.
  • the amino acid at the position corresponding to position 217 of SEQ ID NO: 1 may be leucine or isoleucine, and / or the amino acid at the position corresponding to position 490 of SEQ ID NO: 1 may be lysine.
  • the amino acid at the position corresponding to position 252 of SEQ ID NO: 1 may be methionine.
  • leucine is introduced into a position corresponding to position 217 of SEQ ID NO: 1 and lysine is introduced into a position corresponding to position 490 with respect to wild-type Heike firefly luciferase (SEQ ID NO: 1).
  • Mutant (amino acid sequence is represented by SEQ ID NO: 9); Mutation in which isoleucine was introduced at a position corresponding to position 217 of SEQ ID NO: 1 (position 217 of SEQ ID NO: 3) with respect to wild-type Genji firefly luciferase (SEQ ID NO: 3) And a mutant in which methionine is introduced at a position corresponding to position 252 of SEQ ID NO: 1 (position 249 of SEQ ID NO: 7) with respect to wild-type F. pensilvanica firefly luciferase (SEQ ID NO: 7). These mutants may further include a mutation at the position 393 or a position corresponding to the position 393.
  • the luciferase variant comprises an amino acid mutation at a position corresponding to position 393 of SEQ ID NO: 1 (and optionally other amino acid mutations described herein), and (i) to (iii) below.
  • the amino acid sequence of (iii) is 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more of the entire amino acid sequence of (i). % Or more, 77% or more, 78% or more, 79% or more, preferably 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more % Or more, 89% or more, more preferably 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, still more preferably 95% or more, 96% or more, 97% or more, and even more preferably 98% or more.
  • the “identical region” in the amino acid sequence of (iii) is a region in which the same amino acid residue is conserved in the four firefly luciferases (Heike firefly, Genji firefly, North American firefly, and Fotulis Pennsylvania) shown in FIG. Can be specified as In the amino acid sequence of (iii), the same region and a region corresponding to the same region in the luciferase mutant are 91% or more, 92% or more, 93% or more, 94% or more, preferably 95% or more, 96% or more. It has a sequence identity of 97% or more, more preferably 98% or more, and most preferably 99% or more.
  • the identity of an amino acid sequence and a gene sequence can be calculated by a program such as GENETYX (manufactured by GENETYX) such as maximum matching or search homology, or a program such as multiple alignment of CLUSTAL @ W or pairwise alignment by BLAST. it can.
  • GENETYX manufactured by GENETYX
  • a program such as multiple alignment of CLUSTAL @ W or pairwise alignment by BLAST. it can.
  • % identity refers to a region that could be aligned when two or more amino acid sequences were aligned using BLAST (BLASTP) or the like for the amino acid sequence.
  • the total number of amino acids is used as a denominator, and the number of positions occupied by the same amino acid is used as a numerator. Therefore, usually, when there is a region where no identity is found in two or more amino acid sequences, for example, when there is an additional sequence in the C-terminus where no identity is found in one amino acid sequence, the region where the identity is not found Is not used in the calculation of% identity since it cannot be aligned.
  • the range of "one or several” is 1 to 10, preferably 1 to 7, more preferably 1 to 5, particularly preferably 1 to 3, or 1 or 2 is there.
  • the luciferase variant comprises an amino acid mutation at a position corresponding to position 393 of SEQ ID NO: 1 (and optionally other amino acid mutations described herein), and (i) to (iii) below.
  • Amino acid sequence selected from the group consisting of: (I) the amino acid sequence of SEQ ID NO: 1, (Ii) an amino acid sequence in which one or several amino acids are substituted, deleted or added at a position other than the position corresponding to position 393 of SEQ ID NO: 1 in the amino acid sequence of (i), and (iii) 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, preferably 95% or more, 96% or more, 97% or more, more preferably 98% or more, An amino acid sequence having preferably 99% or more sequence identity, including.
  • the luciferase variant of the present invention has luciferase activity.
  • the presence or absence of luciferase activity can be measured using, for example, Lumitester C-110 (manufactured by Kikkoman Biochemifa) according to the method described in Examples.
  • thermostability can be evaluated, for example, using the remaining activity when a firefly luciferase is heat-treated at a predetermined temperature for a predetermined time as an index.
  • the thermostability of the firefly luciferase in the present invention is determined by heating the firefly luciferase under high temperature conditions, for example, usually at a reaction temperature of 30 to 50 ° C., for example, 35 to 45 ° C. or 35 to 40 ° C., for a certain period of time.
