WO2018186702A1 - Copolypeptide à séquences multiples, qui est composé d'un peptide à base d'élastine et d'une protéine de pied de moule et qui présente une bonne sensibilité aux stimuli et une bonne adhérence de surface, son procédé de préparation et son utilisation - Google Patents

Copolypeptide à séquences multiples, qui est composé d'un peptide à base d'élastine et d'une protéine de pied de moule et qui présente une bonne sensibilité aux stimuli et une bonne adhérence de surface, son procédé de préparation et son utilisation Download PDF

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WO2018186702A1
WO2018186702A1 PCT/KR2018/004029 KR2018004029W WO2018186702A1 WO 2018186702 A1 WO2018186702 A1 WO 2018186702A1 KR 2018004029 W KR2018004029 W KR 2018004029W WO 2018186702 A1 WO2018186702 A1 WO 2018186702A1
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mfp
block
copolypeptide
ebppi
ratio
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PCT/KR2018/004029
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English (en)
Korean (ko)
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임동우
이재희
이재상
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한양대학교 에리카산학협력단
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Priority to US16/500,177 priority Critical patent/US12049491B2/en
Priority claimed from KR1020180039787A external-priority patent/KR102070124B1/ko
Publication of WO2018186702A1 publication Critical patent/WO2018186702A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/82—Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/04—Macromolecular materials
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]

Definitions

  • the present invention relates to a multi-block copolypeptide having a stimulus responsiveness and surface adhesion, and more particularly, to a multi-block copolypeptide consisting of an elastin-based peptide and a mussel foot protein, a method of preparing the multi-block copolypeptide, A core-shell self-assembled nanostructure comprising a block copolypeptide and a hydrogel comprising the multi-block copolypeptide.
  • Protein-based copolymer blocks self-assembled into core-shell micelles have received considerable attention as drug delivery systems, and in particular, triple block polypeptides have been studied for tissue engineering applications because sol-gel transitions due to physical or chemical crosslinking occur. come.
  • various protein-based materials have been developed for drug delivery and tissue engineering applications.
  • Bioadhesives refer to substances that have adhesion properties to various biological samples such as proteins, DNA, growth factors, cells, etc., such as cell membranes, cell walls, lipids, proteins, DNA, growth factors, cells, and tissues.
  • Various biomedical applications are possible, such as engineering supports, drug delivery carriers, tissue fillers, wound healing, or intestinal adhesion prevention.
  • Bioadhesives require strong adhesion and crosslinking capabilities and must maintain their function in vivo for a long time.
  • Bioadhesives currently commercially available or commercially available include cyanoacrylate instant adhesives, fibrin glues, gelatin glues and polyurethane-based adhesives.
  • bioadhesives using synthetic polymers show very weak strength in the presence of an aqueous solution in vivo, and cyanoacrylate-based bioadhesives have been pointed out as causing great side effects such as immune responses in the human body.
  • fibrin-based bioadhesives currently used in actual patients have little side effects, but their use is limited because of their very low adhesion.
  • formalin or glutaaldehyde which is used as a crosslinking agent, also causes cross-linking reaction with proteins in vivo, resulting in tissue toxicity, and polyurethane-based tissue adhesive has a problem in that aromatic diisocyanate, which is a synthetic raw material, is biotoxic. .
  • MFP mussel foot protein
  • DOPA makes important chemical contributions to adhesion through intermolecular and intramolecular crosslinking (Silverman HG et al., Marine biotechnology, 9 (6), 661-681, 2007; Lee Haeshin. Et al., Proceedings of the National Academy of Sciences, 103 (35), 12999-13003, 2006).
  • Waveguides with catechol side chains in which tyrosine residues are hydroxylated by tyrosinase can bind metal ions, oxides and semimetals through coordination bonds or hydrogen bonds (Sever, MJ et al., Angewandte). Chemie, 116 (4), 454-456, 2004).
  • the present inventors have made efforts to find a polypeptide having a stimulus responsiveness and surface adhesion that can be used in biomedical applications, elastin-based peptide (Elastin-based Polypeptide, EBP); And using a multiblock copolypeptide (MFP) consisting of mussel quartet protein (MFP), forms a self-assembled core-shell structure and hydrogel, reversible change with temperature stimulation, surface adhesion It confirmed that it was remarkably excellent, and completed this invention.
  • EBP elastin-based Polypeptide
  • Another object of the present invention is a gene encoding the multiblock copolypeptide, a recombinant vector comprising the gene, a recombinant microorganism into which the gene or the recombinant vector is introduced, and a method for producing a multiblock copolypeptide using the recombinant microorganism.
  • Still another object of the present invention is a self-assembling nanostructure and a self-assembling nanostructure of the core-shell structure in which the multiblock copolypeptide is a temperature structure, the EBP block forms a core structure, and the MFP block forms a shell structure.
  • the multiblock copolypeptide is a temperature structure
  • the EBP block forms a core structure
  • the MFP block forms a shell structure.
  • Still another object of the present invention is to prepare a multi-block copolypeptide cross-linking between block polypeptides by temperature stimulation, hydrogel, bioadhesive composition comprising the hydrogel and surgery comprising the hydrogel To provide a suture.
  • the present invention provides an elastin-based peptide (EBP); And multi-block copolypeptides consisting of mussel foot protein (MFP).
  • EBP elastin-based peptide
  • MFP mussel foot protein
  • the present invention also provides a gene encoding the multiblock copolypeptide, a recombinant vector comprising the gene, a recombinant microorganism into which the gene or the recombinant vector is introduced.
  • the present invention also comprises the steps of (a) culturing a recombinant microorganism to produce the multi-block copolypeptide; And (b) obtaining the generated multiblock copolypeptide.
  • the present invention also provides a self-assembled nanostructure and a self-assembled nanostructure of the core-shell structure wherein the multi-block copolypeptide is a temperature structure, the EBP block forms a core structure, the MFP block forms a shell structure It provides a drug delivery composition comprising.
  • the present invention also provides a hydrogel in which the multiblock copolypeptides are prepared by forming crosslinks between block polypeptides by temperature stimulation.
  • the present invention also provides a bioadhesive composition and a surgical suture containing the hydrogel.
  • the multiblock copolypeptides of the present invention form self-assembled core-shell structures and hydrogels that can be reversibly changed with temperature stimulation, and have excellent surface adhesion, which makes them useful in biomedical applications. Can be.
  • FIG. 1 is a molecular schematic of various block copolypeptides composed of EBP and MFP ((A): double and triple block copolypeptides forming a core-shell (micelle) structure, (B): MFP forming a hydrogel) -EBP-MFP triple block copolypeptide, (C): EBP-MFP-EBP triple block copolypeptide to form hydrogel, (D): Stimulation responsiveness and surface adhesion of EBP-MFP-EBP triple block copolypeptide Sex mechanism).
  • Figure 2 is a result of confirming the MFP DNA of the present invention by agarose gel electrophoresis ((A): Mcfp5, (B) Mgfp5, lane (M): size marker, lane (1): 1 MFP repeat unit, lane (2): 2 MFP repeat units, lane (3): 4 MFP repeat units).
  • Figure 3 illustrates the cloning of the EBP-MFP block copolypeptide gene of the present invention ((A): multiplex cloning of MFP, (B): EBP-MFP block copolypeptide cloning).
  • FIG. 4 is a schematic of the cloning of the tyrosinase and ORF438 genes of the present invention ((A): tyrosinase cloning, (B): ORF438 cloning).
  • Figure 5 shows the results of (A) tyrosinase and (B) ORF438DNA of the present invention by agarose gel electrophoresis (lane (1) and (2) DNA template: 25ng, lane (3) and (4) DNA Template: 50 ng, lane (N): negative control).
