WO2023048262A1 - Ligature de peptide à l'aide d'une enzyme - Google Patents

Ligature de peptide à l'aide d'une enzyme Download PDF

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WO2023048262A1
WO2023048262A1 PCT/JP2022/035498 JP2022035498W WO2023048262A1 WO 2023048262 A1 WO2023048262 A1 WO 2023048262A1 JP 2022035498 W JP2022035498 W JP 2022035498W WO 2023048262 A1 WO2023048262 A1 WO 2023048262A1
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amino acid
acid sequence
seq
enzyme
sequence shown
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敏幸 脇本
研一 松田
雅和 小林
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国立大学法人北海道大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/02—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length in solution
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea

Definitions

  • the present invention relates to enzymatic production of peptides and modification of peptides.
  • Peptides are fundamental biomolecules that support life activities, and techniques for visualizing or controlling functions through artificial modification will greatly contribute to the elucidation of life phenomena. . Peptides themselves are also important drug discovery modalities, as typified by antibodies and insulin. Therefore, techniques for site-specific modification of peptides have been vigorously studied so far (Non-Patent Document 1), and attempts have also been made to create new peptides. In general, peptides have low resistance to high temperature conditions and organic solvents, and have many exposed reaction sites with similar chemical characteristics. Enzyme catalysts with mild reaction conditions and high molecular recognition ability are expected to be applied as protein modification tools. has been proposed as (Non-Patent Document 2). However, each of these has advantages and disadvantages (excessively high substrate specificity and stability of the substrate itself), and targets that can be targeted are limited.
  • PBP-type TE penicillin-binding protein-type thioesterase
  • PBP-type TE can catalyze not only intramolecular amidation but also intermolecular amidation. Specifically, it was found that PBP-type TE can regioselectively bind a donor peptide substrate (referred to as a donor substrate) to the N-terminus of an acceptor peptide substrate (referred to as an acceptor substrate) via an amide bond. . Furthermore, the inventors have found that ligation products are also obtained when donor substrates with diols as leaving groups are used. The present inventors completed the present invention based on such findings.
  • a method for producing a ligated peptide which comprises forming an amide bond between the C-terminus of a donor substrate and the N-terminus of an acceptor substrate using PBP-type TE as a catalyst.
  • PBP-type TE is an enzyme having the amino acid sequence shown in SEQ ID NO: 2, or a mutant enzyme thereof, and the mutant enzyme has any of the following amino acid sequences: (a) an amino acid sequence having 38% or more identity to the amino acid sequence shown in SEQ ID NO: 2; (b) an amino acid sequence in which several or several tens of amino acids are substituted, deleted, inserted or added in the amino acid sequence shown in SEQ ID NO: 2, or (c) complementary to the base sequence shown in SEQ ID NO: 1 has an amino acid sequence encoded by a nucleotide sequence that hybridizes to a specific nucleotide sequence under stringent conditions, and has a peptide ligation activity equal to or greater than that of an enzyme having the amino acid sequence shown in SEQ ID NO: 2 (1).
  • PBP-type TE is an enzyme having the amino acid sequence shown in SEQ ID NO: 7, or a mutant enzyme thereof, and the mutant enzyme has any of the following amino acid sequences: (a) an amino acid sequence having 38% or more identity to the amino acid sequence shown in SEQ ID NO: 7; (b) an amino acid sequence in which several or several tens of amino acids are substituted, deleted, inserted or added in the amino acid sequence shown in SEQ ID NO: 7, or (c) complementary to the nucleotide sequence shown in SEQ ID NO: 6 has an amino acid sequence encoded by a nucleotide sequence that hybridizes to a specific nucleotide sequence under stringent conditions, and has a peptide ligation activity equal to or greater than that of an enzyme having the amino acid sequence shown in SEQ ID NO: 7 (1).
  • the second residue from the C-terminal side of the donor substrate and the C-terminal residue are a cationic amino acid and a hydrophobic D-amino acid, respectively, and the carboxyl group of the C-terminal hydrophobic D-amino acid is activated;
  • PBP-type TE is an enzyme having the amino acid sequence shown in SEQ ID NO: 2, or a mutant enzyme thereof, and the mutant enzyme has any of the following amino acid sequences: (a) an amino acid sequence having 38% or more identity to the amino acid sequence shown in SEQ ID NO: 2; (b) an amino acid sequence in which several or several tens of amino acids are substituted, deleted, inserted or added in the amino acid sequence shown in SEQ ID NO: 2, or (c) complementary to the base sequence shown in SEQ ID NO: 1 has an amino acid sequence encoded by a nucleotide sequence that hybridizes to a specific nucleotide sequence under stringent conditions, and has a peptide ligation activity equal to or greater than that of an enzyme having the amino acid sequence shown in SEQ ID NO: 2 (7).
  • PBP-type TE is an enzyme having the amino acid sequence shown in SEQ ID NO: 7, or a mutant enzyme thereof, and the mutant enzyme has any of the following amino acid sequences: (a) an amino acid sequence having 38% or more identity to the amino acid sequence shown in SEQ ID NO: 7; (b) an amino acid sequence in which several or several tens of amino acids are substituted, deleted, inserted or added in the amino acid sequence shown in SEQ ID NO: 7, or (c) complementary to the nucleotide sequence shown in SEQ ID NO: 6 has an amino acid sequence encoded by a nucleotide sequence that hybridizes to a specific nucleotide sequence under stringent conditions, and has a peptide ligation activity equal to or greater than that of an enzyme having the amino acid sequence shown in SEQ ID NO: 7 (7).
