WO2022196538A1 - 改変アルカリホスファターゼ - Google Patents

改変アルカリホスファターゼ Download PDF

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WO2022196538A1
WO2022196538A1 PCT/JP2022/010743 JP2022010743W WO2022196538A1 WO 2022196538 A1 WO2022196538 A1 WO 2022196538A1 JP 2022010743 W JP2022010743 W JP 2022010743W WO 2022196538 A1 WO2022196538 A1 WO 2022196538A1
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alkaline phosphatase
amino acid
modified alkaline
acid sequence
modified
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French (fr)
Japanese (ja)
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佑介 萩原
康博 三原
智晴 本山
祥吾 中野
創平 伊藤
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Ajinomoto Co Inc
University of Shizuoka
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Ajinomoto Co Inc
University of Shizuoka
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase

Definitions

  • the present invention relates to modified alkaline phosphatase and the like.
  • Alkaline phosphatase (EC 3.1.3.1) is a phosphomonoesterase with low substrate selectivity that has an alkaline pH optimum. Alkaline phosphatase is widely used in laboratory and clinical test reagents. For example, in experiments, alkaline phosphatase has been used to dephosphorylate the 5' ends of DNA or RNA and as a reporter in reporter assays. In clinical test reagents, alkaline phosphatase is used for the detection or quantification of target molecules in the form of alkaline phosphatase-labeled substances (eg, affinity substances such as antibodies).
  • alkaline phosphatase-labeled substances eg, affinity substances such as antibodies.
  • bovine small intestine alkaline phosphatase Bovine Intestinal Alkaline Phosphatase: bIAP
  • bIAPII Bovine Intestinal Alkaline Phosphatase
  • bIAPII which is isomer II
  • a bIAPII mutant having serine, glycine, or asparagine at position 430 and alanine at position 430 exhibits high thermostability while maintaining activity equivalent to that of native bIAPII (Patent Document 1).
  • An object of the present invention is to provide a highly active alkaline phosphatase with excellent thermostability.
  • Modified alkaline phosphatase which is the following protein (A), (B), or (C): (A) a protein comprising the amino acid sequence of SEQ ID NO: 3, 5, 7, or 9; (B) a protein comprising an amino acid sequence having 95% or more identity to the amino acid sequence of SEQ ID NOs: 3, 5, 7, or 9 and having dephosphorylation activity; or (C) SEQ ID NOs: 3, 5 , 7, or 9 amino acid sequences, including substitutions, deletions, insertions, additions, and amino acid sequences containing mutations of 1 to 23 amino acid residues selected from the group consisting of combinations thereof, and dephosphorylation A protein with oxidative activity.
  • the amino acid sequence of (B) or (C) maintains mutations of one or more amino acid residues selected from the group consisting of I332V, E343Q, I352T, G402S, T413E, E416D, E455Q, and I461V; The modified alkaline phosphatase of [1].
  • the amino acid sequence of (B) or (C) maintains mutation of one or more amino acid residues selected from the group consisting of I332V, E343Q, G402S, T413E, E416D, and E455Q of [2] Modified alkaline phosphatase.
  • the mutation of one or more amino acid residues is a mutation of one or more amino acid residues selected from the group consisting of I332V, E343Q, I352T, G402S, T413E, E416D, E455Q, and I461V, [ 5] modified alkaline phosphatase.
  • modified alkaline phosphatase of [5] wherein the mutation of one or more amino acid residues is mutation of one or more amino acid residues selected from the group consisting of E343, I352, G402, and E455.
  • bovine intestinal alkaline phosphatase is the following protein (A), (B), or (C): (A) a protein comprising the amino acid sequence of SEQ ID NO: 1; (B) a protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 1 and having dephosphorylation activity; or (C) a substitution or deletion in the amino acid sequence of SEQ ID NO: 1 , insertions, additions, and combinations thereof, and has dephosphorylation activity.
  • A a protein comprising the amino acid sequence of SEQ ID NO: 1
  • B a protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 1 and having dephosphorylation activity
  • C substitution or deletion in the amino acid sequence of SEQ ID NO: 1 , insertions, additions, and combinations thereof, and has dephosphorylation activity.
  • modified alkaline phosphatase of any one of [1] to [8], wherein the modified alkaline phosphatase is a protein further comprising a portion encoding another functional peptide in addition to the portion encoding the modified alkaline phosphatase.
  • a method for producing modified alkaline phosphatase comprising: Introducing mutations of one or more amino acid residues selected from the group consisting of I332, E343, I352, G402, T413, E416, E455, and I461 into bovine intestinal alkaline phosphatase, In the amino acid sequence encoding bovine small intestine alkaline phosphatase, an amino acid sequence containing mutations of one or more amino acid residues selected from the group consisting of I332, E343, I352, G402, T413, E416, E455, and I461, and producing a modified alkaline phosphatase having dephosphorylation activity.
  • [11] A substance labeled with the modified alkaline phosphatase of any one of [1] to [9] or the modified alkaline phosphatase produced by the method of [10].
  • the method for measuring alkaline phosphatase activity is the modified alkaline phosphatase of any of [1] to [9], the modified alkaline phosphatase produced by the method of [10], or any of [11] to [13]
  • the method of [14] which is a method of detecting or quantifying a substance to be measured using the substance of and a substrate.
  • Phosphorylation using the modified alkaline phosphatase of any of [1] to [9], the modified alkaline phosphatase produced by the method of [10], or the substance of any of [11] to [13] A method of producing a dephosphorylated material comprising dephosphorylating a material to produce a dephosphorylated material.
  • [17] A polynucleotide encoding the modified alkaline phosphatase of any one of [1] to [9] or the modified alkaline phosphatase produced by the method of [10].
  • an expression vector comprising the polynucleotide of [17];
  • a transformed cell comprising an expression unit of a polynucleotide encoding the modified alkaline phosphatase of any one of [1] to [9] or the modified alkaline phosphatase produced by the method of [10].
  • the modified alkaline phosphatase mutant of the present invention is excellent in both activity and thermostability.
  • the modified alkaline phosphatase variants of the invention are useful as reagents (eg, clinical test reagents, laboratory reagents).
  • FIG. 1 shows the amino acid sequence of native alkaline phosphatase bIAPII (SEQ ID NO: 1).
  • FIG. 2 shows the amino acid sequence (SEQ ID NO: 3) of modified alkaline phosphatase Mut1.
  • FIG. 3 shows the amino acid sequence (SEQ ID NO: 5) of modified alkaline phosphatase Mut2.
  • Figure 4 shows the amino acid sequence of modified alkaline phosphatase Mut3 (SEQ ID NO:7).
  • Figure 5 shows the amino acid sequence of modified alkaline phosphatase Mut4 (SEQ ID NO: 9).
  • FIG. 6 is a diagram showing the thermostability evaluation (residual activity after heating for 10 minutes) of the purified enzyme.