  • FIG. 6 shows (A) DNA agarose gel electrophoresis (1.2%) of tyrosinase gene (824bp) and orf438 gene (438bp); (B) SDS-PAGE results of tyrosinase ( ⁇ 35 kDa), EBP-MFP double block copolypeptide ( ⁇ 24 kDa) and orf438 ( ⁇ 15 kDa) co-expressed in E. coli (lane (1): EBP-MFP double block) Copolypeptide, lane (2): tyrosinase and orf438, lane (3): double block copolypeptide, tyrosinase and orf438 co-expressed in E.
  • EBP-MFP-EBP triple block copolypeptide ⁇ 41 kDa
  • tyrosinase ⁇ 35 kDa
  • orf438 ⁇ 15 kDa
  • E NBT staining hydroxylation via a simultaneous expression system of tyrosinase, ORF438 and double block copolypeptides in E. coli of EBPPI [G 1 A 4 F 1 ] 6 -MFP [Mgfp5] 1 double block copolypeptides The result is confirmed.
  • Figure 7 is a result of NBT / Glycinate staining after treatment with mushroom-derived tyrosinase in order to confirm the tyrosine residue modification of the double block copolypeptide of the present invention.
  • FIG. 8 shows SDS-PAGE results of copper staining of the block copolypeptide of the present invention ((A): EBPPI [G 1 A 4 F 1 ] 6 -MFP [Mgfp5] 1 double block treated with mushroom-derived tyrosinase.
  • FIG. 11 shows (A) mushroom-derived tyrosinase-catalyzed EBPPI [G 1 A 4 F 1 ] 6 -MFP [Mgfp5] 1 -EBPPI [G 1 A 4 F 1 ] 6 triple block nose treated with 10 mM NaIO 4 Polypeptide (10% by weight) photo; (B) The photo on the left shows mushroom-derived tyrosinase-catalyzed EBPPI [G 1 A 4 F 1 ] 6 -MFP [Mgfp5] 1 -EBPPI [G 1 A 4 F 1 ] 6 triple block under (A) conditions Surface attachment picture of the copolypeptide, the right is a 20% by weight EBP triple block copolypeptide picture as a control treated with (A) conditions.
  • FIG. 12 is a photograph of 10 mM NaIO 4 treated with hydroxylated block copolypeptide (10 wt.%) In a coexpression system; (B) Photos of adhesion experiments in the presence of water of hydroxylated blockcopolypeptides in a coexpression system.
  • FIG. 13 was measured by the DLS instrument with the hydrodynamic radius of (A) mushroom-derived tyrosinase-catalyzed hydroxylation with or without modification.
  • the hydrodynamic radius of the block copolypeptides was measured at 12.5 uM in 10 mM phosphate buffer (pH 5).
  • the hydrodynamic radius of the block copolypeptides after phase transition is 50 nm to 70 nm, indicating that the block copolypeptides are in the form of specific structures.
  • FIG. 14 shows transmission electron micrographs of the temperature of EBPPI [G 1 A 4 F 1 ] 6 -MFP [Mgfp5] 1 double block copolypeptides with mushroom-derived tyrosinase-catalyzed hydroxylation ((A, B , C) nanostructures of EBPPI [G 1 A 4 F 1 ] 6 -MFP [Mgfp5] 1 double block copolypeptides observed at 10 ° C., with accumulations of 0.2 ⁇ m, 50 nm and 50 nm, respectively (D, E , F) constructs of EBPPI [G 1 A 4 F 1 ] 6 -MFP [Mgfp5] 1 double block copolypeptides observed at 37 ° C., with accumulations of 0.5 mm, 100 nm and 50 nm, respectively (G, H, I) The structure of EBPPI [G 1 A 4 F 1 ] 6 -MFP [Mgfp5] 1 double block copoly
  • EBP elastin-based peptide
  • MFPs multiblock copolypeptides
  • the present invention provides an elastin-based peptide (EBP); And it relates to a multiblock copolypeptide (multiblock copolypeptide) consisting of mussels musa protein (MFP).
  • EBP elastin-based peptide
  • MFP mussels musa protein
  • copolypeptide means a polypeptide that is a copolymer.
  • polypeptide means any polymer chain of amino acids.
  • peptide and protein are interchangeable with the term “polypeptide”, which also refers to a polymer chain of amino acids.
  • polypeptide includes “polypeptide” analogs of natural or synthetic proteins, protein fragments and protein sequences.
  • the polypeptide may be a monomer or a polymer.
  • phase transition means that the state of a substance changes, such as when water turns into water vapor or ice turns into water.
  • Polypeptides having the phase change behavior according to the present invention are basically stimulin reactive elastin-based polypeptides (EBPs).
  • EBPs stimulin reactive elastin-based polypeptides
  • the "elastin-based polypeptides” are also called “elastin-like polypeptides (ELPs).” It is a term widely used in the technical field of the present invention.
  • the EBP undergoes a reversible phase transition at lower critical solution temperature (LCST), also referred to as transition temperature (T t ). These have large water solubility below T t , but become insoluble when the temperature exceeds T t .
  • LCST critical solution temperature
  • the physicochemical properties of EBP are mainly controlled by the combination of pentapeptide repeat units Val-Pro- (Gly or Ala) -X aa -Gly [VP (G or A) XG].
  • the third amino acid of that repeat unit determines the relative mechanical properties.
  • the third amino acid Gly determines elasticity
  • Ala determines plasticity. The elasticity or plasticity is a property that appears after the transition.
  • both the hydrophobicity and the multimerization of the pentapeptide repeating unit of the guest residue X aa as the fourth amino acid affect T t .
  • Mussel triglyceride protein of the present invention can be attached to various surfaces through waveguide.
  • DOPA having a catechol side chain imparts surface adhesion through hydrogen bonds and coordinate bonds with surface molecules
  • quinones an oxidized form of DOPA
  • Intramolecular and intermolecular crosslinking forms to provide strong underwater cohesion. Crosslinking through quinone formation produces a hardened sheath and exhibits moisture-resistance.
  • DOPA and quinone are essential for surface adhesion, which are determined by pH conditions.
  • pH conditions around mussel quiescents are lower than pH 3.0, limiting the oxidation of DOPA to adsorb to surface oxides through hydrogen bonding and metal ion coordination.
  • MFP is exposed to seawater (pH-8.3) to induce oxidation from DOPA to quinones, crosslinking and protein coagulation.
  • hydrophilic amino acids of MFPs such as Ser and Gly participate in cohesive interactions by hydrogen bonding, cation-pi interactions, electrostatic and hydrophobic interactions (Waite, JH, Journal of Experimental) Biology, 220 (4), 517-530, 2017).
  • a novel type of block copolypeptide consisting of a multifunctional EBP block and an MFP block is reasonably designed, synthesized, and characterized.
  • the present invention was intended to combine with MFP and EBP block, a stimulatory reactive protein, to observe self-assembly structure with biomimetic underwater adhesion, and to apply it to the biomedical field.
  • MFP and EBP block a stimulatory reactive protein
  • the mussel californianus foot protein 5 (Mcfp5) and the Mediterranean have a high percentage of tyrosine content ( ⁇ 30%) in all mussel foot protein types.
  • Mgfp5 The gene sequence of mussel galloprovincialis foot protein 5 (Mgfp5) was selected. Tyrosine content of MFP is related to surface adhesion efficiency and strength (Silverman H. G. et al., Marine biotechnology, 9 (6), 661-681, 2007).
  • the multiblock copolypeptide is in the group consisting of (EBP) n (MFP) n, (EBP) n (MFP) n (EBP) n and (MFP) n (EBP) n (MFP) n It is composed of any one arrangement, wherein n is an integer of 1 or more, it may be characterized in that the repetition number of EBP or MFP.