  • the second residue from the C-terminal side of the donor substrate and the C-terminal residue are a cationic amino acid and a hydrophobic D-amino acid, respectively, and the carboxyl group of the C-terminal hydrophobic D-amino acid is activated;
  • (11) The method according to (10), wherein the activation of the carboxyl group of the C-terminal hydrophobic D-amino acid is performed by imparting a thioester-type leaving group to the carboxyl group.
  • (12) The method according to (10), wherein the activation of the carboxyl group of the C-terminal hydrophobic D-amino acid is performed by adding a diol to the carboxyl group.
  • a peptide ligation kit containing PBP-type TE (13) A kit for making modified peptides containing PBP-type TE. (15) The kit according to (14), further comprising a donor substrate containing a modifying group and/or an acceptor substrate containing a modifying group.
  • the present invention provides new enzymatic tools for peptide synthesis and new peptide modification techniques. Since the ligated peptide obtained by the present invention has an amide bond containing D-amino acids, it has improved resistance to degradative enzymes as compared with peptides consisting only of L-amino acids. Since the peptide ligation of the present invention is enzymatic, it does not require complicated operations such as attachment of a protecting group and deprotection as in chemical synthesis. Furthermore, the present invention provides a technique for selectively modifying the N-terminus of peptides and proteins. This modification approach can also be used to introduce non-peptidic structures at the N-terminus of peptides and proteins.
  • a ligation product can also be obtained when a donor substrate having a diol as a leaving group is used. Since diols are generally inexpensive and can be easily introduced to the C-terminus of a donor substrate by solid-phase synthesis, the present invention provides an inexpensive and convenient method for peptide ligation.
  • FIG. 3 shows the results of a ligation reaction using peptide 1 having biotin attached to the N-terminus as a donor substrate and different types of peptides 2, 4 and 5 as acceptor substrates using the enzyme SurE.
  • a chart obtained by analysis is shown. +SurE indicates charts of products obtained from reactions containing the enzyme SurE, and -SurE indicates charts of products obtained from reactions without the enzyme SurE.
  • FIG. 4 shows the products obtained when the ligation reaction was performed using the enzyme SurE using peptide 8 having propiolic acid attached to the N-terminus as a donor substrate and different types of peptides 2, 4 and 5 as acceptor substrates.
  • a chart obtained by HPLC analysis is shown.
  • +SurE indicates charts of products obtained from reactions containing the enzyme SurE
  • -SurE indicates charts of products obtained from reactions without the enzyme SurE.
  • a ligated peptide is characterized by using a PBP-type TE as a catalyst to generate an amide bond between the C-terminus of a donor substrate and the N-terminus of an acceptor substrate. to provide a method of manufacturing
  • PBP-type TE is a novel enzyme family discovered by the present inventors from soil bacteria (actinomycetes), and is a cyclizing enzyme family involved in non-ribosomal peptide biosynthesis (Kuranaga, T. et al., Angew Chem. Int. Ed. 2018, 57, 9447-9451 and Matsuda, K. et al. Nat. Catal. 2020, 3, 507-515).
  • the organisms derived from the PBP-type TE used in the present invention are not particularly limited, they are preferably bacteria, more preferably soil bacteria, and even more preferably actinomycetes.
  • Actinomycete-derived PBP-type TEs include, but are not limited to, enzymes derived from actinomycetes such as Streptomyces and Goodfellowiella.
  • PBP-type TEs may be derived from actinomycetes or non-actinomycete bacteria other than the above genera.
  • the PBP-type TE used for the peptide ligation of the present invention may be selected from known ones. Examples thereof include, but are not limited to, the enzyme Nsm16 from Streptomyces noursei NBRC 15452 and the enzyme SurE from Streptomyces albidoflavus NBRC 12854. In this specification, the enzyme Nsm16 is sometimes referred to as "Nsm16" and the enzyme SurE as "SurE".
  • peptide ligation refers to forming an amide bond (peptide bond) between the C-terminal amino acid residue of the donor substrate and the N-terminal amino acid residue of the acceptor substrate.
  • a donor substrate refers to a substrate having a C-terminal carboxyl group that donates to the N-terminal amino group of an acceptor substrate
  • an acceptor substrate is a substrate having an N-terminal amino group that accepts the C-terminal carboxyl group of a donor substrate.
  • PBP-type TEs that can be used in the present invention are capable of catalyzing peptide ligation reactions.
  • the term "ligation" does not include cyclization of peptides.
  • Examples of enzymes preferably used in the present invention include the enzyme Nsm16, but are not limited thereto.
  • the nucleotide sequence of DNA encoding the enzyme Nsm16 is shown in SEQ ID NO:1.
  • the amino acid sequence of enzyme Nsm16 is shown in SEQ ID NO:2. That is, in the present invention, an enzyme having the amino acid sequence shown in SEQ ID NO:2 or an enzyme having an amino acid sequence encoded by the base sequence shown in SEQ ID NO:1 is preferably used.