  • it can be evaluated by comparing the residual activity ratio after heat treatment for 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 calculated by the ratio of the activity after heat treatment to the firefly luciferase activity before acting under the above-mentioned high temperature conditions.
  • the improvement of the thermostability according to the present invention refers to a luciferase which does not introduce the mutation of the present invention (mutation at the position corresponding to position 393 in SEQ ID NO: 1) when the firefly luciferase mutant is allowed to act under the above conditions.
  • the present invention relates to a polynucleotide encoding the luciferase mutant of the present invention (hereinafter, also referred to as “luciferase gene”).
  • the sequence of a polynucleotide can be readily determined based on the amino acid sequence of a firefly luciferase variant.
  • polynucleotides encoding the amino acid sequences of SEQ ID NOs: 1, 3, 5, and 7 include the polynucleotides of SEQ ID NOs: 2, 4, 6, and 8, respectively.
  • the polynucleotide of the present invention for example, (I) a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, and 8; (Ii) a nucleotide sequence in which one or several nucleotides are substituted, deleted or added in any one of the nucleotide sequences of the above (i); and (iii) a nucleotide sequence of any one of the above (i) 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, preferably 80% or more, 81% in total length 82% or more, 83% or more, 84% or more, 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 % Or more, 94% or more, more preferably 95% or more
  • chromosomal DNA or mRNA can be extracted from a firefly tissue or cell having a luciferase-producing ability by a conventional method, for example, a method described in Current Protocols in Molecular Biology (WILEY Interscience, 1989).
  • cDNA can be synthesized using mRNA as a template. Using the chromosomal DNA or cDNA thus obtained, a library of chromosomal DNA or cDNA can be prepared.
  • a suitable primer DNA is prepared, and a DNA containing a target nucleotide fragment encoding luciferase is amplified by a suitable polymerase chain reaction (Polymerase Chain Reaction, PCR method) such as 5′RACE method or 3′RACE method, By linking these DNA fragments, a DNA containing the full length of the nucleotide encoding the target luciferase can be obtained.
  • a suitable polymerase chain reaction Polymerase Chain Reaction, PCR method
  • 5′RACE method or 3′RACE method By linking these DNA fragments, a DNA containing the full length of the nucleotide encoding the target luciferase can be obtained.
  • nucleotide sequence encoding luciferase when the nucleotide sequence encoding luciferase is known, such as the nucleotide sequences shown in SEQ ID NOs: 2, 4, 6, and 8, the nucleotide sequence may be artificially synthesized.
  • an artificial gene synthesis service is provided, for example, by Integrated ⁇ DNA ⁇ Technologies.
  • Mutation treatment of the luciferase gene can be performed by any known method according to the intended mutation form. That is, a method of bringing a luciferase gene or a recombinant DNA into which the gene is incorporated into contact with a mutagenic agent and causing it to act; an ultraviolet irradiation method; a genetic engineering technique; or a method utilizing a protein engineering technique is widely used. Can be used.
  • Examples of the mutagenic agent used in the above mutagenesis treatment include hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine, nitrous acid, sulfurous acid, hydrazine, formic acid, and 5-bromouracil. be able to.
  • the conditions for the contact and action can be set according to the type of the drug to be used and the like, and are not particularly limited as long as the desired mutation can be actually induced in the luciferase gene.
  • a desired mutation can be induced by contacting and acting at a reaction temperature of 20 to 80 ° C. for 10 minutes or more, preferably 10 to 180 minutes, preferably at the drug concentration of 0.5 to 12 M.
  • Ultraviolet irradiation can also be performed according to a conventional method as described above (Hyundai Kagaku, 024-30, June 1989).
  • Site-Specific Mutagenesis As a method that makes full use of protein engineering techniques, a technique generally known as Site-Specific Mutagenesis can be used.
  • the Kramer method Nucleic Acids Res., 12, 12, 9441 (1984): Methods Enzymol., 154, 350 (1987): Gene, 37, 73 (1985)
  • Eckstein method Nucleic, 1349, 1987) 1985: cNucleic Acids Res., 13, 8755 (1985): cNucleic Acids Res, 14, 9679 (1986)
  • Kunkel method Proc. Natl. Aci., Sci.