  • the elastin-based peptide is selected from any one of a group consisting of [VPGXG VPGXG VPGXG VPGXG VPGXG VPGXG] block, [VPAXG VPAXG VPAXG VPAXG VPAXG VPAXG] block and [IPAXG IPAXG IPAXG IPAXG IPAXG] block. It is composed of the amino acid sequence represented by the formula, X may be characterized in that the amino acid except proline.
  • X (or X aa ) is referred to as a "guest residue".
  • Various kinds of X aa may be introduced to prepare various kinds of EBP according to the present invention.
  • the polypeptide may have multi-stimuli responsiveness.
  • multi-stimulatory responsiveness means to be responsive to one or more stimuli.
  • the stimulus may be one or more selected from the group consisting of temperature, pH, ionic strength and ligand.
  • Ligand in the present invention is a substance that specifically binds to a desired substance, for example, various antibodies, antigens, enzymes, substrates, receptors, peptides, DNA, RNA, aptamers, protein A, protein G, avidin, Biotin, chelate compounds, various metal ions (e.g., calcium ions) and the like.
  • a desired substance for example, various antibodies, antigens, enzymes, substrates, receptors, peptides, DNA, RNA, aptamers, protein A, protein G, avidin, Biotin, chelate compounds, various metal ions (e.g., calcium ions) and the like.
  • the [VPGXG VPGXG VPGXG VPGXG VPGXG VPGXG] block is characterized by the amino acid sequence of SEQ ID NO: 1, wherein the [VPAXG VPAXG VPAXG VPAXG VPAXG VPAXG] block is represented by the amino acid sequence of SEQ ID NO: 2 And the [IPAXG IPAXG IPAXG IPAXG IPAXG IPAXG IPAXG IPAXG IPAXG IPAXG] block is represented by the amino acid sequence of SEQ ID NO.
  • amino acid means a natural “amino acid” or “artificial” amino acid, and preferably means a natural "amino acid”.
  • amino acid refers to glycine, alanine, serine, valine, leucine, isoleucine, methionine, glutamine, asparagine, cysteine, histidine, phenylalanine, arginine, tyrosine or tryptophan.
  • amino acids The nature of these amino acids is well known in the art. Specifically, it shows hydrophilicity (negative charge or positive charge) or hydrophobicity, and also shows the properties of aliphatic or aromatic.
  • Abbreviations such as Gly (G) and Ala (A) used in the present invention are amino acid abbreviations.
  • Amino acid abbreviations are glycine (Gly, G), alanine (Ala, A), valine (Val, V), leucine (Leu, L), isoleucine (Ile, I), proline (Pro, P), phenylalanine (Phe, F ), Tyrosine (Tyr, Y), tryptophan (Trp, W), cysteine (Cys, C), methionine (Met, M), serine (Ser, S), threonine (Thr, T), lysine (Lys, K) It is represented by arginine (Arg, R), histidine (His, H), aspartic acid (Asp, D), glutamic acid (Glu, E), asparagine (Asn, N), glutamine (Gln, Q).
  • the abbreviation is a widely used expression in
  • hydrophilic amino acid is an amino acid exhibiting hydrophilic properties, such as lysine, arginine, and the like
  • hydrophobic amino acid is an amino acid showing hydrophobic properties, such as phenylalanine, leucine and the like.
  • X in the [VPGXG VPGXG VPGXG VPGXG VPGXG VPGXG] block is A (Ala), G (Gly), I (Ile) in a ratio of 1: 4: 1; K (Lys), G (Gly), I (Ile) are in a ratio of 1: 4: 1; Or D (Asp), G (Gly), I (Ile) in a ratio of 1: 4: 1; E (Glu), G (Gly), I (Ile) consists of a ratio of 1: 4: 1; G (Gly), A (Ala), F (Phe) consists of 1: 3: 2 ratio; K (Lys), A (Ala), F (Phe) consists of a ratio of 1: 3: 2; D (Asp), A (Ala), F (Phe) consists of a ratio of 1: 3: 2; K (Lys), F (Phe) consist of 3: 3 ratio; D (Asp), A (Ala), F
  • X in the [VPAXG VPAXG VPAXG VPAXG VPAXG VPAXG VPAXG VPAX] block is A (Ala), G (Gly), I (Ile) in a ratio of 1: 4: 1; K (Lys), G (Gly), I (Ile) are in a ratio of 1: 4: 1; Or D (Asp), G (Gly), I (Ile) in a ratio of 1: 4: 1; E (Glu), G (Gly), I (Ile) consists of a ratio of 1: 4: 1; Or G (Gly), A (Ala), F (Phe) in a ratio of 1: 3: 2.
  • X in the [IPAXG IPAXG IPAXG IPAXG IPAXG IPAXG] block is characterized by G (Gly), A (Ala), and F (Phe) in a ratio of 1: 4: 1 or 1: 3: 2. You can do
  • EBPs having a pentapeptide repeating unit Val-Pro- (Gly or Ala) -X aa -Gly [VP (G or A) XG] are named as follows.
  • X aa may be any amino acid except Pro.
  • the pentapeptide repeat of the plastic Val-Pro-Ala-X aa- Gly (VPAXG) is defined as the plastic elastin-based polypeptide with plasticity (EBPP).
  • EBPP plastic elastin-based polypeptide with plasticity
  • VPGXG the pentapeptide repeat of Val-Pro-Gly-X aa -Gly
  • EBPE elastin-based polypeptide with elasticity
  • the pentapeptide repeat of Ile-Pro-Ala-X aa- Gly is also defined as a plastic elastin-based polypeptide (EBPPI) with the first position replaced by Ile.
  • EBPPI plastic elastin-based polypeptide
  • [XiYjZk] n the capital letters in parentheses are the short-term amino acid code of the guest residue, ie, the amino acid at position 4 (X aa or X) of the EBP pentapeptide, and their corresponding subscripts are EBP monomers as repeat units. Shows the ratio of guest residues in the gene.
  • n of [XiYjZk] n is SEQ ID NO: 1 [VPGXG VPGXG VPGXG VPGXG VPGXG VPGXG], SEQ ID NO: 2 [VPAXG VPAXG VPAXG VPAXG VPAXG VPAXG VPAXG], or SEQ ID NO: 3 [IPAXG IPAXG IPAXG IPAXG IPAXG] The total number of repetitions of the EBP is shown.
  • EBPP [G1A3F2] 12 is an EBPP block consisting of 12 repeating units of SEQ ID NO: 2 [VPAXG VPAXG VPAXG VPAXG VPAXG], wherein Gly, Ala, and at the fourth guest residue position (X aa ) The ratio of Phe is 1: 3: 2.
  • the gene and amino acid sequences of the EBP block of the present invention are shown in Tables 1 and 2, respectively.
  • the mussel triglyceride protein may be characterized in that the mussel californianus foot protein 5 (Msselp5) or the mussel galloprovincialis foot protein 5 (Mgfp5). have.
  • MFP [Mgfp5] n and MFP [Mcfp5] n represents the type of mussel species and ligature proteins in the letters and numbers in parentheses, subscript 'n' indicates the number of repetitions of the MFP block.
  • MFP [Mgfp5] 1 means Mediterranean mussel (mussel galloprovincialis), MFP type 5, and one MFP block repeat unit.
  • double and triple block copolys consisting of EBP and MFP Peptides are represented by the construction of an MFP block and an EBP block with a hyphen between the two blocks, for example, a double block copeptide is an EBPPI [G1A4F1] n -MFP [Mgfp5] n, and a triple block copolypeptide is an EBPPI [ G1A4F1] n -MFP [Mgfp5] n -EBPPI [G1A4F1] n .
  • the multi-block copolypeptide is characterized by the amino acid sequence of SEQ ID NO: 50 to 70, it may be characterized by the nucleotide sequence of SEQ ID NO: 71 to 91.
  • the present invention relates to a gene encoding the multiblock copolypeptide.