  • An enzyme that is a mutant of the enzyme Nsm16 and has a peptide ligation activity equal to or higher than that of the enzyme Nsm16 is also preferably used in the present invention.
  • the peptide ligation activity equivalent to or higher than that of the enzyme Nsm16 is about 50% or more of the enzyme Nsm16, preferably about 70% or more, more preferably about 80% or more, even more preferably about 90% or more. refers to the activity of
  • the peptide ligation activity of an enzyme can be measured by reacting donor and acceptor substrates and analyzing the products.
  • a test enzyme, a donor substrate and an acceptor substrate are allowed to react according to the procedures described in the Examples of the present specification, and the resulting reaction solution is subjected to LC-MS analysis to measure the amount of ligation product, thereby obtaining a peptide.
  • Ligation activity may be measured.
  • a specific example of a mutant of the enzyme Nsm16 is about 35% or more, for example, about 38% or more, preferably about 50% or more, more preferably about 70% or more (e.g., 75% or more) relative to the amino acid sequence shown in SEQ ID NO: 2. % or more, 80% or more, 85% or more), even more preferably about 90% or more (e.g. 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more), and have a ligation activity equal to or greater than that of Nsm16, but are not limited thereto.
  • the identity of amino acid sequences can be determined using known search means such as BLASTP.
  • mutants of the enzyme Nsm16 include an enzyme having an amino acid sequence in which several or several tens of amino acids are substituted, deleted, inserted or added to the amino acid sequence shown in SEQ ID NO: 2, , enzymes that have peptide ligation activity equal to or greater than that of the enzyme Nsm16, but are not limited to these.
  • Several tens refer to about 10 to about 90, for example, about 20, about 30, about 40, about 50, about 60, about 70, about 80 or about 90, Alternatively, it may be a number between these numerical values.
  • Substitution of amino acids in the amino acid sequence may be with any amino acid, but preferably with amino acids having similar properties and/or structures.
  • bracketed amino acids may be substituted for each other: (G, A), (K, R, H), (D, E), (N, Q), (S, T, Y), (C, M), (F, W, Y, H), (V, L, I).
  • a further specific example of a mutant of the enzyme Nsm16 is an enzyme having an amino acid sequence encoded by a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 1. , enzymes that have peptide ligation activity equal to or greater than that of the enzyme Nsm16, but are not limited to these.
  • Stringent conditions include, for example: in a buffer containing 0.25 M Na 2 HPO 4 , pH 7.2, 7% SDS, 1 mM EDTA, 1 ⁇ Denhardt's solution at a temperature of 60 to 68° C., preferably 65° C., more preferably 68° C. Hybridize for 16 to 24 hours, then in a buffer containing 20 mM Na 2 HPO 4 , pH 7.2, 1% SDS, 1 mM EDTA at a temperature of 60 to 68°C, preferably 65°C, more preferably 68°C.
  • a mutant of the enzyme Nsm16 may be naturally derived or non-naturally derived, for example, artificially produced using genetic engineering techniques.
  • the mutant of the enzyme Nsm16 may be an enzyme in which amino acid substitutions, deletions, insertions or additions have been made in the amino acid sequence of PBP-type TE of Streptomyces noursei NBRC 15452 by known methods.
  • the mutant of the enzyme Nsm16 may be a PBP-type TE possessed by actinomycetes other than Streptomyces noursei NBRC 15452, or a PBP-type TE possessed by bacteria other than actinomycetes.
  • Nsm16 and its variants can be obtained using known methods.
  • Nsm16 and variants thereof can be produced by cloning the Nsm16 gene or its homologues or orthologues using PCR, ligating into an expression vector, introducing the expression vector into host cells, and culturing the host cells. good.
  • Recombinant Nsm16 may be obtained using the methods described in the Examples herein.
  • a known method such as site-directed mutagenesis may be used to modify the nucleotide sequence of the gene encoding Nsm16, and the modified gene may be used to prepare Nsm16 mutants.
  • enzymes preferably used in the present invention include, but are not limited to, the enzyme SurE.
  • the base sequence of DNA encoding the enzyme SurE is shown in SEQ ID NO:6.
  • the amino acid sequence of enzyme SurE is shown in SEQ ID NO:7. That is, in the present invention, an enzyme having the amino acid sequence shown in SEQ ID NO: 7 or an enzyme having an amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 6 is preferably used.
  • An enzyme that is a mutant of the enzyme SurE and has a peptide ligation activity equal to or greater than that of the enzyme SurE is also preferably used in the present invention.
  • Peptide ligation activity equal to or greater than that of the enzyme SurE is about 50% or more of the enzyme SurE, preferably about 70% or more, more preferably about 80% or more, even more preferably about 90% or more. refers to the activity of The peptide ligation activity of an enzyme can be measured by reacting donor and acceptor substrates and analyzing the products.
  • a test enzyme, a donor substrate and an acceptor substrate are allowed to react according to the procedures described in the Examples of the present specification, and the resulting reaction solution is subjected to LC-MS analysis to measure the amount of ligation product, thereby obtaining a peptide.
  • Ligation activity may be measured.