  • Modified alkaline phosphatase or modified alkaline phosphatase-labeled substance (1) Modified alkaline phosphatase
  • the present invention provides a modified alkaline phosphatase, which is a protein (A), (B), or (C) below: (A) a protein comprising the amino acid sequence of SEQ ID NO: 3, 5, 7, or 9; (B) a protein comprising an amino acid sequence having 95% or more identity to the amino acid sequence of SEQ ID NOs: 3, 5, 7, or 9 and having dephosphorylation activity; or (C) SEQ ID NOs: 3, 5 , 7, or 9 amino acid sequences, including substitutions, deletions, insertions, additions, and amino acid sequences containing mutations of 1 to 23 amino acid residues selected from the group consisting of combinations thereof, and dephosphorylation A protein with oxidative activity.
  • A a protein comprising the amino acid sequence of SEQ ID NO: 3, 5, 7, or 9
  • B a protein comprising an amino acid sequence having 95% or more
  • amino acid sequences of SEQ ID NOs: 3, 5, 7, and 9 are the amino acid sequences of the bIAPII variants Mut1, Mut2, Mut3, and Mut4, respectively, disclosed in the Examples.
  • the percent identity of the above amino acid sequences is 95% or more. Preferably, the identity may be 96% or greater, 97% or greater, 98% or greater or 99% or greater.
  • the above amino acid residue mutations are selected from the group consisting of amino acid residue deletions, substitutions, insertions, additions, and combinations thereof.
  • the number of mutations is a number that does not impair the properties (eg, activity, thermostability) of the modified alkaline phosphatase, and is 1 to 23.
  • the number of mutations is preferably 1 to 20, more preferably 1 to 15, still more preferably 1 to 10, particularly preferably 1 to 5 (eg, 1, 2, 3, 4 , or 5).
  • the modified alkaline phosphatase of the present invention may have one or more amino acid residue mutations as long as the desired percent identity and properties are maintained.
  • the positions of amino acid residues that may be mutated are obvious to those skilled in the art. For example, one skilled in the art can 1) compare the amino acid sequences of multiple proteins with similar properties (e.g., SEQ ID NOS: 3, 5, 7, and 9); 2) regions that are relatively conserved; 3) identify regions that are not significantly conserved, and then 3) identify regions that may play an important role in function and regions that are important for function from relatively conserved and relatively non-conserved regions, respectively. Since it can predict regions that cannot play a role, it is possible to recognize the correlation between structure and function.
  • amino acid residues that may be mutated in the amino acid sequence of the modified alkaline phosphatase of the present invention.
  • the amino acid residue after mutation at such position is the desired natural ⁇ -amino acid residue that is different from the amino acid residue before mutation.
  • Such desirable natural ⁇ -amino acid residues are L-alanine (A), L-asparagine (N), L-cysteine (C), L-glutamine (Q), L-isoleucine (I), L- Leucine (L), L-Methionine (M), L-Phenylalanine (F), L-Proline (P), L-Serine (S), L-Threonine (T), L-Tryptophan (W), L-Tyrosine (Y), L-valine (V), L-aspartic acid (D), L-glutamic acid (E), L-arginine (R), L-histidine (H), L-lysine (K), or glycine ( G).
  • amino acid residue mutations may be conservative substitutions.
  • conservative substitution refers to the replacement of a given amino acid residue with an amino acid residue having a similar side chain.
  • Families of amino acid residues with similar side chains are well known in the art. For example, such families include amino acids with basic side chains (e.g. lysine, arginine, histidine), amino acids with acidic side chains (e.g. aspartic acid, glutamic acid), amino acids with uncharged polar side chains. (e.g. asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (e.g.
  • amino acids with aromatic side chains e.g. tyrosine, phenylalanine, tryptophan, histidine
  • amino acids with hydroxyl group e.g. alcoholic, phenolic
  • amino acids with sulfur-containing side chains e.g. cysteine, methionine
  • Amino acids with uncharged polar side chains and amino acids with non-polar side chains are sometimes collectively referred to as neutral amino acids.
  • conservative amino acid substitutions are between aspartic acid and glutamic acid, between arginine and lysine and histidine, between tryptophan and phenylalanine, between phenylalanine and valine. , between leucine, isoleucine and alanine, and between glycine and alanine.
  • the dephosphorylation activity can be determined by measuring the conversion activity from p-nitrophenol phosphate to p-nitrophenol. For example, the modified alkaline phosphatase containing the amino acid sequences of (B) and (C) is compared with the modified alkaline phosphatase containing the amino acid sequence of the corresponding SEQ ID NO in (A) above when the activity is measured under the same conditions. As a standard, it is preferred to have an activity of 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 95% or more.
  • the modified alkaline phosphatase of the present invention can also have excellent thermostability.
  • the modified alkaline phosphatase containing the amino acid sequences of (B) and (C) contains the amino acid sequence of the corresponding SEQ ID NO in (A) above when subjected to heat treatment and subsequent activity measurement under the same conditions.
  • Has a residual activity of 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 95% or more based on the modified alkaline phosphatase is preferred.
  • Such conditions include heat treatment of a cell-free extract of alkaline phosphatase at 70° C.
  • the modified alkaline phosphatase of the present invention having excellent thermostability has excellent liquid stability and/or long-term storage stability. It can be said that Accordingly, the modified alkaline phosphatase of the present invention is useful as a reagent.
  • the amino acid sequence of (B) or (C) has mutations of one or more amino acid residues selected from the group consisting of I332V, E343Q, I352T, G402S, T413E, E416D, E455Q, and I461V. may be maintained.
  • the number of amino acid residue mutations to be maintained may be 1, or 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or 8. good too.
  • the amino acid sequence (B) or (C) is, among the above mutations, one or more amino acid residue mutations selected from the group consisting of I332V, E343Q, G402S, T413E, E416D, and E455Q may be maintained.
  • the number of amino acid residue mutations maintained may be one, or may be two or more, three or more, four or more, five or more, or six.
  • the amino acid sequence (B) or (C) has one or more amino acid residue mutations selected from the group consisting of I332V, E343Q, G402S, T413E, and E416D among the above mutations. may be maintained.
  • the number of amino acid residue mutations maintained may be one, or two or more, three or more, four or more, or five.
  • the amino acid sequence of (B) or (C) maintains mutations of one or more amino acid residues selected from the group consisting of I332V, G402S, T413E, and E416D among the above mutations. You may have The number of amino acid residue mutations maintained may be one, or two or more, three or more, or four.
  • the amino acid sequence of (B) or (C) maintains one or more amino acid residue mutations selected from the group consisting of E343Q, I352T, G402S, and E455Q among the above mutations.
  • the number of amino acid residue mutations maintained may be one, or two or more, three or more, or four.