  • the present invention relates to a recombinant vector comprising the gene.
  • the present invention relates to a recombinant microorganism into which the gene or the recombinant vector is introduced.
  • the recombinant microorganism may be characterized in that the expression vector containing a gene encoding a tyrosinase (tyrosinase) or a gene encoding a tyrosinase is further introduced and co-expressed.
  • tyrosinase tyrosinase
  • the mussel quartet protein forms a waveguide having a catechol side chain in which tyrosine residues are hydroxylated by tyrosinase, and the waveguide is a metal ion or oxide through coordination bond or hydrogen bond. And semimetals.
  • the waveguide is specifically dyed in the NBT and glycinate solution due to the redox reaction.
  • orf438 gene may be further included in the expression vector.
  • the present invention provides a method for producing a multi-block copolypeptide by culturing the recombinant microorganism; And (b) obtaining the generated multiblock copolypeptide.
  • the recombinant microorganism of step (a), the expression vector containing a gene encoding a tyrosinase (tyrosinase) or a gene encoding tyrosinase is further introduced to the multi-block copolypeptide and tyro It may be characterized by the co-expression of cinases.
  • the tyrosine residue of the multi-block copolypeptide may be characterized in that the tyrosinase is modified into a dopa (3,4-dihydroxyphenylalanine) residue.
  • tyrosinase (tyrosinase) is expensive, according to the method for producing a multi-block copolypeptide of the present invention, it is economical because it can express a large amount of tyrosinase in bacteria.
  • the vector refers to a DNA preparation containing a nucleotide sequence of the polynucleotide encoding the target protein operably linked to a suitable control sequence to express the target protein in a suitable host cell.
  • the regulatory sequence may comprise a promoter capable of initiating transcription, any operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence regulating the termination of transcription and translation, as desired It can be manufactured in various ways.
  • the promoter of the vector may be constitutive or inducible. After being transformed into a suitable host, the vector can replicate or function independently of the host genome and integrate into the genome itself.
  • the vector used in the present invention is not particularly limited as long as it can be replicated in a host cell, and any vector known in the art may be used.
  • Examples of commonly used vectors include natural or recombinant plasmids, phagemids, cosmids, viruses and bacteriophages.
  • pWE15, M13, ⁇ MBL3, ⁇ MBL4, ⁇ IXII, ⁇ ASHII, ⁇ APII, ⁇ t10, ⁇ t11, Charon4A, and Charon21A can be used as a phage vector or cosmid vector
  • pBR, pUC, pBluescriptII, pGEM system, pTZ system, pCL system and pET system can be used.
  • the vector usable in the present invention is not particularly limited and known expression vectors can be used.
  • expression control sequence refers to a DNA sequence that is essential for the expression of a coding sequence operably linked in a particular host organism.
  • Such regulatory sequence is a promoter for transcription, for controlling such transcription.
  • Any operator sequence, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence that controls termination of transcription and translation, for example, a regulatory sequence suitable for prokaryotes includes a promoter, optionally an operator sequence, and a ribosomal binding site
  • Eukaryotic cells include promoters, polyadenylation signals, and enhancers, the most influential factors affecting the expression levels of genes in the plasmids, the promoters for high expression, the SR ⁇ promoter and cytomegalovirus. Derived promoters and the like are preferably used.
  • any of a wide variety of expression control sequences can be used in the vector.
  • useful expression control sequences include, for example, early and late promoters of SV40 or adenovirus, lac system, trp system, TAC or TRC system, T3 and T7 promoters, major operator and promoter region of phage lambda, fd Regulatory regions of the code protein, promoters for 3-phosphoglycerate kinase or other glycolysis enzymes, promoters of the phosphatase such as Pho5, promoters of the yeast alpha-crossing system and prokaryotic or eukaryotic cells or viruses thereof And other sequences of constitution and induction known to modulate the expression of the genes, and various combinations thereof.
  • the T7 RNA polymerase promoter ⁇ 10 may be usefully used to express protein NSP in E. coli.
  • Nucleic acids are "operably linked” when placed in a functional relationship with other nucleic acid sequences. This may be genes and regulatory sequence (s) linked in such a way as to allow gene expression when appropriate molecules (eg, transcriptional activating proteins) bind to regulatory sequence (s).
  • DNA for a pre-sequence or secretion leader is operably linked to DNA for a polypeptide when expressed as a shear protein that participates in the secretion of the polypeptide;
  • a promoter or enhancer is operably linked to a coding sequence when it affects the transcription of the sequence;
  • the ribosomal binding site is operably linked to a coding sequence when it affects the transcription of the sequence;
  • the ribosomal binding site is operably linked to a coding sequence when positioned to facilitate translation.
  • "operably linked” means that the linked DNA sequence is in contact, and in the case of a secretory leader, is in contact and present within the reading frame.
  • enhancers do not need to touch. Linking of these sequences is performed by ligation (linking) at convenient restriction enzyme sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers according to conventional methods are used.
  • expression vector generally refers to a fragment of DNA that is generally double stranded as a recombinant carrier into which fragments of heterologous DNA have been inserted.
  • heterologous DNA refers to heterologous DNA, which is DNA not naturally found in host cells.
  • the gene must be operably linked to transcriptional and translational expression control sequences that function in the selected expression host.
  • the expression control sequence and the gene of interest are included in one expression vector including the bacterial selection marker and the replication origin. If the expression host is a eukaryotic cell, the expression vector must further comprise an expression marker useful in the eukaryotic expression host.
  • expression host / vector combinations can be used to express the genes encoding the polypeptides of the invention.
  • Suitable expression vectors for eukaryotic hosts include, for example, expression control sequences derived from SV40, bovine papilloma virus, adenovirus, adeno-associated virus, cytomegalovirus and retrovirus.
  • Expression vectors that can be used in bacterial hosts include a broader host range, such as bacterial plasmids, RP4, which can be exemplified in E. coli such as pBluescript, pGEX2T, pUC vectors, colE1, pCR1, pBR322, pMB9 and derivatives thereof.
  • Phage plasmids phage DNA that can be exemplified by a wide variety of phage lambda derivatives such as ⁇ gt10 and ⁇ gt11, NM989, and other DNA phages such as M13 and filamentary single-stranded DNA phages.
  • Useful expression vectors for yeast cells are 2 ⁇ plasmids and derivatives thereof.
  • a useful vector for insect cells is pVL 941.
  • Host cells transformed or transfected with the expression vectors described above constitute another aspect of the present invention.
  • transformation means introducing DNA into a host so that the DNA is replicable as an extrachromosomal factor or by chromosomal integration.
  • transfection means that the expression vector is accepted by the host cell whether or not any coding sequence is actually expressed.
  • the host cell of the invention is a recombinant microorganism into which a vector having a polynucleotide encoding at least one target protein is introduced, or a polynucleotide encoding at least one target protein is introduced into the microorganism so that the polynucleotide is integrated into a chromosome to express the target protein.
  • a recombinant microorganism infected with a trait It may be a prokaryotic or eukaryotic cell.
  • a host having a high DNA introduction efficiency and a high expression efficiency of the introduced DNA is usually used.
  • Known eukaryotic and prokaryotic hosts such as Escherichia coli, Pseudomonas, Bacillus, Streptomyces, fungi, yeast, insect cells such as Spodoptera pruperferda (SF9), animal cells such as CHO and mouse cells, COS 1, COS African green monkey cells such as 7, BSC 1, BSC 40 and BMT 10, and tissue cultured human cells are examples of host cells that can be used.
  • COS cells since SV40 large T antigen is expressed in COS cells, the plasmid having the origin of replication of SV40 is present as a large number of copies of the episome in the cells. And expression can be expected.
  • the introduced DNA sequence may be obtained from the same species as the host cell, may be of a different species than the host cell, or it may be a hybrid DNA sequence comprising any heterologous or homologous DNA.