  • mutants of the enzyme SurE include about 35% or more, for example, about 38% or more, preferably about 50% or more, more preferably about 70% or more (e.g., 75% or more) relative to the amino acid sequence shown in SEQ ID NO: 7. % or more, 80% or more, 85% or more), even more preferably about 90% or more (e.g. 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or greater, 99% or greater), and have ligation activity equal to or greater than that of SurE, but are not limited thereto.
  • the identity of amino acid sequences can be determined using known search means such as BLASTP.
  • a further specific example of the mutant of the enzyme SurE is an enzyme having an amino acid sequence in which several or several tens of amino acids are substituted, deleted, inserted or added to the amino acid sequence shown in SEQ ID NO: 7, , enzymes that have peptide ligation activity equal to or greater than that of the enzyme SurE, but are not limited to these.
  • Several tens refer to about 10 to about 90, for example, about 20, about 30, about 40, about 50, about 60, about 70, about 80 or about 90, Alternatively, it may be a number between these numerical values.
  • Amino acid substitutions in amino acid sequences may be with any amino acid, but are preferably with amino acids having similar properties and/or structures.
  • bracketed amino acids may be substituted for each other: (G, A), (K, R, H), (D, E), (N, Q), (S, T, Y), (C, M), (F, W, Y, H), (V, L, I).
  • a further specific example of a variant of the enzyme SurE is an enzyme having an amino acid sequence encoded by a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 6. , enzymes that have peptide ligation activity equal to or greater than that of the enzyme SurE, but are not limited to these.
  • Mutants of the enzyme SurE may be naturally derived or non-naturally derived, for example, artificially produced using genetic engineering techniques.
  • the enzyme SurE mutant may be an enzyme in which amino acid substitutions, deletions, insertions or additions have been made by known methods in the amino acid sequence of PBP-type TE of Streptomyces albidoflavus NBRC 12854.
  • the mutant of the enzyme SurE may be a PBP-type TE possessed by actinomycetes other than Streptomyces albidoflavus NBRC 12854, or a PBP-type TE possessed by bacteria other than actinomycetes.
  • SurE and its mutants can be obtained using known methods.
  • SurE and variants thereof can be produced by cloning the SurE gene or its homologues or orthologues using PCR, ligating into an expression vector, introducing the expression vector into host cells, and culturing the host cells. good.
  • Recombinant SurE may be obtained using the methods described in the Examples herein.
  • a known method such as site-directed mutagenesis may be used to modify the base sequence of the gene encoding SurE, and the modified gene may be used to prepare a SurE mutant.
  • enzymes preferably used in the present invention include, but are not limited to, the enzyme SurE.
  • the base sequence of DNA encoding the enzyme SurE is shown in SEQ ID NO:6.
  • the amino acid sequence of enzyme SurE is shown in SEQ ID NO:7. That is, in the present invention, an enzyme having the amino acid sequence shown in SEQ ID NO: 7 or an enzyme having an amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 6 is preferably used.
  • Mutants of the enzyme SurE may be naturally derived or non-naturally derived, for example, artificially produced using genetic engineering techniques.
  • the enzyme SurE mutant may be an enzyme in which amino acid substitutions, deletions, insertions or additions have been made by known methods in the amino acid sequence of PBP-type TE of Streptomyces albidoflavus NBRC 12854.
  • the mutant of the enzyme SurE may be a PBP-type TE possessed by actinomycetes other than Streptomyces albidoflavus NBRC 12854, or a PBP-type TE possessed by bacteria other than actinomycetes.
  • Nsm16 applies to the enzyme SurE, its variants, and their acquisition and production.
  • Nsm16 shall be replaced with SurE, SEQ ID NO: 1 with SEQ ID NO: 6, and SEQ ID NO: 2 with SEQ ID NO: 7.
  • a preferred amino acid sequence of PBP-type TE used in the present invention has the following characteristics:
  • the amino acid sequence of the portion corresponding to amino acid residues at positions 71 to 74 of SEQ ID NO: 2 is Ser-X 1 -X 2 -Lys, and/or amino acid residues at positions 164 to 169 of SEQ ID NO: 2
  • the amino acid sequence of the corresponding portion is Ser-Tyr-Ser-Asn-X 3 -Gly, and/or
  • the amino acid sequence of the portion corresponding to amino acid residues at positions 280-283 of SEQ ID NO: 2 is Gly-His- X 4 -Gly, and/or the amino acid sequence of the portion corresponding to amino acid residues at positions 357-362 of SEQ ID NO: 2 is Gly-X 5 -X 6 -X 7 -Asn-Gly.
  • preferred amino acid sequences of PBP-type TEs used in the present invention have the following characteristics:
  • the amino acid sequence of the portion corresponding to amino acid residues at positions 63-66 of SEQ ID NO: 7 is Ser-X 1 -X 2 -Lys, and/or amino acid residues at positions 153-158 of SEQ ID NO: 7 the amino acid sequence of the corresponding portion is Ser-Tyr-Ser-Asn-X 3 -Gly, and/or the amino acid sequence of the portion corresponding to amino acid residues at positions 304-307 of SEQ ID NO: 7 is Gly-His- X 4 -Gly, and/or the amino acid sequence of the portion corresponding to amino acid residues at positions 374-379 of SEQ ID NO: 7 is Gly-X 5 -X 6 -X 7 -Asn-Gly.