  • the present invention also includes mutations of one or more amino acid residues selected from the group consisting of I332, E343, I352, G402, T413, E416, E455, and I461 in the amino acid sequence encoding bovine intestinal alkaline phosphatase.
  • a modified alkaline phosphatase comprising an amino acid sequence and having dephosphorylation activity is provided.
  • the amino acid residue after mutation at these sites is the desired natural ⁇ -amino acid residue that is different from the amino acid residue before mutation.
  • Such desirable natural ⁇ -amino acid residues are glycine, L-alanine (A), L-asparagine (N), L-cysteine (C), L-glutamine (Q), L-isoleucine (I), L-leucine (L), L-methionine (M), L-phenylalanine (F), L-proline (P), L-serine (S), L-threonine (T), L-tryptophan (W), L - from tyrosine (Y), L-valine (V), L-aspartic acid (D), L-glutamic acid (E), L-arginine (R), L-histidine (H), and L-lysine (K)
  • the mutation may be a mutation of one or more amino acid residues selected from the group consisting of I332V, E343Q, I352T, G402S, T413E, E416D, E455
  • the mutation may be mutation of one or more amino acid residues selected from the group consisting of I332, E343, G402, T413, E416, and E455.
  • the mutation may be mutation of one or more amino acid residues selected from the group consisting of I332V, E343Q, G402S, T413E, E416D, and E455Q.
  • the number of amino acid residues to be mutated may be one, or two or more, three or more, four or more, five or more, or six.
  • the mutation may be a mutation of one or more amino acid residues selected from the group consisting of I332, E343, G402, T413, and E416.
  • the mutation may be mutation of one or more amino acid residues selected from the group consisting of I332V, E343Q, G402S, T413E, and E416D.
  • the number of amino acid residues to be mutated may be one, or two or more, three or more, four or more, or five.
  • the mutation may be a mutation of one or more amino acid residues selected from the group consisting of I332, G402, T413, and E416.
  • the mutation may be mutation of one or more amino acid residues selected from the group consisting of I332V, G402S, T413E, and E416D.
  • the number of amino acid residues to be mutated may be one, or two or more, three or more, or four.
  • the mutation may be mutation of one or more amino acid residues selected from the group consisting of E343, I352, G402, and E455.
  • the mutation may be mutation of one or more amino acid residues selected from the group consisting of I332V, G402S, T413E, and E416D.
  • the number of amino acid residues to be mutated may be one, or two or more, three or more, or four.
  • the calf intestinal alkaline phosphatase may be the following protein (A), (B), or (C): (A) a protein comprising the amino acid sequence of SEQ ID NO: 1; (B) a protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 1 and having dephosphorylation activity; or (C) a substitution or deletion in the amino acid sequence of SEQ ID NO: 1 , insertions, additions, and combinations thereof, and has dephosphorylation activity.
  • the identity of the above amino acid sequences is 90% or more.
  • the identity may be 91% or greater, 92% or greater, 93% or greater, 94% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater. Calculation of such percent identity can be performed in the same manner as described above.
  • the above amino acid residue mutations are selected from the group consisting of amino acid residue deletions, substitutions, insertions, additions, and combinations thereof.
  • the number of mutations is a number that does not impair the properties (eg, activity, thermostability) of the modified alkaline phosphatase, and is 1 to 47.
  • the number of mutations is preferably 1 to 45, more preferably 1 to 40, still more preferably 1 to 30, particularly preferably 1 to 20, 1 to 15, 1 to 10, or 1 to 5 (eg, 1, 2, 3, 4, or 5).
  • the modified alkaline phosphatase of the present invention may have one or more amino acid residue mutations as long as the desired percent identity and properties are maintained.
  • the positions of amino acid residues that may be mutated are obvious to those skilled in the art.
  • a person skilled in the art can 1) compare the amino acid sequences of a plurality of proteins having similar properties [for example, the amino acid sequence of SEQ ID NO: 1 (bovine intestinal alkaline phosphatase) and another bovine intestinal alkaline phosphatase (e.g., bovine comparison with the amino acid sequences encoding small intestine-derived alkaline phosphatase I, III, IV, V, VI, and/or VII) or comparison of the amino acid sequences of SEQ ID NOs: 1, 3, 5, 7, and 9], 2) Identify relatively conserved and relatively non-conserved regions, and then 3) from the relatively conserved and relatively non-conserved regions, respectively, Since it can predict regions that can play important roles and regions that cannot play important roles in function
  • amino acid residues that may be mutated in the amino acid sequence of the modified alkaline phosphatase of the present invention.
  • the amino acid residue after mutation at such position is the desired natural ⁇ -amino acid residue that is different from the amino acid residue before mutation.
  • desired natural ⁇ -amino acid residues are the same as those described above.
  • Amino acid residue mutations may be conservative substitutions as described above.
  • the dephosphorylation activity and thermal stability are also the same as those described above.
  • the modified alkaline phosphatase of the present invention may be a fusion protein further comprising a portion encoding another functional peptide in addition to the portion encoding the modified alkaline phosphatase.
  • the modified alkaline phosphatase of the present invention may have a signal sequence at its N-terminus to promote extracellular secretion from transformed cells. Examples of such signal sequences include MglB signal sequence, PelB signal sequence, OmpA signal sequence, PhoA signal sequence, OmpF signal sequence, PhoE signal sequence, MalE signal sequence, OmpC signal sequence, Lpp signal sequence and LamB signal sequence. mentioned.
  • the modified alkaline phosphatase of the invention may also have other peptide moieties (eg, tag moieties) at the C-terminus or N-terminus, or both (preferably the C-terminus).
  • Other peptide components that can be added to the modified alkaline phosphatase of the present invention include, for example, peptide components that facilitate purification of the target protein (e.g., histidine tag, tag portion such as Strep-tag II; glutathione-S-transferase, proteins commonly used for purification of target proteins such as maltose binding protein), peptide components that improve the solubility of target proteins (e.g., Nus-tag), peptide components that act as chaperones (e.g., trigger factors), and other functions Proteins or domains of proteins or peptide components as linkers connecting them are included.
  • target protein e.g., histidine tag, tag portion such as Strep-tag II; glutathione-S-transferas
  • the fusion protein of the present invention may be a fusion protein that includes a portion encoding an affinity peptide or an antigenic peptide as a portion encoding another functional peptide.
  • Target molecules for such affinity peptides include oligopeptides or polypeptides (i.e. proteins), oligosaccharides or polysaccharides, nucleic acids or (e.g. DNA, RNA or artificial nucleic acids), lipids, low molecular weight compounds (e.g. amino acids, monosaccharides, nucleotides, nucleosides, other biological components present in body fluids such as blood, and pharmaceuticals).
  • affinity peptides include antibodies or fragments thereof (eg, single-chain antibodies) and antibody mimetics as described below.