  • the relative strength of the sequence, the controllability, and the compatibility with the DNA sequences of the present invention should be considered, particularly with regard to possible secondary structures.
  • Single cell hosts may be selected from a host for the selected vector, the toxicity of the product encoded by the DNA sequence of the invention, the secretory properties, the ability to accurately fold the protein, the culture and fermentation requirements, the product encoded by the DNA sequence of the invention from the host. It should be selected in consideration of factors such as the ease of purification. Within the scope of these variables, one skilled in the art can select a variety of vector / expression control sequence / host combinations capable of expressing the DNA sequences of the invention in fermentation or large scale animal culture.
  • binding method binding method
  • panning method panning method
  • film emulsion method film emulsion method
  • a conventionally known genetic manipulation method may be used, and the non-viral delivery method may be cell perforation, lipofection, microinjection, ballistic method, virosome, liposome.
  • Immunoliposomes, polyvalent cations or lipid nucleic acid conjugates, naked DNA, artificial virons, and chemical promoted DNA influx.
  • Sonorization for example methods using the Sonitron 2000 system (Rich-Mar), can also be used for the delivery of nucleic acids.
  • Other representative nucleic acid delivery systems are Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc. (Rockville, Maryland).
  • Suitable cations or neutral lipids for effective receptor-recognition lipofection of polynucleotides include lipids from Felgner (WO91 / 17424 and WO91 / 16024) and can be delivered to cells via in vitro introduction and to target tissues via in vivo introduction. have.
  • nucleic acid complexes including target liposomes, such as immunolipid complexes
  • Methods of preparing lipid: nucleic acid complexes, including target liposomes, such as immunolipid complexes, are well known in the art (Crystal, Science., 270: 404-410, 1995; Blaese et al., Cancer Gene Ther., 2: 291).
  • Lentiviral vectors are retroviral vectors that generate high viral titers by transducing or infecting non-dividing cells.
  • the target tissue determines the retroviral gene persistence system.
  • Retroviral vectors contain cis acting long terminal repeats that can pack 6-10 kb outer sequences. Minimal cis acting LTRs sufficient for replication and packaging of the vector can be used to integrate the therapeutic gene into target cells for permanent transgene expression.
  • Widely used retroviral vectors include murine leukemia virus (MuLV), gibbon leukemia virus (GaLV), monkey immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combination viruses thereof (Buchscher et al. , J. Virol., 66: 2731-2739, 1992; Johann et al., J. Virol., 66: 1635-1640 1992; Sommerfelt et al., Virol., 176: 58-59, 1990; Wilson et al. , J. Virol., 63: 2374-2378, 1989; Miller et al., J. Virol., 65: 2220-2224, 1991; PCT / US94 / 05700.
  • MiLV murine leukemia virus
  • GaLV gibbon leukemia virus
  • SIV monkey immunodeficiency virus
  • HAV human immunodeficiency virus
  • sucrose phosphorylase proteins are more common with adenovirus-based systems, and adenovirus-based vectors cause high efficiency transduction in many cells but do not require cell division.
  • the vector allows for high titers and high levels of expression and can be produced in large quantities in a simple system.
  • Adeno accessory virus (AAV) vectors are also used to transduce into cells with target nucleic acids, for example for the production of nucleic acids and peptides in vitro and for gene therapy in vivo and in vitro (West et al., Virology., 160: 38-47, 1987; US Pat. No.
  • pLASN and MFG-S are examples of retroviruses used in clinical trials (Dunbar et al., Blood., 85: 3048-305, 1995; Kohn et al., Nat.
  • PA317 / pLASN was the first therapeutic vector used in gene therapy (Blaese et al., Science., 270: 475-480, 1995) Transduction efficiency of MFG-S packaging vector was 50% or higher (Ellem et al., Immunol Immunother., 44 (1): 10-20, 1997; Dranoff et al., Hum. Gene Ther. , 1: 111-2, 1997).
  • rAAV Recombinant adeno-associated virus vectors
  • All vectors are derived from plasmids with AAV 145 bp inverted terminal repeats flanking the transgene expression cassette. Efficient gene delivery and stable transgene delivery due to integration into the genome of transduced cells is a great advantage of the vector system (Wagner et al., Lancet., 351: 9117-17023, 1998; Kearns et al., Gene Ther., 9: 748-55, 1996).
  • co-expression means that two or more genes are expressed at the same time, in the present invention is expressed as co-expression.
  • tyrosine residues of the MFP block were hydroxylated in two ways ((1) bacterial coexpression system (orf438 and tyrosinase and block copolypeptides co-expressed in E. coli) and (2 ) Mushroom-derived tyrosinase catalysis).
  • Double and triple block copeptides composed of EBP (A block) and MFP (B block) are designed in AB-, ABA- and BAB-types to form self-assembled micellar structures and injectable hydrogels, It can be used as a bio coating and a bioadhesive material having.
  • Dopa (DOPA) in MFP plays an important role in surface adhesion, and quinone, an oxidized form of DOPA, imparts cohesion through intermolecular crosslinking.
  • EBP blocks with phase transitions above LCST exhibit improved cohesion for physically crosslinked hydrogelation and surface adhesion.
  • the EBP-MFP block copolypeptide and orf438 and tyrosinase were co-expressed in E. coli to hydroxylate the tyrosine residues of the block copolypeptides without further treatment.
  • the EBP-MFP block copolypeptide has superior adhesion compared to the EBP-MFP block copolypeptide modified by mushroom-derived tyrosinase, it can be seen that it is useful for industrial scale up (Fig. 12).
  • the surface adhesion strength of the block copolypeptides co-expressed in the form of micelles and hydrogels was investigated.
  • the EBP-MFP block copolypeptide of the present invention has great potential as micelles and hydrogels having surface adhesion (Figs. 10, 11, 13 and 14).
  • the present invention relates to a self-assembled nanostructure of a core-shell structure in which the multiblock copolypeptide is a temperature stimulus so that the EBP block forms a core structure and the MFP block forms a shell structure.
  • the core-shell structure refers to a micelle structure
  • micelles generally refer to a thermodynamically stable and uniform spherical structure formed by low molecular weight materials having both amphiphilic, for example, hydrophilic and hydrophobic groups.
  • a non-aqueous drug is dissolved and added to the compound having the micelle structure, the drug is present in the micelle, and the micelle can release the target-oriented drug in response to a change in temperature or pH in the body, thereby serving as a carrier for drug delivery.
  • the possibility of application is very high.
  • MFPs with different block lengths were fused with EBPPI to form self-assembled nanostructures by thermal stimulation.
  • Molecules of the EBPPI-MFP biblock copolypeptide can self assemble into core-shell nanostructures in response to temperature (FIG. 1 (A)).
  • MFP is fused to the N-terminus or C-terminus of EBPPI [G1A4F1] 6 to exhibit surface adhesion under moisture conditions.
  • Surface adherent micelles can be applied to the surface-coated nanostructures on the stent, which can be inserted into the lumen of anatomical vessels to maintain passage and drug delivery carriers.
  • the present invention relates to a drug delivery composition comprising the self-assembled nanostructure.
  • the self-assembled nanostructures according to the present invention can be used as an extracellular matrix as an effective scaffold for drug delivery.
  • the drug is not particularly limited and includes chemicals, small molecules, peptide or protein medicines, nucleic acids, viruses, antibacterial agents, anticancer agents, anti-inflammatory agents and the like.
  • the small molecules may be, for example, but not limited to, contrast agents (eg, T1 contrast agents, T2 contrast agents such as superparamagnetics, radioisotopes, etc.), fluorescent markers, dyeing materials, and the like.
  • contrast agents eg, T1 contrast agents, T2 contrast agents such as superparamagnetics, radioisotopes, etc.