  • the amino acid sequences of more preferred PBP-type TEs have the following characteristics:
  • the amino acid sequence of the portion corresponding to amino acid residues at positions 357 to 362 of SEQ ID NO: 2 is Gly-X 5 -X 6 -X 7 -Asn-Gly, or positions 374 to 379 of SEQ ID NO: 7
  • the amino acid sequence of the portion corresponding to the amino acid residues is Gly-X 5 -X 6 -X 7 -Asn-Gly.
  • X 1 to X 7 each independently represent an arbitrary amino acid residue.
  • the corresponding portion may be a portion having the same amino acid residue number as SEQ ID NO: 2 or SEQ ID NO: 7, or a portion in the vicinity thereof.
  • Corresponding portions can be found by comparing the amino acid sequence of the PBP-type TE to be compared with the amino acid sequence of SEQ ID NO:2 or the amino acid sequence of SEQ ID NO:7. Such comparisons may be performed by alignment using known programs such as BLAST and ClustalW.
  • PBP-type TEs examples include, but are not limited to, mutants of the enzyme Nsm16 and mutants of the enzyme SurE.
  • a peptide refers to a molecule in which amino acid residues are linked by peptide bonds. The number of amino acid residues is 2 or more.
  • peptides include oligopeptides, polypeptides and proteins. As used herein, not all bonds between amino acid residues in a peptide need to be peptide bonds.
  • Amino acids that constitute peptides may be those contained in natural proteins or those not contained in natural proteins (eg, ⁇ -alanine, ⁇ -aminobutyric acid, ornithine, homozygous cysteine, etc.). Amino acids constituting a peptide may be in the L-form or the D-form.
  • Amino acids that constitute a peptide may be those that exist in vivo, or those that do not exist in vivo, such as those that are artificially synthesized.
  • the amino acids that make up the peptide may be modified.
  • the side chain carboxyl group may be esterified
  • the hydrogen of the side chain amino group may be substituted with an alkyl group
  • the side chain SH group may form an S—S bond with another molecule.
  • a ring such as a phenyl group in a side chain may be substituted with OH, halogen, an alkyl group, or the like, and a glycoside may be formed via oxygen or nitrogen in the side chain.
  • Peptides may include non-peptidic structures and modifying groups (discussed below). As used herein, the left end of the peptide is the N-terminus and the right end is the carboxyl-terminus. In this specification, the positions of amino acid residues in peptides are counted from the N-terminus.
  • the acceptor substrate may be any peptide.
  • the type of amino acid residue that constitutes the acceptor substrate is not particularly limited, but the N-terminal amino acid residue of the acceptor substrate is preferably a hydrophobic amino acid.
  • the number of amino acid residues that constitute the acceptor substrate is also not particularly limited.
  • the donor substrate may be a peptide containing any type and number of amino acid residues.
  • the second amino acid residue from the C-terminal side of the donor substrate is a cationic amino acid.
  • the C-terminal amino acid residue of the donor substrate is a hydrophobic amino acid. It is preferred that the C-terminal amino acid residue of the donor substrate is in the D-configuration. It is more preferred that the penultimate and C-terminal residues of the donor substrate are cationic and hydrophobic D-amino acids, respectively.
  • the C-terminal amino acid residue of the donor substrate is D-form, the amide bond generated by ligation also contains a D-amino acid, thus improving resistance to degradative enzymes. Ligation activity tends to be higher when the donor substrate is a shorter peptide. Ligation of a short donor substrate to an acceptor substrate can result in less change in the properties of the acceptor substrate.
  • Cationic amino acids are known and exemplified by, but not limited to, lysine, arginine, histidine, ornithine.
  • a cationic amino acid may be referred to as an amino acid having an isoelectric point higher than seven.
  • Hydrophobic amino acids are also known, exemplified by, but not limited to, leucine, isoleucine, valine, alanine, tryptophan, phenylalanine, and methionine.
  • a hydrophobic amino acid may be referred to as an amino acid having a higher hydrophobicity index than glycine.
  • the carboxyl group of the C-terminal amino acid of the donor substrate is preferably activated.
  • activation include, but are not limited to, esterification.
  • esterification include, but are not limited to, thioesterification, alkylesterification (eg, methylesterification, ethylesterification, etc.). It is preferable to thioesterify the carboxyl group of the C-terminal D-amino acid of the donor substrate for activation, that is, to provide a thioester-type leaving group for activation. Thioesterification with N-acetylcysteamine (formation of SNAC form) is preferred.
  • the carboxyl group of the C-terminal amino acid of the donor substrate may be activated by adding a leaving group such as alcohol, phenol, or thiol.
  • Preferred leaving groups are alcohols, especially diols.
  • a typical example of a diol is an alkyl group having two hydroxyl groups.
  • Ethylene glycol (sometimes referred to as EG) or EG analogs are more preferred leaving groups, with EG being an even more preferred leaving group.
  • Examples of EG analogues include, but are not limited to, diols having 1 to 4 carbon atoms such as methylene glycol, propylene glycol, and 1,3-butanediol.
  • the donor substrate may contain non-peptidic structures.
  • a non-peptidic structure refers to a structure that is not contained in natural peptides, and its type and structure are not particularly limited. Examples of non-peptidic structures include, but are not limited to, labels such as biotin, fluorescein, rhodamine, luciferin, PEG, sugars, lipids, nucleic acids, and the like. Non-peptidic structures may be naturally occurring or artificially produced.