  • Antigenic peptides include, for example, viral antigenic peptides, allergenic peptides, tumor-specific antigenic peptides, tumor-associated antigenic peptides, bacterial antigenic peptides, fungal antigenic peptides, parasite antigenic peptides.
  • the present invention also provides a method for producing modified alkaline phosphatase, comprising: Introducing mutations of one or more amino acid residues selected from the group consisting of I332, E343, I352, G402, T413, E416, E455, and I461 into bovine intestinal alkaline phosphatase, In the amino acid sequence encoding bovine small intestine alkaline phosphatase, an amino acid sequence containing mutations of one or more amino acid residues selected from the group consisting of I332, E343, I352, G402, T413, E416, E455, and I461, and producing a modified alkaline phosphatase having dephosphorylation activity.
  • the bovine small intestine-derived alkaline phosphatase may be a native alkaline phosphatase or a mutant alkaline phosphatase.
  • the bovine intestinal alkaline phosphatase utilized in the present invention is an alkaline phosphatase that does not have the desired mutation at one or more sites as described above.
  • the bovine intestinal alkaline phosphatase used in the present invention has two or more (e.g., 2, 3, 4, 5, 6, 7, or 8) sites as described above. Alkaline phosphatase that does not have the mutation of interest in
  • Mutations of amino acid residues introduced into these sites are mutations of desired natural ⁇ -amino acid residues that are different from the amino acid residues before mutation. Such desired natural ⁇ -amino acid residues are the same as those described above.
  • the introduced mutation may be a mutation of one or more amino acid residues selected from the group consisting of I332V, E343Q, I352T, G402S, T413E, E416D, E455Q, and I461V.
  • the number of mutations to be introduced may be 1, or may be 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or 8. . Mutations can be introduced by any method (eg, site-directed mutagenesis) capable of introducing mutations at desired sites.
  • the introduced mutation may be a mutation of one or more amino acid residues selected from the group consisting of I332, E343, G402, T413, E416, and E455.
  • the introduced mutation may be a mutation of one or more amino acid residues selected from the group consisting of I332V, E343Q, G402S, T413E, E416D, and E455Q.
  • the number of mutations introduced may be one, or two or more, three or more, four or more, five or more, or six.
  • the introduced mutation may be a mutation of one or more amino acid residues selected from the group consisting of I332, E343, G402, T413, and E416.
  • the introduced mutation may be a mutation of one or more amino acid residues selected from the group consisting of I332V, E343Q, G402S, T413E, and E416D.
  • the number of mutations introduced may be one, or two or more, three or more, four or more, or five.
  • the introduced mutation may be a mutation of one or more amino acid residues selected from the group consisting of I332, G402, T413, and E416.
  • the introduced mutation may be a mutation of one or more amino acid residues selected from the group consisting of I332V, G402S, T413E, and E416D.
  • the number of mutations to be introduced may be one, or two or more, three or more, or four.
  • the introduced mutation may be a mutation of one or more amino acid residues selected from the group consisting of E343, I352, G402, and E455.
  • the introduced mutation may be a mutation of one or more amino acid residues selected from the group consisting of I332V, G402S, T413E, and E416D.
  • the number of mutations to be introduced may be one, or two or more, three or more, or four.
  • the calf intestinal alkaline phosphatase may be the following protein (A), (B), or (C): (A) a protein comprising the amino acid sequence of SEQ ID NO: 1; (B) a protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 1 and having dephosphorylation activity; or (C) a substitution or deletion in the amino acid sequence of SEQ ID NO: 1 , insertions, additions, and combinations thereof, and has dephosphorylation activity.
  • the degree of percent identity to the amino acid sequence of SEQ ID NO: 1 and the number of amino acid residue mutations in the amino acid sequence of SEQ ID NO: 1 are the same as described above.
  • the present invention also provides a modified alkaline phosphatase obtained by the method for producing a modified alkaline phosphatase as described above.
  • Substance labeled with modified alkaline phosphatase The present invention also provides a substance labeled with modified alkaline phosphatase.
  • a substance to be labeled with modified alkaline phosphatase is an affinity substance or an antigen for any target molecule.
  • Target molecules for such affinity substances include oligopeptides or polypeptides (i.e., proteins), oligosaccharides or polysaccharides, nucleic acids or (e.g., DNA, RNA, or artificial nucleic acids), lipids, low-molecular-weight compounds (e.g., amino acids, monosaccharides, nucleotides, nucleosides, other biological components present in body fluids such as blood, and pharmaceuticals).
  • affinity substances include antibodies, aptamers, lectins, complementary strands of nucleic acids, and antibody mimetics.
  • alkaline phosphatase-labeled affinity substances have been reported (eg, Berg et al., Labeling Antibodies, http://cshprotocols.cshlp.org/content/2020/7/pdb.top099242 .full, Suzuki et al., Open sandwich ELISA with VH-/VL-alkaline phosphorase fusion proteins, https://doi.org/10.1016/S0022-1759(99)00020-4).
  • Antigens include, for example, virus antigens, allergens, carbohydrate antigens, tumor-specific antigens, tumor-associated antigens, bacterial antigens, fungal antigens, and parasite antigens.
  • Various antigens labeled with alkaline phosphatase have been reported (see, for example, Japanese Patent No. 2525195).
  • the affinity agent may be an antibody.
  • Antibodies may be polyclonal antibodies or monoclonal antibodies.
  • Antibody isotypes include, for example, IgG (eg, IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, IgE, and IgY.
  • Antibodies can be full-length antibodies or antibody fragments (eg, F(ab') 2 , Fab', Fab, Fv, single chain antibodies).
  • the labeling of substances with the modified alkaline phosphatase of the present invention can be carried out in the same manner as the labeling of substances with alkaline phosphatase. Since labeling of substances with alkaline phosphatase is commonly used, those skilled in the art can appropriately label substances with the modified alkaline phosphatase of the present invention. Labeling of substances with alkaline phosphatase includes, for example, chemical labeling and enzymatic labeling.
  • Examples of chemical labeling of substances with alkaline phosphatase include methods using glutaraldehyde as a cross-linking agent (e.g., Winston et al., Conjugation of Enzymes to Antibodies, https://currentprotocols.onlinelibrary.wiley.com/doi/full /10.1002/0471142727.mb1101s50) ⁇ ( ⁇ Watabe et al.,Ultrasensitive enzyme-linked immunosorbent assay (ELISA) of proteins by combination with the thio-NAD cycling method,Biophysics(Nagoya -shi), 2014, 10, 49-54; Japanese Patent No. 5265816) can be used.
  • glutaraldehyde as a cross-linking agent
  • Enzymatic labeling of substances with alkaline phosphatase includes, for example, a method using sortase A as a binding enzyme (e.g., Sakamoto et al., Enzyme-Mediated Site-Specific Antibody-Protein Modification Using a ZZ Domain as a Linker, https:/ /pubs.acs.org/doi/10.1021/bc100206z), a method using lipoic acid ligase as a binding enzyme (e.g., Cohen et al., Site-specific protein modification using lipoic acid ligase and bis-aryl hydrzone, for https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4758125/).