  • the Site-Specific Mutagenesis can be carried out by using a commercially available kit, for example, a QuickChange Site-Directed Mutagenesis Kit (manufactured by Agilent Technologies).
  • the desired modified luciferase gene can also be directly synthesized by an organic synthesis method or an enzyme synthesis method in addition to the above-described gene modification method.
  • the determination or confirmation of the DNA base sequence of the luciferase gene obtained by the above method can be performed, for example, by using a multi-capillary DNA analysis system Applied Biosystems 3130xl Genetic Analyzer (manufactured by Thermo Fisher Scientific).
  • the present invention relates to a vector comprising the above polynucleotide. It is preferable in terms of handling that these luciferase genes are linked to various vectors according to a conventional method.
  • a vector that can be used in the present invention includes a plasmid, and any other vector known to those skilled in the art, such as bacteriophage and cosmid, can be used.
  • the type of vector can be selected according to the host cell, and specifically, for example, pET16-b or pKK223-3 is preferred.
  • the present invention relates to a host cell comprising the polynucleotide or the vector.
  • Host cells include, but are not limited to, bacteria such as Escherichia coli and Bacillus subtilis, yeast cells, insect cells, animal cells (eg, mammalian cells), plant cells, and the like, and preferably bacterial cells such as Escherichia coli.
  • the luciferase gene obtained as described above is incorporated into a vector such as a bacteriophage, a cosmid, or a plasmid used for transformation of a prokaryotic or eukaryotic cell by a conventional method, and a host corresponding to each vector is subjected to a conventional method.
  • a host corresponding to each vector is subjected to a conventional method.
  • the obtained recombinant DNA 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, Escherichia coli strain BL21 (DE3) (both manufactured by Takara Bio Inc.) or the like can be transformed or transduced to obtain each strain.
  • the present invention relates to a method for producing a luciferase mutant having improved thermostability, comprising a step of culturing the host cell.
  • the culture can be performed by various known methods, and may be performed by a solid culture method, but is preferably performed by a liquid culture method.
  • the method of the present invention may include a step of culturing the above host cell under conditions capable of expressing a luciferase protein, and optionally, a step of isolating luciferase from the culture or culture solution.
  • the condition under which the luciferase protein can be expressed means that the luciferase gene is transcribed and translated to produce a polypeptide encoded by the gene.
  • the method of the present invention comprises artificially introducing a mutation at a position corresponding to position 393 of SEQ ID NO: 1 of the luciferase protein before the culturing step. This can be done by artificially introducing a mutation into the sequence of the gene encoding luciferase.
  • Examples of a medium for culturing the host cell include, for example, sodium chloride, phosphoric acid 2 or more in one or more nitrogen sources such as yeast extract, tryptone, peptone, meat extract, corn steep liquor, and soybean or wheat bran exudate.
  • Add one or more inorganic salts such as potassium hydrogen, dipotassium hydrogen phosphate, magnesium sulfate, magnesium chloride, ferric chloride, ferric sulfate or manganese sulfate. The added one is used.
  • the initial pH of the medium is suitably adjusted to pH 7-9.
  • the culture is performed at a culture temperature of 20 to 42 ° C., preferably at a culture temperature of about 25 to 37 ° C. for 4 to 24 hours, and more preferably at a culture temperature of about 25 to 37 ° C. for 8 to 16 hours. It is preferably carried out by culturing, stationary culturing, or the like.
  • luciferase can be collected from the culture using a conventional enzyme collecting means.
  • the cells are subjected to ultrasonic destruction treatment, grinding treatment or the like by a conventional method, or the present enzyme is extracted using a lytic enzyme such as lysozyme, or lysed by shaking or standing in the presence of toluene or the like.
  • a lytic enzyme such as lysozyme, or lysed by shaking or standing in the presence of toluene or the like.
  • the solution is filtered, centrifuged or the like to remove a solid portion, and if necessary, nucleic acids are removed with streptomycin sulfate, protamine sulfate, manganese sulfate, or the like, and then ammonium sulfate, alcohol, acetone, or the like is added thereto. Then, the precipitate is collected to obtain a crude enzyme of luciferase.
  • a luciferase purified enzyme from the above luciferase crude enzyme, for example, gel filtration method using Sephadex, Superdex or Ultrogel, ion exchange carrier, hydrophobic carrier, adsorption elution method using hydroxyapatite, polyacrylamide Electrophoresis using a gel or the like, sedimentation such as sucrose density gradient centrifugation, affinity chromatography, fractionation using a molecular sieving membrane or a hollow fiber membrane, etc., are appropriately selected or performed in combination. As a result, a purified luciferase enzyme can be obtained. Thus, a desired luciferase can be obtained.