  • a PBP-type TE and a donor substrate containing a non-peptidic structure can be used to impart a non-peptidic structure to the acceptor substrate.
  • the donor substrate may contain a peptidic structure.
  • a peptidic structure is a structure comprising two or more amino acids linked by amide bonds.
  • a PBP-type TE and a donor substrate containing a peptidic structure can be used to impart a peptidic structure to the acceptor substrate.
  • the acceptor substrate may contain a non-peptidic structure and/or a peptidic structure.
  • a PBP-type TE and an acceptor substrate containing a non-peptidic structure may be used to impart a non-peptidic structure to the donor substrate.
  • a PBP-type TE and an acceptor substrate containing a peptidic structure may be used to impart a peptidic structure to the donor substrate.
  • non-peptidic structures and peptidic structures may be used as modifying groups.
  • the present invention uses a PBP-type TE as a catalyst to generate an amide bond between the C-terminus of a donor substrate containing a modifying group and the N-terminus of an acceptor substrate, or Provided is a method for producing a modified peptide, characterized by forming an amide bond between the C-terminus of a substrate and the N-terminus of an acceptor substrate having a modifying group.
  • the above method can be used as a technique for selectively modifying the N-terminus of the target peptide (acceptor substrate). Moreover, the above method can be used as a means for selectively modifying the C-terminus of a target peptide (donor substrate).
  • a modifying group refers to a group that modifies a target peptide, and its type and structure are not particularly limited. The modifying group may have a peptidic structure or a non-peptidic structure. Examples of modifying groups include fluorescent labels, luminescent labels, radioactive labels, biotin, avidin, peptides or proteins (e.g.
  • CPP Cell Penetrating Peptide, membrane permeable peptide
  • signal sequences epitope tags, histidine tags, maltose binding proteins, glutathione-S-transferase, luciferase, antibodies or portions thereof), antigens or portions thereof, saccharides, lipids, nucleic acids and the like.
  • the modifying group may be naturally occurring or artificially produced.
  • the modifying group can be bound to the substrate using known means and methods.
  • the modifying group can be attached to the substrate by covalent bonding, ionic bonding, electrostatic bonding, hydrophobic bonding, and the like.
  • the modifying group may be covalently attached to the N-terminal amino group of the donor substrate, and the modifying group may be covalently attached to the C-terminal carboxyl group of the acceptor substrate.
  • the modifying group may be covalently bonded via a functional group (carboxyl group, amino group, hydroxyl group, SH group, etc.) in the side chain of the amino acid residue constituting the substrate.
  • a CPP may be introduced into the peptide of interest to improve its membrane permeability.
  • a signal sequence may be introduced into the peptide of interest to facilitate its subcellular localization.
  • An affinity tag sequence may be introduced into the peptide of interest to facilitate its purification.
  • ADC Antibody-Drug Conjugate
  • PDC Peptide-Drug Conjugate
  • the present invention provides a peptide ligation kit containing PBP-type TE.
  • the kit is usually accompanied by an instruction manual.
  • the kit may contain a donor substrate and/or an acceptor substrate.
  • the kit may contain, for example, freeze-dried PBP-type TE in terms of storage stability and stability.
  • kits for producing modified peptides containing PBP-type TEs are usually accompanied by an instruction manual.
  • the kit may comprise a donor substrate containing modifying groups and/or an acceptor substrate containing modifying groups.
  • the kit may contain, for example, freeze-dried PBP-type TE in terms of storage stability and stability.
  • the present invention provides a donor substrate used for peptide ligation with PBP-type TE.
  • the penultimate and C-terminal residues of the donor substrate are cationic and hydrophobic D-amino acids, respectively.
  • the C-terminal amino acid of said donor substrate is activated. More preferably, the C-terminal amino acid of the donor substrate is thioesterified (eg SNAC-formed) at its carboxyl group or activated by the addition of diols (eg EG).
  • Nsm16 1 Peptide ligation using Nsm16 1 .
  • Preparation of Recombinant Enzyme Nsm16 A DNA fragment encoding the enzyme Nsm16 was amplified from the genomic DNA of Streptomyces noursei NBRC 15452 using polymerase KOD One (Toyobo). The set of primers used for amplification was as follows (capital letters). An EcoRI recognition site was added to the forward primer, and a HindIII recognition site was added to the reverse primer (underlined parts of SEQ ID NOs: 3 and 4).
  • Nsm16_Fw 5'- ccggaattc GTGCACGGGGACTCAGCGGATCC-3' (SEQ ID NO: 3)
  • Nsm16_Rv 5'-ccc aagctt TTAGTGCGGCCGTGCGCCGTGG-3' (SEQ ID NO: 4)
  • the amplified fragment was inserted into the EcoRI/HindIII sites in the multiple cloning site of pET-28a(+) (Novagen) to obtain the recombinase Nsm16 expression vector (Nsm16-pET28a).
  • the expression vector obtained above was transformed into E. E. coli BL231 (DE3), a single colony was inoculated into 10 mL of 2xYT medium (1.6% Bacto peptone, 1.0% Bacto Yeast Extract, 0.5% NaCl) containing 50 ⁇ g/mL of kanamycin, and 37 C. overnight to obtain a seed culture.