  • sortase A e.g., Sakamoto et al., Enzyme-Mediated Site-Specific Antibody-Protein Modification Using a ZZ Domain as a Linker, https:/ /pubs.a
  • a substance labeled with the modified alkaline phosphatase of the present invention is useful for detection or quantification of a substance to be measured.
  • immunological methods for detecting or quantifying target molecules using affinity substances as substances are widely used.
  • the affinity substance is an antibody and the target molecule (antigen) of the antibody is the substance to be measured
  • the antibody labeled with the modified alkaline phosphatase of the present invention is a detection antibody (e.g., , a labeled antibody in a sandwich assay using at least two kinds of antibodies including a solid-phase antibody against a substance to be measured and a labeled antibody against the substance to be measured, and a labeled antibody in a non-sandwich assay).
  • the affinity substance is an antibody and the target molecule of the antibody is another antibody against the substance to be measured
  • the antibody labeled with the modified alkaline phosphatase of the present invention binds to the other antibody against the substance to be measured.
  • Immunological methods for detecting or quantifying target molecules are also commonly used when the substance is an antibody.
  • the modified alkaline phosphatase of the invention is produced using a transformed cell comprising an expression unit comprising a polynucleotide encoding the modified alkaline phosphatase of the invention and a promoter operably linked thereto. or using a cell-free system or the like.
  • the present invention also provides such polynucleotides and transformed cells, as well as expression vectors that can be used to produce transformants.
  • the polynucleotide of the present invention is a polynucleotide encoding the modified alkaline phosphatase of the present invention.
  • Polynucleotides of the invention may be DNA or RNA, although DNA is preferred.
  • the transformed cells of the present invention can be produced, for example, by methods using expression vectors containing the polynucleotides of the present invention (eg, competent cell method, electroporation method), or genome modification techniques.
  • the expression vector is an integrative vector that undergoes homologous recombination with the host cell's genomic DNA
  • the expression unit can be integrated into the host cell's genomic DNA upon transformation.
  • the expression vector is a non-integrating vector that does not undergo homologous recombination with the genomic DNA of the host cell
  • the expression unit is not integrated into the genomic DNA of the host cell by transformation, and the expression vector is incorporated into the host cell. It can exist independently of genomic DNA while remaining intact.
  • the expression unit is incorporated into the host cell's genomic DNA, and the expression unit inherent in the host cell is modified. It is possible to
  • the present invention also provides an expression vector containing the polynucleotide of the present invention.
  • the expression vector of the present invention may further contain elements such as terminators, ribosome binding sites, and drug resistance genes that function in host cells.
  • Drug resistance genes include, for example, resistance genes to drugs such as tetracycline, ampicillin, kanamycin, hygromycin, and phosphinothricin.
  • the expression vector may also contain a region that allows homologous recombination with the genome of the host cell for homologous recombination with the genome DNA of the host cell.
  • an expression vector may be designed so that the expression unit it contains is located between a pair of homologous regions (e.g., homology arms, loxP, FRT, homologous to a particular sequence in the genome of the host cell).
  • the genomic region of the host cell into which the expression unit is to be introduced is not particularly limited, but may be a locus of a gene highly expressed in the host cell.
  • the expression vector may be a plasmid, viral vector, phage, or artificial chromosome.
  • Expression vectors may also be integrative or non-integrative vectors.
  • An integrating vector may be a type of vector that integrates in its entirety into the genome of the host cell.
  • an integrating vector may be a type of vector whose only part (eg, an expression unit) is integrated into the genome of the host cell.
  • Expression vectors can also be DNA vectors or RNA vectors (eg, retroviruses).
  • the expression vector may also be a commonly used expression vector.
  • Such expression vectors include, for example, pUC (e.g., pUC19, pUC18), pSTV, pBR (e.g., pBR322), pHSG (e.g., pHSG299, pHSG298, pHSG399, pHSG398), RSF (e.g., RSF1010), pACYC (e.g., pACYC177, pACYC184), pMW (eg pMW119, pMW118, pMW219, pMW218), pQE (eg pQE30), and derivatives thereof.
  • pUC e.g., pUC19, pUC18
  • pSTV e.g., pBR322
  • pHSG e.g., pHSG299, pHSG298, pHSG399, pHSG398
  • RSF e.g., RSF101010
  • pACYC e.g., p
  • Examples of hosts for expressing the modified alkaline phosphatase of the present invention include bacteria belonging to the genus Escherichia such as Escherichia coli, bacteria belonging to the genus Corynebacterium (e.g., Corynebacterium glutamicum), and bacilli.
  • prokaryotic cells including bacteria of the genus [e.g., Bacillus subtilis], bacteria of the genus Saccharomyces [e.g., Saccharomyces cerevisiae], bacteria of the genus Pichia [e.g., Pichia stipitis ], bacteria of the genus Aspergillus [eg, Aspergillus oryzae], and various other eukaryotic cells can be used.
  • insect cells, plant cells, animal cells (eg, mammalian cells such as Chinese hamster ovary (CHO) cells) can be used as hosts.
  • a strain lacking a given gene may be used as a host.
  • Transformed cells include, for example, transformed cells harboring an expression vector in the cytoplasm, and transformed cells into which a gene of interest has been introduced onto the genome.
  • the transformed cells of the present invention can be cultured, for example, in a medium having the composition described below, using a predetermined culture device (eg, test tube, flask, jar fermenter).
  • Culture conditions can be appropriately set. Specifically, the culture temperature may be 10° C. to 37° C., the pH may be 6.5 to 7.5, and the culture time may be 1 hour to 100 hours.
  • the culture may be performed while controlling the dissolved oxygen concentration.
  • the dissolved oxygen concentration (DO value) in the culture medium may be used as an index for control.
  • Aeration and agitation conditions should be controlled so that the relative dissolved oxygen concentration DO value when the atmospheric oxygen concentration is 21% does not fall below, for example, 1 to 10%, preferably 3% to 8%. can be done.
  • the culture may be batch culture or fed-batch culture. In the case of fed-batch culture, the culture can be continued by continuously or discontinuously adding a sugar source solution or a phosphoric acid-containing solution to the culture medium.
  • the host to be transformed is as described above, but when it comes to Escherichia coli in detail, it can be selected from Escherichia coli JM109 strain, DH5 ⁇ strain, HB101 strain, BL21 (DE3) strain, etc. of Escherichia coli K12 strain subspecies. Methods for performing transformation and methods for selecting transformed cells are also described in Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Press (2001/01/15), and the like. A method for producing a transformed E. coli and using it to produce a desired enzyme will be described in more detail below as an example.