  • Luciferase produced by the method of the present invention can be used in the kit described herein or in a method for detecting at least one of ATP, ADP and AMP.
  • the invention relates to a kit for detecting at least one of ATP, ADP, and AMP, comprising a luciferase variant described herein.
  • the kit of the present invention may include luciferin in addition to the luciferase mutant.
  • metal ions such as magnesium, manganese, and calcium may be included in the kit.
  • concentration of metal ion depending on the enzyme used.
  • ATP, O 2 and luciferin are converted by luciferase to AMP, pyrophosphate, CO 2 and oxyluciferin, which results in luminescence. The reaction that occurs at this time is represented as follows.
  • the kit of the present invention further comprises an enzyme that catalyzes a reaction for producing ATP from ADP.
  • Enzymes that catalyze the reaction of producing ATP from ADP include pyruvate kinase (PK), acetate kinase (AK), creatine kinase (CK), polyphosphate kinase (PPK), hexokinase, glucokinase, glycerol kinase, fructokin Kinase, phosphofructokinase, riboflavin kinase, and fructose bisphosphatase can be selected from the group consisting of:
  • the kit of the present invention further comprises pyruvate orthophosphate dikinase (PPDK), adenylate kinase (ADK) or pyruvate water dikinase (PWDK).
  • ATP is contained in the sample, it is converted to AMP by luciferase and emits light.
  • ADP is contained in a sample in a system in which an enzyme that catalyzes a reaction for producing ATP from ADP is present, ADP is converted to ATP by the enzyme, and then ATP is subjected to a luminescence reaction. This makes it possible to measure the total amount of ATP and ADP present in the system.
  • AMP is contained in a sample in a system in which PPDK is present, this is converted to ATP by PPDK, PEP, and PPi.
  • AMP when AMP is contained in the sample, this is converted to ATP by PWDK, PEP, and phosphoric acid.
  • the generated ATP emits light again by luciferase. Light emission is stably maintained, and the amount of light emission correlates with the total amount of ATP and AMP present in the system, so that ATP and AMP can be quantified.
  • an enzyme that catalyzes a reaction for producing ATP from ADP and PPDK, ADK or PWDK are present, the total amount of ATP, ADP and AMP can be measured.
  • the luciferin may be any as long as it is recognized as a substrate by the luciferase used, and may be a natural or chemically synthesized one. Also, any known luciferin derivative can be used.
  • the basic skeleton of luciferin is imidazopyrazinone, and there are many tautomers.
  • Luciferin includes firefly luciferin. Firefly luciferin is a substrate for firefly luciferase (EC 1.13.12.7).
  • the luciferin derivatives may be those described in JP-A-2007-91695, JP-T-2010-523149 (WO 2008/127677), and the like.
  • the kit of the present invention may include at least one of a stabilizer, a buffer, and instructions.
  • the invention relates to a method for detecting at least one of ATP, ADP, and AMP, comprising using a luciferase variant described herein.
  • the method may include catalyzing a luciferin oxidation reaction using the luciferase variant described herein, and measuring luminescence generated by the oxidation reaction.
  • the catalyst for the luciferin oxidation reaction by the luciferase mutant is as described in “Kit for detecting at least one of ATP, ADP, and AMP”.
  • the reaction can be performed by reacting the sample with the luciferase mutant and luciferin described herein.
  • ATP is contained in the sample, it is converted to AMP by luciferase and emits light, so that ATP can be measured.
  • the total amount of ATP and ADP present in the system can be measured.
  • PPDK or PWDK is present, ATP and AMP can be quantified.
  • an enzyme that catalyzes a reaction for producing ATP from ADP and PPDK, ADK or PWDK are present, the total amount of ATP, ADP and AMP can be measured.
  • the luminescence of luciferase can be measured by a known method.
  • a suitable luminescence measuring device for example, a luminometer (Centro LB960 or Lumat3 @ LB9508, manufactured by Berthold, Lumitester C-110, manufactured by Kikkoman Biochemifa) can be used. It can be evaluated using the relative luminescence intensity (RLU) obtained using Lumitester C-100, Lumitester PD-20, Lumitester PD-30, etc. as an index. Usually, the luminescence generated during the conversion of luciferin to oxyluciferin is measured.