  • fluorescent markers e.g., fluorescent markers, dyeing materials, and the like.
  • the peptide or protein drug product may be a hormone, a hormone analog, an enzyme, an enzyme inhibitor, a signaling protein or a part thereof, an antibody or a part thereof, a single chain antibody, a binding protein or a binding domain, an antigen, an adhesion protein, a structural protein, a regulatory protein, a toxin Proteins, cytokines, transcriptional regulators, blood clotting factors, vaccines, and the like.
  • FGF fibroblast growth factor
  • VEGF vascular endothelial growth factor
  • TGF transforming growth factor
  • bone morphogenetic protein BMP human growth hormone
  • pGH pig growth hormone
  • G-CSF leukocyte growth factor
  • EPO red blood cell growth factor
  • M-CSF macrophage growth factor
  • TNF tumor necrosis factor
  • EGF Epidermal growth factor
  • PDGF platelet-induced growth factor
  • NGF nerve growth factor
  • growth hormone releasing factor growth hormone releasing factor
  • engiotensin luteinizing hormone releasing hormone (LHRH)
  • corpus luteum LHRH agonist insulin
  • TRH thyroid-stimulating hormone releasing hormone
  • TRH endostatin, endostatin, somatostatin, glucagon, endorphins, vacitracin, mergain, colistin, single antibodies, vaccines or these Containing a mixture of , but it is not limited there
  • the nucleic acid can be, for example, RNA, DNA or cDNA, and the sequence of nucleic acids can be a coding site sequence or a non-coding site sequence (eg antisense oligonucleotide or siRNA).
  • the virus may be a virus core (ie, a nucleic acid of a virus packaged without a virus envelope) comprising the virus or the nucleic acid of the virus.
  • viruses and viral cores that can be transported include, but are not limited to, papilloma virus, adenovirus, baculovirus, retroviral core and semilky virus core.
  • the antimicrobial agent may be minocycline, tetracycline, oploxacin, phosphomycin, mergaine, profloxacin, ampicillin, penicillin, doxycycline, thienamycin, cephalosporin, norcardycin, gentamicin, neomycin, kanamycin , Paromomycin, micronomycin, amikacin, tobramycin, dibecasin, cytotaxin, cefacller, erythromycin, ciprofloxacin, levofloxacin, endoxacin, vancomycin, imipenem, fusidic acid and mixtures thereof It may be, but is not limited thereto.
  • the anticancer agents include paclitaxel, taxotier, adriamycin, endostatin, angiostatin, mitomycin, bleomycin, cisplatin, carboplatin, doxorubicin, daunorubicin, idarubicin, 5-fluorouracil, methotrexate, exec Tinomycin-D and mixtures thereof, but is not limited thereto.
  • the anti-inflammatory agents are acetaminophen, aspirin, ibuprofen, diclofenac, indomethacin, pyricampam, phenopropene, flubiprofen, ketoprofen, naproxen, suprofen, roxofene, synoxycamp , Tenoxycam, and mixtures thereof, but is not limited thereto.
  • the present invention relates to a hydrogel in which the multiblock copolypeptide is prepared by forming crosslinks between block polypeptides by temperature stimulation.
  • Hydrogel of the present invention has a mechanical flexibility similar to the actual tissue, and contains a lot of water, but since the bond of the gel is not broken by water, it requires adhesion with a wet biological surface containing water and external moisture Applications to medical adhesives and the like that must have resistance to Edo have been actively made. Accordingly, the hydrogel having excellent tissue adhesion according to the present invention is capable of various biomedical applications such as tissue adhesives or hemostatic agents, tissue engineering supports, drug delivery carriers, tissue fillers, wound healing, and intestinal adhesion prevention.
  • Triple block copolypeptides were prepared as injectable hydrogels with surface adhesion (FIGS. 1B and 1C).
  • the EBP block is used as a physical crosslinker with stimulatory reactivity
  • the MFP block is used for the introduction of chemical crosslinking and surface adhesion by quinone formation.
  • Triple block copolypeptides including MFP-EBP-MFP and EBP-MFP-EBP, can be self-assembled into hydrogels by oxidation by temperature change and NaIO 4 treatment.
  • 1 (D) shows the mechanism of surface adhesion, intermolecular crosslinking and stimulus reactivity of the triple block copolypeptides.
  • the hydrogel may be formed by the oxidative or non-covalent interaction of the dopa (3,4-dihydroxyphenylalanine) residue contained in the mussel musa protein (MFP).
  • MFP mussel musa protein
  • the present invention relates to a bioadhesive composition comprising the hydrogel.
  • the bioadhesive composition of the present invention can be used in various areas such as skin, blood vessels, digestive organs, cranial nerves, plastic surgery, orthopedics, and the like, by replacing cyanoacrylic adhesives or fibrin adhesives that are mainly used in the market.
  • the biocompatible biotissue adhesive of the present invention can replace surgical sutures, can be used to block unnecessary blood vessels, and can be used for hemostasis and suturing of soft tissues such as facial tissues and cartilage, and hard tissues such as bones and teeth. It is possible to apply as a home medicine.
  • Various application fields of the biocompatible bioadhesive composition of the present invention are summarized as follows.
  • the bioadhesive of the present invention may be applied to the internal and external surfaces of the human body, that is, the bioadhesive of the present invention may be applied to the external surface of the human body such as skin or the surface of an internal organ exposed during a surgical procedure. Can be applied as In addition, the bioadhesives of the present invention can be used to bond damaged parts of a tissue, to seal leakage of air / fluid from the tissue, to adhere a medical device to the tissue, or to fill a defective portion of the tissue.
  • biological tissue herein is not particularly limited and includes, for example, skin, bones, nerves, axons, cartilage, blood vessels, corneas, muscles, fascia, brain, prostate, breast, endometrium, lung, spleen, small intestine. , Liver, testes, ovaries, cervix, rectum, stomach, lymph nodes, bone marrow and kidneys.
  • the bioadhesive of the present invention may be used for wound healing.
  • the biocompatible bioadhesive of the present invention can be used as a dressing applied to a wound.
  • the bioadhesive of the present invention can be used for skin closure. That is, the bioadhesive of the present invention may be applied topically and used to seal the wound, replacing the suture.
  • the bioadhesive of the present invention can be applied to restoring hernia, for example, can be used for the surface coating of the mesh used for restoring hernia.
  • the bioadhesive of the present invention can also be used to prevent closure and leakage of tubular structures such as blood vessels.
  • the bioadhesive of the present invention can also be used for hemostasis.
  • the bioadhesive of the present invention may be used as an anti-adhesion agent after surgery.
  • Adhesion occurs at all surgical sites, where other tissues stick around the wound around the surgical site. Adhesion occurs 97% after surgery, especially 5-7% of which causes serious problems.
  • the wound may be minimized during surgery or anti-inflammatory agents may be used. It also activates tissue plasminogen activators (TPAs) or physical barriers such as crystalline solutions, polymer solutions, and solid membranes to prevent fibrin formation, but these methods can be toxic in vivo and have other side effects. have.
  • TPAs tissue plasminogen activators
  • the bioadhesive of the present invention can be applied to exposed tissue after surgery to be used to prevent adhesions occurring between the tissue and surrounding tissue.
  • the present invention relates to a surgical suture comprising the hydrogel.
  • a support for tissue engineering comprising a hydrogel of the present invention.
  • Tissue engineering technology refers to a method of culturing cells isolated from a patient's tissue in a support to prepare a cell-support complex, and then transplanting the prepared cell-support complex back into the human body. It is applied to the regeneration of almost all organs of the human body such as cartilage, artificial cornea, artificial blood vessel, artificial muscle.
  • Bioadhesive hydrogels of the present invention can provide scaffolds similar to living tissue to optimize the regeneration of living tissue and organs in tissue engineering techniques.