  • E. Promoters used for heterologous protein production in E. coli can be used, such as PhoA, PhoC, T7 promoter, lac promoter, trp promoter, trc promoter, tac promoter, PR promoter of lambda phage, PL promoter, T5 promoter, etc. Strong promoters are included, with PhoA, PhoC and lac being preferred.
  • vectors examples include pUC (e.g., pUC19, pUC18), pSTV, pBR (e.g., pBR322), pHSG (e.g., pHSG299, pHSG298, pHSG399, pHSG398), RSF (e.g., RSF1010), pACYC (e.g., pACYC177, pACYC184), pMW (eg, pMW119, pMW118, pMW219, pMW218), pQE (eg, pQE30), derivatives thereof, and the like may be used.
  • Phage DNA vectors may be used as other vectors.
  • expression vectors containing promoters and capable of expressing the inserted DNA sequences may be used.
  • the vector may be pUC, pSTV, pMW.
  • a terminator which is a transcription termination sequence, may also be ligated downstream of the polynucleotide of the present invention.
  • terminators include T7 terminator, fd phage terminator, T4 terminator, tetracycline resistance gene terminator, and E. coli trpA gene terminator.
  • a so-called multicopy type is preferable, and examples thereof include plasmids having a replication origin derived from ColE1, such as pUC-based plasmids, pBR322-based plasmids, and derivatives thereof. be done.
  • the term "derivative" means a plasmid modified by base substitution, deletion, insertion and/or addition.
  • the vector preferably has a marker such as an ampicillin resistance gene in order to select transformed cells.
  • Expression vectors with strong promoters are commercially available as such plasmids [eg, pUC system (manufactured by Takara Bio), pPROK system (manufactured by Clontech), pKK233-2 (manufactured by Clontech)].
  • the modified alkaline phosphatase of the present invention can be obtained by transforming E. coli with the obtained expression vector of the present invention and culturing this E. coli.
  • the medium a medium commonly used for culturing E. coli, such as M9-casamino acid medium and LB medium, may be used.
  • the medium may contain a given carbon source, nitrogen source and coenzyme (eg, pyridoxine hydrochloride).
  • coenzyme eg, pyridoxine hydrochloride
  • peptone, yeast extract, NaCl, glucose, MgSO4 , ammonium sulfate, potassium dihydrogen phosphate, ferric sulfate, manganese sulfate, and the like may be used.
  • culture conditions and production induction conditions are appropriately selected according to the types of vector markers, promoters, host bacteria, and the like used.
  • the modified alkaline phosphatase of the present invention can be obtained by disrupting (e.g., sonication, homogenization) or lysing (e.g., lysozyme treatment) the cells to produce a homogenate and a lysate.
  • disrupting e.g., sonication, homogenization
  • lysing e.g., lysozyme treatment
  • alkaline phosphatase is secreted or leaked out of the cells
  • a sterilized solution containing alkaline phosphatase can be obtained from the culture solution by centrifugation or membrane filtration.
  • the modified alkaline phosphatase of the present invention can be obtained by subjecting such crushed product, lysate and sterilized solution to techniques such as extraction, precipitation, filtration and column chromatography.
  • Method using modified alkaline phosphatase or modified alkaline phosphatase-labeled substance (1) Measurement of alkaline phosphatase activity
  • a method for measuring alkaline phosphatase activity comprising measuring alkaline phosphatase activity.
  • any substrate that can be used to measure alkaline phosphatase activity can be used as the substrate.
  • substrates include phosphorylated substances [e.g., p-nitrophenol phosphate, 5-bromo-4-chloro-3-indolyl phosphate, 2-chloro-5-(4-methoxyspiro[1 ,2-dioxetane-3,2′-(5-chlorotricyclo[3.3.1.1 3.7 ]decane])-4-yl]-1-phenyl phosphate, naphthol phosphate AS-TR, Phenolphthalein phosphate, indoxyl phosphate, naphthol AS-BI phosphate, naphthol AS-E phosphate, naphthol AS-MX phosphate, bis(p-nitrophenyl) phosphate, phenolphthalein bisphosphate, 5- A luminescent substrate comprising bromo-4-chloro-3-indolyl phosphate, 1-naphth
  • alkaline phosphatase activity e.g., pH 8-11
  • appropriate temperature e.g., 4-40 ° C.
  • index value of dephosphorylated substance e.g., absorbance
  • biological assays such as reporter assays.
  • the method for measuring alkaline phosphatase activity may be a method of detecting or quantifying a target molecule using a substance labeled with the modified alkaline phosphatase of the present invention.
  • the substance labeled with modified alkaline phosphatase and its target molecule are the same as those described above.
  • a substance labeled with the modified alkaline phosphatase of the present invention can be used as a detection antibody that can bind to the substance to be measured, or a secondary antibody that can bind to another antibody to the substance to be measured, as described above. Therefore, measurement of alkaline phosphatase activity enables detection or quantification of the substance to be measured.
  • the modified alkaline phosphatase of the present invention or a modified alkaline phosphatase-labeled substance is used to dephosphorylate a phosphorylated substance to produce a dephosphorylated substance.
  • a method for producing a dephosphorylated substance comprising:
  • the phosphorylated substances are dephosphorylated by reacting the phosphorylated substances with modified alkaline phosphatase or modified alkaline phosphatase-labeled substances under conditions for measuring alkaline phosphatase activity as described above. Phosphorylated substances can be produced.
  • dephosphorylation of a phosphorylated substance may be dephosphorylation of the 5' or 3' terminal phosphate of a nucleic acid.
  • Nucleic acids may be single-stranded or double-stranded (with overhanging or blunt ends). According to such a method, undesired ligation of nucleic acids (eg, vector self-ligation) can be prevented.
  • Example 1 Design of Alkaline Phosphatase and Production of Expression Plasmid Plasmids (expression vectors) used in this study were produced by the methods described below.
  • sequences having these motif-like sequences were selected and further analyzed with ClustalW to remove sequences with low homology and sequences containing gaps, finally obtaining 10 sequences.
  • 10 sequences were used to design artificial alkaline phosphatase sequences designated Mut1, Mut2, Mut3, and Mut4 as shown in the amino acid sequences of SEQ ID NOs: 3, 5, 7, 9 (FIGS. 2-5).
  • (A) bIAPII expression plasmid The amino acid sequence (SEQ ID NO: 1) obtained by removing the N-terminal secretory signal sequence and the C-terminal GPI anchor region from bovine intestinal alkaline phosphatase (bIAPII) was expressed in E. coli. After optimizing the codons of E. coli and converting it into a base sequence, a plasmid containing this base sequence (SEQ ID NO: 2) was synthesized by Eurofins Genomics.
  • the bIAPII-encoding region was amplified by PCR using the primers of SEQ ID NO: 11 and SEQ ID NO: 12, and the resulting fragment was cloned with NcoI and XhoI using the In-fusion HD cloning kit (Takara Bio). Cloned into digested pET-28a(+) vector.