  • a 2.0 mL culture was transferred to 200 mL 2xYT medium containing 50 ⁇ g/mL kanamycin and incubated at 37° C. for 3 hours. The culture was chilled on ice and 0.1 mL of IPTG was added to induce expression of the recombinant enzyme Nsm16. E. coli was grown overnight at 16°C.
  • the cells were collected by centrifugation (3500xg, 10 minutes) and disrupted using an ultrasonic homogenizer. After removing debris by centrifugation (17000 ⁇ g, 10 minutes), fractions containing soluble proteins were applied to a Ni-NTA affinity column ( Merck Millipore). The column was washed with wash buffer and eluted with 500 mM imidazole wash buffer. The eluate from the column was concentrated with an Amicon Ultra 0.5 mL filter (Merck Millipore). The concentration of the protein solution was measured using a Bio-Rad protein assay kit. In this experiment, the recombinant enzyme Nsm16 was obtained as a protein having a histidine tag attached to the N-terminal side. Its amino acid sequence is shown in SEQ ID NO:5.
  • the recombinant enzyme Nsm16 thus obtained was used for the following peptide ligation experiments.
  • Solid Phase Peptide Synthesis Protocol Step 1 The Fmoc group of the solid phase supported peptide was removed by using a 20% piperidine/DMF solution (10 min, room temperature).
  • Step 2 The resin in the reaction vessel was washed with DMF (x3) and CH2Cl2 ( x3 ).
  • Step 3 To a solution of F-moc protected building block (4 eq) was added DIC (4 eq) in NMP and Oxyma (4 eq in DMF). After 2-3 minutes of preactivation, the mixture was poured into the reaction vessel.
  • Step 4 The resin in the reaction vessel was washed with DMF (x3) and CH2Cl2 ( x3 ). Amino acids were enriched onto the solid phase support by repeating steps 1-4. Thioesterification of the N-terminal amino acid of the donor substrate was performed using N-acetylcysteamine in a conventional manner.
  • the donor substrates used in this experiment were: It was a biotinylated glycine-D-tryptophan-D-arginine-D-phenylalanine SNAC form.
  • the acceptor substrates used in this experiment were: D-phenylalanine-glycine-glycine-D-tryptophan-D-arginine-D-phenylalanine.
  • Peptide ligation (1) The donor substrate and acceptor substrate shown in the upper part of FIG. 1 were ligated with the enzyme Nsm16. Reactions were initiated by adding 1.0 ⁇ M enzyme Nsm16 to reaction mixtures containing 200 ⁇ M donor substrate and 800 ⁇ M acceptor substrate in 20 mM Tris-HCl (pH 8.0). After enzyme addition, the mixture was incubated at 30° C. for 2 hours, and the reaction was stopped by adding an equal volume of 0.1% TFA. After diluting the sample with an equal volume of methanol, it was centrifuged at 20000 ⁇ g for 10 minutes. The resulting supernatant was analyzed by LC-MS (amaZon SL-NPC) operating in positive mode.
  • LC-MS amaZon SL-NPC
  • LC-MS was coupled with a Shimadzu HPLC system. Separation was performed using a Cosmosil 5C 18 -MS-II 2.0 ⁇ 150 mm column (Nacalai Tesque). Water + 0.05% TFA and acetonitrile + 0.05% TFA were used as mobile phases A and B, respectively. Samples were eluted in gradient mode from 10% to 90% mobile phase B over 20 minutes at a flow rate of 0.2 ml ⁇ min ⁇ 1 .
  • Peptide ligation (2) Next, in addition to the donor substrate used in peptide ligation (1) above, a smaller donor substrate (see FIG. 2) was used to examine the peptide ligation reaction.
  • the small donor substrates used in this experiment were: Biotinylated D-Tryptophan-D-Arginine-D-Phenylalanine SNAC Biotinylated D-Arginine-D-Phenylalanine SNAC Biotinylated D-Phenylalanine SNAC.
  • the acceptor substrate used in this experiment was the same as for peptide ligation (1) above. Reaction conditions were the same as for peptide ligation (1) above.
  • coli was grown overnight at 16°C. Cells were collected by centrifugation (3500 ⁇ g, 10 minutes) and disrupted with an ultrasonic homogenizer. After removing debris by centrifugation (17000 ⁇ g, 10 min), the fraction containing soluble protein was applied to a Ni-NTA affinity column (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 20 mM imidazole) equilibrated with wash buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 20 mM imidazole). Merck Millipore). The column was washed with wash buffer and eluted with 500 mM imidazole wash buffer.
  • the recombinant enzyme SurE thus obtained was used for the following peptide ligation experiments.
  • Step 1 The Fmoc group of the solid phase supported peptide was removed by using a 20% piperidine/DMF solution (10 min, room temperature).
  • Step 2 The resin in the reaction vessel was washed with DMF (x3) and CH2Cl2 ( x3 ).
  • Step 3 To a solution of F-moc protected building block (4 eq) was added DIC (4 eq) in NMP and Oxyma (4 eq in DMF). After 2-3 minutes of preactivation, the mixture was poured into the reaction vessel. The resulting mixture was stirred for 30 minutes.