  • the support of the present invention can easily implement an artificial extracellular matrix, it can be used as a medical material such as cosmetics, wound dressings, dental matrix.
  • Hydrogels of the present invention can be easily attached to various bioactive substances involved in the action of promoting the growth and differentiation of cells through the interaction with the cells or tissues of the human body and help the regeneration and recovery of tissues.
  • the physiologically active substance may collectively refer to various biomolecules that may be included to implement an artificial extracellular matrix having a structure similar to a natural extracellular matrix.
  • Bioactive substances include cells, proteins, nucleic acids, sugars, enzymes, and the like, and examples thereof include cells, proteins, polypeptides, polysaccharides, monosaccharides, oligosaccharides, fatty acids, nucleic acids, and the like. have.
  • the cell may be any cell including prokaryotic and eukaryotic cells.
  • the physiologically active substance includes, but is not limited to, plasmid nucleic acid as a nucleic acid material, hyaluronic acid as a sugar substance, heparin sulfate, chondroitin sulfate, alginate, and hormonal protein as a protein substance.
  • PET-21a vector and BL21 (DE3) E. coli cells were purchased from Novagen Inc. (Madison, WI, U.S.). Top 10 competent cells were purchased from Invitrogen (Carlsbad, CA, U.S.). Oligonucleotides were chemically synthesized by Cosmo Gene Tech (Seoul, South Korea). Fermentas (Ontario, Canada) was purchased from Fast AP, a thermosensitive alkaline phosphatase, and restriction endonucleases including BamHI and XbaI. Other restriction endonucleases were obtained from New England Biolabs (Ipswich, MA, U.S.), including BseRI, AcuI, and other restriction enzymes.
  • T4 DNA ligase was obtained from Elpis Bio-tech (Taejeon, South Korea). All kits for DNA mini-preparation, gel extraction, and PCR purification were obtained from Geneall Biotechnology (Seoul, South Korea). Dyne Agarose High was obtained from DYNE BIO (Seongnam, South Korea). All Top10 cells were grown in TB DRY medium (MO BIO Laboratories, Carlsbad, CA, U.S.) and super optimal broth with catabolite repression (SOC) medium supplemented with 20 mM glucose (Formedium, UK). All BL21 (DE3) cells were grown in circular growth medium obtained from MP Biomedicals (Solon, OH, U.S.).
  • Tris-HCI 2-20%
  • a precast gel was obtained from Bio-Rad (Hercules, CA, U.S.).
  • Phosphate buffered saline (PBS, pH 7.4), ampicillin and polyethyleneimine (PEI) were purchased from Sigma-Aldrich (St Louis, MO).
  • MFP gene was obtained from M. galloprovincialis- and M. californianus-foot protein 5.
  • the pUC plasmid containing the MFP gene sequence was treated with a buffer containing 10 U of XbaI and 15 U of Acu1 at 37 ° C. for 30 to 60 minutes, and the mpET-21a plasmid vector was also subjected to restriction enzymes of 10 U of XbaI and 15 U of BseRI. Treated. Thereafter, 90 pmol of MFP dsDNA and 30 pmol of linearized mpET-21a cloning vector were incubated in T4 DNA ligase buffer containing 1 U of T4 DNA ligase for 30 minutes at 16 ° C. for ligation. The ligated plasmids were transformed into Top 10 chemical receptor cells and then applied onto SOC plates supplemented with 50 ⁇ g / ml ampicillin. The inserted sequence was then confirmed by DNA sequencing (Table 3).
  • the gene sequence of MFP was multiplied up to four repeat units. Gene sequences and sizes encoding multimerized MFP were confirmed using DNA sequencing and DNA agarose gel electrophoresis.
  • Block copolypeptide libraries consisting of EBP and MFP were synthesized using plasmids with EBP or MFP single block genes.
  • the plasmid containing EBP represented by the gene and amino acid sequence shown in Table 1 and Table 2 was treated with a buffer containing 10 U of XbaI and 15 U of BseRI for 30-60 minutes at 37 ° C., followed by PCR purification kit.
  • Purification with Plasmids with MFP block gene were treated with a buffer containing 10 U of XbaI and 15 U of AcuI for 30-60 minutes at 37 ° C.
  • the MFP gene represented by the amino acid sequence of Table 3 was isolated using agarose gel electrophoresis and purified by gel purification kit.
  • Plasmids with EBP blocks were used as vectors and fused with the MFP gene as insert. Ligation was performed by incubating 90 pmol of purified insert and 30 pmol of linearized vector in ligase buffer containing 1 U of T4 DNA ligase for 30-60 minutes at 16 ° C. The product was then transformed into Top10 recipient cells and then streaked onto SOC plates supplemented with 50 ⁇ g / ml ampicillin. In order to synthesize double and triple block copolypeptides of EBP and MFP, the double block gene of EBPPI-MFP was synthesized by inserting the MFP gene at the 5 'or 3' end of the EBPPI gene.
  • the MFP-EBPPI-MFP or EBPPI-MFP-EBPPI triple block gene was synthesized by inserting the MFP or EBPPI gene into the 5 'or 3' end of the previously synthesized EBPPI-MFP double block gene.
  • Other double and triple block copolypeptide genes were synthesized by varying the block order and length of EBP and MFP.
  • Triple block copolypeptides were synthesized by a recursive directional ligation (RDL) method using double block copolypeptides as building blocks (FIG. 3 (B)).
  • the EBPPI [G1A4F1] 6 gene was seamlessly fused to the N-terminus of the MFP [Mgfp5] 1-EBPPI [G1A4F1] 6 double block copolypeptide gene by RDL, resulting in EBPPI [G1A4F1] 6-MFP [Mgfp5] 1-EBPPI [G1A4F1] 6 triple block copolypeptide genes were synthesized.
  • the length and molecular weight of the double block copolypeptides are shown in Table 5 below.
  • Di-block copolypeptides Nucleotide chain length (bp) M.W (kDa) MFP [Mgfp5] 1 -EBPP [G 1 A 3 F 2 ] 24 (SEQ ID NO: 50) 2400 69.55 MFP [Mgfp5] 2 -EBPP [G 1 A 3 F 2 ] 24 (SEQ ID NO: 51) 2631 78.47 MFP [Mgfp5] 4 -EBPP [G 1 A 3 F 2 ] 24 (SEQ ID NO: 52) 3093 96.31 MFP [Mgfp5] 1 -EBPPI [G 1 A 3 F 2 ] 6 (SEQ ID NO: 53) 780 25.23 MFP [Mgfp5] 2 -EBPPI [G 1 A 3 F 2 ] 6 (SEQ ID NO: 54) 1011 34.15 MFP [Mgfp5] 4 -EBPPI [G 1 A 3 F 2 ] 6 (SEQ ID NO: 55) 1470 51
  • the length and molecular weight of the triple block copolypeptides are shown in Table 6 below.
  • Example 4 PCR and vector construction of tyrosinase and orf438 for bacterial co-expression
  • E. coli cells were grown in TB dry medium containing 50 ⁇ g / mL ampicillin.
  • pIJ702 a plasmid containing tyrosinase and S. lividans comprising orf438 were obtained from the American Type Culture Collection (ATCC, 35387). Single colonies of S. lividans were grown at 30 ° C. in R2 YE medium. Plasmids containing both tyrosinase and orf438 were purified from S.lividans.
  • the tyrosinase gene was amplified by polymerase chain reaction (PCR) using primers of pSA-tyr-5p and pSA-tyr-3 '(pSA-tyr-5p (SEQ ID NO: 46): 5′-g gaG GAT CCg acc gtc cgc aag aac cag-3 ′ and pSA-tyr-3 ′ (SEQ ID NO: 47): 5 ′ gga AAG CTT gac gtc gaa ggt gta gtg ccg ⁇ 3 ′. Amplified PCR products were treated with BamI and HindIII.