  • (C) The nucleotide sequence (SEQ ID NO: 4) encoding the artificially designed ALP expression plasmid Mut1 (SEQ ID NO: 3) to which the MglB signal sequence was added was inserted into the NcoI/XhoI site of the pET-28a (+) vector. Synthesized by Genomics. Using this plasmid as a template and the primers of SEQ ID NO: 18 and SEQ ID NO: 16, linear DNA was amplified by PCR and circularized with an In-fusion HD cloning kit. In this plasmid, Mut1 with an MglB signal sequence added to the N-terminus and a His tag added to the C-terminus is expressed.
  • Nucleotide sequences encoding each are SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10.
  • Example 2 Preparation of cell-free extract
  • the plasmids constructed in (B) and (C) of Example 1 were transferred to E. coli.
  • E. coli BL21(DE3) was transformed to obtain various ALP-expressing strains. Each expression strain was grown overnight at 37° C. on LB agar medium containing 50 mg/L of kanamycin.
  • the obtained cells were inoculated into 100 mL of 4 ⁇ TB medium containing 50 mg/L kanamycin, 5 mM magnesium sulfate and 10 ⁇ M zinc chloride, and cultured with shaking at 37° C. using a Sakaguchi flask.
  • IPTG was added to a final concentration of 0.1 mM to induce expression, and cultured at 25° C. for 18 hours.
  • bacterial cells were collected from 1 mL of the resulting culture solution by centrifugation. Next, it was suspended in 100 ⁇ L of xTractor buffer (Takara Bio), magnesium sulfate was added to a final concentration of 1 mM and zinc chloride was added to a final concentration of 100 ⁇ M, and the cells were lysed by incubating at room temperature for 20 minutes. Cell residues were removed from the lysate by centrifugation, and the resulting supernatant was used as a cell-free extract.
  • xTractor buffer Tala Bio
  • Example 3 Activity Measurement of Cell-Free Extract The ALP activity of the cell-free extract was measured by conversion activity from p-nitrophenol phosphate to p-nitrophenol.
  • serial dilutions of the cell-free extract were prepared with a dilution buffer (100 mM Tris-HCl, 150 mM sodium chloride, 1 mM magnesium sulfate, 0.05 mM zinc chloride, 0.1% bovine serum albumin, pH 7.0).
  • the amount of enzyme that produces 1 ⁇ mol of p-nitrophenol per minute is defined as 1 U, and the activity of the cell-free extract was measured by the following formula.
  • Activity of cell-free extract (U / L) ( ⁇ A test - ⁇ A blank ) ⁇ 1000 ⁇ 200 / (18.75 ⁇ 0.5 ⁇ 4) 18.75: Molar extinction coefficient of p-nitrophenol (mM -1 cm -1 ) 0.5: optical path length (cm)
  • Table 3 shows the ALP activity of the obtained cell-free extract.
  • the cell-free extract of the artificially designed ALP-expressing strain showed higher ALP activity than that of the bIAPII-expressing strain (Table 3).
  • Example 4 Evaluation of Thermal Stability Based on the values measured in Example 3, each cell-free extract prepared in Example 2 was diluted with the dilution buffer of Example 3 so that the activity was 300 U/L. Diluted. Next, 20 ⁇ L of the enzyme dilution solution was dispensed into microtubes, heat-treated at 70° C. for 10 minutes using TaKaRa PCR Thermal Cycler Dice (Takara Bio), transferred to ice and cooled. In the same manner as in Example 3, ALP activity was measured three times for each of the unheated enzyme dilution and the heat treated enzyme dilution.
  • Residual activity (%) ALP activity of diluted enzyme solution after heat treatment/ALP activity of untreated diluted enzyme solution ⁇ 100
  • a vector fragment was prepared by PCR using the primers of SEQ ID NO: 15 and SEQ ID NO: 16, using the pET-28a(+) vector as a template.
  • the prepared insert and vector fragment were ligated using the In-fusion HD cloning kit.
  • the resulting plasmid expresses the 385G mutant of bIAPII with an MglB signal sequence at the N-terminus and a His tag at the C-terminus.
  • a vector fragment was prepared by PCR using the primers of SEQ ID NO: 27 and SEQ ID NO: 16, using the pET-28a(+) vector as a template.
  • the prepared insert and vector fragment were ligated using the In-fusion HD cloning kit.
  • the resulting plasmid expresses the 322D/385N/430A mutant of bIAPII with an N-terminal MglB signal sequence and a C-terminal His tag.
  • a cell-free extract was prepared by the method of Example 2 using the constructed plasmid, and ALP activity was measured by the method of Example 3. All activities of the cell-free extracts were lower than those of the artificially designed ALP.
  • Example 5 Comparison of expression levels
  • A Construction of wild-type and artificially designed ALP-expressing bacteria Using the plasmids prepared by gene synthesis in (A) and (C) of Example 1 as templates, the regions encoding each ALP were Amplified by PCR. Using the pET-21b(+) vector as a template, a linear vector was amplified by PCR. Table 6 shows the combinations of primer sequences used. Both obtained fragments were ligated and circularized using In-fusion HD cloning kit (Takara Bio) to obtain an expression plasmid in which each ALP was inserted into the NdeI and XhoI sites of the pET-21b(+) vector. This plasmid was transferred to E. E. coli Tuner (DE3) strain, and E. Transformed into E. coli Origami B (DE3).
  • (B) Preparation of cell-free extract and activity evaluation
  • the strain constructed in (A) was grown overnight at 37°C in LB liquid medium supplemented with 100 mg/L ampicillin.
  • the resulting cells were added to 3 mL of main culture medium (composition: 20 g / L Bacto tryptone, 15 g / L yeast extract, 1 g / L sodium chloride, 50 mM potassium phosphate buffer (pH 7.15) to which 100 mg / L of ampicillin was added. , 5 mM magnesium sulfate and 10 ⁇ M zinc chloride) was inoculated to 30 ⁇ L, and cultured with shaking at 37° C. using a test tube. When the OD660 reached 0.5 to 0.7, IPTG was added to a final concentration of 1 mM to induce expression, and cultured at 18°C.
  • main culture medium composition: 20 g / L Bacto tryptone, 15 g / L yeast extract, 1 g / L sodium chloride, 50 mM
  • ammonium sulfate i.e., ammonium sulfate
  • the enzyme solution was appropriately diluted, and the ALP activity was measured according to Example 3, and converted to the activity value per liter of the culture solution.
  • Example 6 Evaluation of specific activity and thermostability of purified enzyme
  • A Construction of wild-type and artificially designed ALP-expressing bacteria
  • the plasmid prepared by gene synthesis in (C) of Example 1 has a His tag at the C-terminus of ALP. was designed to be added and expressed. A stop codon was inserted between the ALP sequence and the His tag to construct a plasmid expressing ALP without a His tag.