  • RLU relative luminescence intensity
  • the luminescence measurement device high sensitivity measurement is possible, and a device equipped with a photomultiplier tube (manufactured by 3M) or a device equipped with a photodiode (manufactured by Hygiena, Neogen, etc.) can be used. .
  • Example 1 Heat resistance test of mutant of Heike firefly luciferase> (Materials and methods) Vector construction pET16-b (Novagen) MCS (multi-cloning site, Nde1-BamH1 site) with HLK (wild-type Heike firefly luciferase (SEQ ID NO: 1) introduced with A217L and E490K mutations for improving thermostability) PCR using a plasmid (HLK pET-16b) into which the gene sequence of (amino acid sequence: SEQ ID NO: 9, nucleotide sequence: SEQ ID NO: 10) was inserted as a template and primers for amplifying the sequence encoding each mutant
  • a plasmid vector encoding each mutant in which the cysteine at position C393 was substituted with various amino acids was prepared.
  • the sequence of the reverse primer used for preparing each plasmid vector is common, and is SEQ ID NO: 11 (AACTTCTCCACGTCTGTTCGGGCCCAAAG).
  • the following table shows the sequence and sequence number of each forward primer used to prepare a plasmid vector encoding luciferase containing each amino acid at position 393.
  • luciferase buffer 5% trehalose, 10 mM Tris, 4.4 mM succinic acid, 1 mM EDTA, 1 mM DTT (pH 7.6).
  • the cells were disrupted (10 s pulse, 20 s rest, total pulse 1 min) using a sonicator astrason ULTRASONIC PROCESSOR XL (manufactured by Misonix).
  • the supernatant obtained by centrifugation was filtered through a 0.45 ⁇ m or 0.20 ⁇ m PVDF membrane to obtain a crude enzyme solution.
  • H217 SEQ ID NO: 9 into which A217L and E490K mutations were introduced was expressed according to the method described in JP-A-8-98680, and purified beforehand.
  • Thermostability test Before storing the prepared enzyme at 37 ° C, it was pre-incubated at 25 ° C for 5 minutes to return from refrigeration to room temperature. Subsequently, the enzyme was stored in a water bath at 37 ° C. for 90 minutes, and diluted with buffer (4.48 g Tricine, 185 mg EDTA ⁇ Na 2 .2H 2 O, 25 g glycerol, 5 g BSA (pH 7.8) per 500 ml). Was used to dilute as needed, and the enzyme was prepared so as to fall within the measurement range of Lumitester C-110.
  • a solution obtained by mixing 2.0 ml of 50 mM Tricine-NaOH buffer (pH 7.8), 0.5 ml of 40 mM ATP solution, 2.0 ml of 5.0 mM Luciferin, and 0.5 ml of 0.1 M MgSO 4 was used.
  • Measurement start time 10 seconds after adding the luminescent reagent to the enzyme solution
  • Measurement time 10 seconds in total
  • HLK used as a control is a purified enzyme containing no His tag
  • C393 mutant is a crude enzyme containing 10 His tags at the N-terminal of the sequence.
  • a purified enzyme of HLK and a purified or crudely purified HLK containing 10 His tags at the N-terminus were prepared in the same manner as described above. The heat resistance was tested by heating at 42 ° C. for 30 minutes or 60 minutes. As a result, there was no difference in stability among the purified enzyme of HLK, the purified enzyme of HLK containing His tag, and the crude purified enzyme of HLK containing His tag. It did not appear to affect stability (data not shown).
  • Example 2 Thermostability test of other firefly luciferase variants> (Materials and methods)
  • the gene sequence of wild type North American firefly luciferase (amino acid sequence: SEQ ID NO: 5, nucleotide sequence: SEQ ID NO: 6) was introduced into the MCS site (Nde1-BamH1 site) of pET16-b.
  • the resulting plasmid was designated as Ppy pET16-b.
  • a wild-type Genji firefly luciferase (amino acid sequence: SEQ ID NO: 3, nucleotide sequence: SEQ ID NO: 4) into which a T217I mutation has been introduced, and a wild-type Foturis pencilvani luciferase (amino acid sequence: SEQ ID NO: 7, nucleotide sequence: sequence)
  • the gene sequence obtained by introducing the T249M mutation into No. 8) was introduced into the MCS site (EcoR1-HindIII site) of pKK223-3.