  • Step 4 The resin in the reaction vessel was washed with DMF (x3) and CH2Cl2 ( x3 ).
  • Amino acids were enriched onto the solid phase support by repeating steps 1-4.
  • D-Phe, D-Arg, and biotin were sequentially added to resin S2 according to a solid-phase peptide synthesis protocol, and donor substrate 1 was obtained by cleaving the resin from the resin by a conventional method.
  • Peptide ligation (1) Peptide 1 having biotin added to the N-terminus and EG added to the C-terminus was used as a donor substrate, and three types of peptides 2, 4 and 5 were used as acceptor substrates, and an enzyme SurE was used to perform a ligation reaction.
  • Either the donor substrate 1 and acceptor substrates 2, 4, 5 shown above were ligated with the enzyme SurE. Reactions were initiated by adding 1.0 ⁇ M enzyme SurE to reaction mixtures containing 200 ⁇ M donor substrate and 800 ⁇ M acceptor substrate in 20 mM Tris-HCl (pH 8.0). After enzyme addition, the mixture was incubated at 30° C. for 2 hours, and the reaction was stopped by adding an equal volume of 0.1% TFA. After diluting the sample with an equal volume of methanol, it was centrifuged at 20000 ⁇ g for 10 minutes. The resulting supernatant was analyzed by LC-MS (amaZon SL-NPC) operating in positive mode. LC-MS was coupled with a Shimadzu HPLC system.
  • Peptide ligation (2) Peptide 8 having pro-opiolic acid added to the N-terminus and EG added to the C-terminus was used as a donor substrate, three types of peptides 2, 4, and 5 were used as acceptor substrates, and an enzyme SurE was used to perform a ligation reaction. . The reaction procedure is the same as in 3 above. Peptide ligation (1) was followed.
  • the present invention provides new enzymatic tools for peptide synthesis and new peptide modification techniques, it can be used in fields such as chemistry, biology, biochemistry, medicine, and pharmacy. INDUSTRIAL APPLICABILITY
  • the present invention can be used, for example, for the development and manufacture of new drugs and new research reagents.
  • SEQ ID NO: 1 shows the base sequence of DNA encoding the enzyme Nsm16 derived from Streptomyces noursei NBRC 15452.
  • SEQ ID NO: 2 shows the amino acid sequence of the enzyme Nsm16 from Streptomyces noursei NBRC 15452.
  • SEQ ID NO: 3 shows the nucleotide sequence of the forward primer (including the EcoRI recognition site) for amplifying the DNA fragment encoding the enzyme Nsm16.
  • SEQ ID NO: 4 shows the nucleotide sequence of the reverse primer (including the HindIII recognition site) for amplifying the DNA fragment encoding the enzyme Nsm16.
  • SEQ ID NO: 5 shows the amino acid sequence of the recombinant enzyme Nsm16 (including histidine tag) obtained in Example.
  • SEQ ID NO: 6 shows the base sequence of DNA encoding the enzyme SurE derived from Streptomyces albidoflavus NBRC 12854.
  • SEQ ID NO: 7 shows the amino acid sequence of the enzyme SurE from Streptomyces albidoflavus NBRC 12854.
  • SEQ ID NO: 8 shows the nucleotide sequence of the forward primer used to prepare the recombinant enzyme SurE.
  • SEQ ID NO: 9 shows the base sequence of the reverse primer used to prepare the recombinant enzyme SurE.
  • SEQ ID NO: 10 shows the amino acid sequence of the recombinant enzyme SurE obtained in Examples.
  • SEQ ID NO: 1 The sequences of SEQ ID NO: 1 to SEQ ID NO: 10 are shown below.

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Abstract

L'invention concerne : un procédé de production d'un peptide ligaturé, caractérisé par la formation d'une liaison amide entre l'extrémité C-terminale d'un substrat de peptide donneur (substrat donneur) et l'extrémité N-terminale d'un substrat de peptide accepteur (substrat accepteur) à l'aide d'une thioestérase de type protéine de liaison à la pénicilline (TE de type PBP) en tant que catalyseur; et un procédé de production d'un peptide modifié, caractérisé par la formation d'une liaison amide entre l'extrémité C-terminale d'un substrat donneur contenant un groupe de modification et l'extrémité N-terminale d'un substrat accepteur à l'aide d'une TE de type PBP en tant que catalyseur.　
PCT/JP2022/035498 2021-09-27 2022-09-22 Ligature de peptide à l'aide d'une enzyme WO2023048262A1 (fr)

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WO2019216248A1 (fr) * 2018-05-07 2019-11-14 国立大学法人北海道大学 Macrocyclase peptidique

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WO2019216248A1 (fr) * 2018-05-07 2019-11-14 国立大学法人北海道大学 Macrocyclase peptidique

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Title
MATSUDA KENICHI, FUJITA KEI, WAKIMOTO TOSHIYUKI: "PenA, a penicillin-binding protein-type thioesterase specialized for small peptide cyclization", JOURNAL OF INDUSTRIAL MICROBIOLOGY & BIOTECHNOLOGY, BASINGSTOKE, GB, vol. 48, no. 3-4, 4 June 2021 (2021-06-04), GB , pages 23, XP093055066, ISSN: 1367-5435, DOI: 10.1093/jimb/kuab023 *

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