  • the plasmid prepared by gene synthesis in Example 1 (C) was used as a template and amplified by PCR using the primers shown in Table 8.
  • the resulting linear double-stranded DNA was circularized using the In-fusion HD cloning kit (Takara Bio) to obtain an expression plasmid in which each ALP was inserted into the NcoI/XhoI site of the pET-28a(+) vector. .
  • This plasmid and the bIAPII expression plasmid constructed in (A) of Example 1 were transferred to E. coli. Transformed into E. coli Origami 2 (DE3).
  • (B) Preparation of cell-free extract The strain constructed in (A) was grown overnight at 37°C on LB agar medium supplemented with 50 mg/L of kanamycin. 100 mL of main culture medium (composition: 20 g / L Bacto tryptone, 15 g / L yeast extract, 1 g / L sodium chloride, 50 mM potassium phosphate buffer (pH 7.15) to which kanamycin 50 mg / L was added to the obtained cells , 5 mM magnesium sulfate and 10 ⁇ M zinc chloride), and cultured with shaking at 37° C. using a Sakaguchi flask.
  • main culture medium composition: 20 g / L Bacto tryptone, 15 g / L yeast extract, 1 g / L sodium chloride, 50 mM potassium phosphate buffer (pH 7.15) to which kanamycin 50 mg / L was added to the obtained cells , 5 mM magnesium sulfate and 10 ⁇ M zinc chloride
  • IPTG IPTG was added to a final concentration of 1 mM to induce expression, and cultured at 18°C. After 24 hours, the cells were collected from the culture broth by centrifugation, suspended in 20 mM Tris-HCl buffer (pH 8.0) containing 1 mM MgCl 2 and 0.1 mM ZnCl 2 , and sonicated. Cell residues were removed from the lysate by centrifugation, and the resulting supernatant was used as a cell-free extract.
  • ALP activity was measured by conversion activity from p-nitrophenol phosphate to p-nitrophenol using QuantiChrom Alkaline Phosphatase Assay Kit (BioAssay Systems). First, serial dilutions of each ALP solution were prepared with a dilution buffer (100 mM Tris-HCl, 150 mM sodium chloride, 1 mM magnesium sulfate, 0.05 mM zinc chloride, 0.1% bovine serum albumin, pH 7.0).
  • a dilution buffer 100 mM Tris-HCl, 150 mM sodium chloride, 1 mM magnesium sulfate, 0.05 mM zinc chloride, 0.1% bovine serum albumin, pH 7.0.
  • ALP activity (U / L) ( ⁇ A test - ⁇ A blank ) ⁇ 1000 ⁇ 200 / (18.75 ⁇ 0.5 ⁇ 20) 18.75: Molar extinction coefficient of p-nitrophenol (mM -1 cm -1 ) 0.5: optical path length (cm)
  • each enzyme solution was diluted appropriately and the absorbance at 280 nm was measured with an ultratrace spectrometer Nanodrop (Thermo Fisher Scientific). Taking the absorbance of ALP at 1 mg/mL as 1, the concentration of each ALP solution was calculated.
  • the specific activity of each ALP was calculated from the ALP activity and ALP concentration (Table 9).
  • thermostability Each purified ALP was diluted with a dilution buffer and then dispensed into microtubes. After heating at each temperature of 55° C. to 85° C. for 10 minutes with a thermal cycler TaKaRa PCR Thermal Cycler Dice (Takara Bio), it was ice-cooled. The ALP activity of these samples was measured three times each according to Example 3, and the activity value at the time of heating at 55° C. was defined as 100%, and the residual activity after heating at each temperature was calculated (FIG. 6). Furthermore, the temperature (T50) at which the residual activity becomes 50% was calculated (Table 9).
  • the artificially designed ALP was found to improve T50 by 13°C or more compared to bIAPII.
  • Example 7 Evaluation of Single Mutants Twenty-eight common mutation points (Table 2) in four artificially designed ALPs were introduced singly into bIAPII, and the effects of each were evaluated.
  • the amino acid sequence (SEQ ID NO: 1) obtained by removing the N-terminal secretory signal sequence and the C-terminal GPI anchor region from bovine intestinal alkaline phosphatase (bIAPII) was transferred to E. coli. It was optimized for the codons of E. coli and converted into a nucleotide sequence.
  • a plasmid was synthesized by GenScript Co., Ltd.
  • the ALP expression strain introduced with bIAPII and 28 single mutations and the Mut2 expression strain prepared in (B) of Example 6 were grown overnight at 37°C in LB liquid medium supplemented with 50 mg/L of kanamycin. let me The resulting cells were added to 3 mL of main culture medium supplemented with 50 mg/L of kanamycin (composition: 20 g/L Bactotryptone, 15 g/L yeast extract, 1 g/L sodium chloride, 50 mM potassium phosphate buffer (pH 7.15) , 5 mM magnesium sulfate and 10 ⁇ M zinc chloride) was inoculated to 30 ⁇ L, and cultured with shaking at 37° C. using a test tube. When the OD660 reached 0.5 to 0.7, IPTG was added to a final concentration of 1 mM to induce expression, and cultured at 18°C.
  • ammonium sulfate was added to these cell-free extracts to a final concentration of 1M, diluted appropriately, and the ALP activity remaining after heating at 60°C for 10 minutes was measured by the method of Example 4.
  • Example 8 Comparison of expression level and thermostability using cell-free extract According to the method, the expression level per culture medium and the remaining ALP activity after heating at 60° C. for 10 minutes were measured.

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WO2025036987A1 (en) 2023-08-15 2025-02-20 Novozymes A/S Polypeptides having alkaline phosphatase activity for animal feed
WO2026082822A1 (en) 2024-10-16 2026-04-23 Novozymes A/S Polypeptides having alkaline phosphatase activity for pets
WO2026082819A1 (en) 2024-10-16 2026-04-23 Novozymes A/S Polypeptides having alkaline phosphatase activity for functional food
WO2026082817A1 (en) 2024-10-16 2026-04-23 Novozymes A/S Polypeptides having alkaline phosphatase activity for use in treatment of inflammatory and metabolic diseases

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WO2025036987A1 (en) 2023-08-15 2025-02-20 Novozymes A/S Polypeptides having alkaline phosphatase activity for animal feed
WO2025036988A2 (en) 2023-08-15 2025-02-20 Novozymes A/S Polypeptides having alkaline phosphatase activity for animal feed
WO2026082822A1 (en) 2024-10-16 2026-04-23 Novozymes A/S Polypeptides having alkaline phosphatase activity for pets
WO2026082819A1 (en) 2024-10-16 2026-04-23 Novozymes A/S Polypeptides having alkaline phosphatase activity for functional food
WO2026082817A1 (en) 2024-10-16 2026-04-23 Novozymes A/S Polypeptides having alkaline phosphatase activity for use in treatment of inflammatory and metabolic diseases

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