  • the resulting plasmids were LucT @ pKK223-3 and PpeT249M @ pKK223-3, respectively.
  • a mutation was introduced into the nucleotide sequence of SEQ ID NO: 4 to include the T217I mutation, and a sequence encoding a His tag (6 His) was added immediately before the stop codon.
  • the nucleotide sequence of SEQ ID NO: 74 was used.
  • the nucleotide sequence of SEQ ID NO: 8 was codon-optimized, a mutation was introduced to include the T249M mutation, and the sequence encoding the His tag (6 His) was terminated.
  • the nucleotide sequence of SEQ ID NO: 31 added immediately before the codon was used.
  • a plasmid vector encoding the mutant was prepared according to Example 1 using the following primers.
  • the sequence of the reverse primer used to prepare a plasmid vector encoding luciferase containing each amino acid at position 393 of Genji firefly luciferase is common (SEQ ID NO: 32 (AACTTCTCCACGTCTGTTAGGACCTAAAG)), and the sequence and SEQ ID NO of each forward primer are as follows: Is shown in the table.
  • the sequence of the reverse primer used to prepare a plasmid vector encoding a luciferase containing each amino acid at position 390 of F. pensilvani luciferase is common except that aspartic acid is introduced at position 390 (SEQ ID NO: 52 ( CAGTTCACCGGTTTCGTTCGGGCCCAGG)).
  • SEQ ID NO: 52 CAGTTCACCGGTTTCGTTCGGGCCCAGG
  • the following table shows the sequence of each forward primer and the reverse primer when aspartic acid is introduced at position 390, and the sequence numbers thereof.
  • the sequence of the forward primer used to prepare a plasmid vector in which tryptophan was introduced at position 391 of North American firefly luciferase was SEQ ID NO: 72 (CAGAGAGGCGAATTATGGGTCAGAGGACC), and the sequence of the reverse primer was SEQ ID NO: 73 (TAATTCGCCTCTCTGATTAACGCCCAGCG).
  • thermostable mutants were in accordance with Example 1.
  • the thermal stability test was also in accordance with Example 1, with the following changes.
  • the warming time in the water bath was 90 minutes for Genji firefly luciferase, 5 minutes for F. philippensis vanilla luciferase, and 20 minutes for North American firefly luciferase.
  • the average value of the measured values was determined by performing three tests on F. pensilvani luciferase, and the relative value at 37 ° C storage time of 5 minutes when the value at 37 ° C storage time of 0 minute was set to 1
  • the following table shows the residual activity ratio of each mutant (the residual activity ratio after 5 minutes) when the residual activity after 5 minutes without mutation and 1 was defined as 1.
  • the average value of the measured values was determined by performing three tests, and the relative value at a storage time of 20 minutes at 37 ° C., where the value when the storage time at 37 ° C. was 0 minutes was 1, and The residual activity ratio (residual activity ratio after 20 minutes) of each mutant assuming that the residual activity after 20 minutes without mutation is 1 is shown in the following table.
  • heat resistance is also found in Genji firefly luciferase, F. pentacholine luciferase, and North American firefly luciferase when the amino acid residue at the position corresponding to position 393 of SEQ ID NO: 1 is substituted. It was shown that it could be improved.
  • Genji firefly luciferase was particularly remarkable in the effect of improving thermostability, as for position 393, asparagine, alanine, serine, arginine, leucine, threonine, histidine, valine (residual activity of 70% or more); or phenylalanine, glycine, tryptophan, tyrosine, In this case, the substitution was performed with isoleucine, proline, methionine, or glutamine (residual activity of 60% or more).
  • thermostability of Foturis pensilvani luciferase was particularly remarkable because at position 390, proline, asparagine, arginine, glycine, serine, lysine, phenylalanine, aspartic acid, glutamine, and threonine (residual activity of 90% or more); This was the case where glutamic acid, isoleucine, alanine (residual activity of 80% or more); or valine, methionine, leucine, histidine (residual activity of 70% or more) were substituted.

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JP7385134B2 (ja) 2021-03-12 2023-11-22 東亜ディーケーケー株式会社 変異型甲虫ルシフェラーゼ、遺伝子、組換えベクター、形質転換体、及び変異型甲虫ルシフェラーゼの製造方法
WO2024158050A1 (ja) 2023-01-27 2024-08-02 キッコーマン株式会社 ルシフェラーゼ変異体
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