  • the orf438 gene was amplified by PCR using primers of pSA-438-5 'and pSA-438-3' and the amplified product was treated with EcoRV and KpnI (pSA-438-5 '(SEQ ID NO: 48). ): 5'- c acG ATA TCg ccg gaa ctc acc cgt cgt-3 ', pSA-438-3' (SEQ ID NO: 49): 5'- caa GTT ACC gtt gga ggg gaa ggg gag gag-3 '.
  • the expression vector pACYCDuet-1 plasmid (Merck, Darmstadt, Germany) was also subjected to the same restriction enzyme as the PCR product, and then the cleaved product was introduced. Finally, the DNA sequence was confirmed by DNA sequencing.
  • E. coli BL21 (DE3) cells containing pET21a with block copolypeptides and plasmids with orf438 and pACYC duet of tyrosinase were grown in Circlegrow medium. Colonies were inoculated in 50 mL TB medium supplemented with 50 ⁇ g / ml Ampicillin (Duchefa) and 50 ⁇ g / ml Chloramphenicol (Duchefa) per ml. The preculture was shaken overnight at 37 ° C. and 200 rpm.
  • the precultured medium was inoculated with 500 mL of high nutrition medium (circlegrow) containing 50 ⁇ g / mL of ampicillin and chloramphenicol, and cultured at 37 ° C. and 200 rpm until the OD600 reached 0.6 to 0.8.
  • high nutrition medium (circlegrow) containing 50 ⁇ g / mL of ampicillin and chloramphenicol
  • IPTG Isopropyl- at a final concentration of 1 mM -D-thiogalactopyranoside
  • IPTG 1 mM -D-thiogalactopyranoside
  • cells were obtained by centrifugation at 4 ° C and 4,500 rpm for 10 minutes.
  • the expressed EBPPI-MFP block copolypeptides were purified using inverse transition cycling (ITC).
  • Cell pellets were resuspended in 5% acetic acid containing 8M urea. After sonication (VC-505, Sonic and materials Inc, Danbury, CT) for 10 seconds in an ice bath, cells were destroyed by 30 seconds of cooling (fooling). Cell lysates were centrifuged at 50 ° C. and 13000 rpm for 15 minutes in a 50 mL centrifuge tube to precipitate insoluble debris of the cell lysates. Subsequently, the supernatant containing the water-soluble EBPPI-MFP block copolypeptide was transferred to a new 50 mL centrifuge tube and centrifuged at 4 ° C. and 13000 rpm for 15 minutes to precipitate nucleic acid contaminants.
  • EBPPI-MFP block copolypeptides were aggregated by the salt effect and separated from lysates by centrifugation for 15 minutes at 37 ° C., 13,000 rpm. Aggregated block copolypeptides were resuspended in wells containing 30 mL of sodium acetate buffer (pH 5.0) and 4M urea at 4 ° C. To remove any aggregated protein contaminants, the resuspended protein solution was centrifuged at 4 ° C. and 13,000 rpm for 15 minutes.
  • ITC Inverse transition cycling
  • the aggregation and resuspension treatment was repeated 5 to 10 times until the purity of the block copolypeptide reached about 95%.
  • the purity was measured using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
  • block copolypeptides For the hydroxylation of tyrosine residues of block copolypeptides, block copolypeptides, tyrosinase ( ⁇ 32 kDa) and orf438 ( ⁇ 15 kDa) were co-expressed in soluble form.
  • the recombinant EBPPI [G 1 A 4 F 1 ] 6 -MFP [Mgfp5] 1 double block copolypeptide was prepared from a pET21a vector comprising a double block copolypeptide and a pACYC duet vector comprising tyrosinase and orf438. Co-expressed with tyrosinase and orf438 in E. coli via a dual vector system.
  • tyrosinase and orf438 genes were identified in the amplified tyrosinase and orf438 encoded pACYC duet vectors.
  • Figure 6 (B) shows a double block copolypeptide of (1) bacterial expression of pET21 alone, (2) tyrosinase and orf438 of bacterial expression of pACYC duet alone, and (3) a coexpressed pET21a vector.
  • Tyrosinase and orf438 of copolypeptide and pACYC duet vectors According to the plasmid copy number of each vector, the block copolypeptide in the pET vector has a copy number (-40) that is higher than the copy number (-12) of the tyrosinase and the pACYC duet vector encoding orf438. That is, the block copolypeptide of the pET vector alone was expressed more than the coexpression system.
  • FIG. 6 (C) also shows (1) a triple block copolypeptide of EBPPI [G 1 A 4 F 1 ] 6 -MFP [Mgfp5] 1 -EBPPI [G 1 A 4 F 1 ] 6 alone with pET21, and (2) Tyrosinase and orf438 of the triple-block copolypeptide of pET21a vector and pACYC duet vector were identified.
  • Tyrosine residues of the EBP-MFP block copolypeptide were converted to DOPA via modification by mushroom derived tyrosinase (Sigma Aldrich, T3824) (FIG. 6 (D)).
  • EBP-MFP block copolypeptide was resuspended in 10 mM phosphate buffer supplemented with 10 mM sodium borate and the pH was adjusted to pH 7.0 using ascorbic acid. Then, mushroom-derived tyrosinase at a final concentration of 0.01 mg / ml was added. The solution was gently incubated for 3 hours at room temperature (RT). The tyrosinase-treated EBP-MFP block copolypeptides were phase transitioned at 40 ° C as the temperature increased, and purified by centrifugation at 16,000 rpm for 10 minutes at 40 ° C to remove tyrosinase. It was. Aggregated modified block copolypeptides were resuspended in 10 mM phosphate (pH 5) in an ice bath and the sample was centrifuged at 16,000 rpm for 15 minutes to remove remaining insoluble material.
  • EBP-MFP block copolypeptide was added as a 5% acetic acid (pH 3) solution and lyophilized. This is because quinone, the oxidized form of DOPA, induces intermolecular covalent bonds and reduces interaction with surface molecules in oxidizing conditions.
  • EBPPI [G 1 A 4 F 1 ] 6 -MFP [Mgfp5] 1 double block copolypeptides expressed alone in E. coli were purified by ITC, and tyrosine residues of the double block copolypeptides were mushroom-derived tyrosinase-. It was transformed into DOPA by catalytic reaction.
  • the purity and molecular weight of all block copolypeptides were characterized by SDS-PAGE including copper staining.
  • the phase transition behavior of the block copolypeptides was characterized by UV-visible spectrophotometer.
  • MFPs with different block lengths were fused with EBPPI to form self-assembled nanostructures by thermal stimulation.
  • EBPPI-MFP double block copolypeptides, MFP-EBPPI-MFP triple block copolypeptides, and EBPPI-MFP-EBPPI triple block copolypeptides with concentrations above 12.5 uM have a hydrodynamic radius (R h ) of 20 nm to 40 nm. Eggplants self-assemble into core-shell nanostructures, but the triple block copolypeptides under concentrated conditions formed hydrogels in response to temperature.
  • EBPPI [G 1 A 4 F 1 ] 6 -MFP [Mgfp5] 1 -EBPPI [G 1 A 4 F 1 ] 6 Triple block copolypeptides show copper stained SDS-PAGE photographic images.
  • the MW of the hydroxylated EBPPI [G 1 A 4 F 1 ] 6 -MFP [Mgfp5] 1 double block copolypeptides is similar to the expected MW ( ⁇ 24.6 kDa) and the hydroxylated EBPPI [G 1 A 4 F 1 ] 6 -MFP [Mgfp5] 1 -EBPPI [G 1 A 4 F 1 ] 6
  • the triple block copolypeptides are also similar to the expected MW ( ⁇ 40.1 kDa).
  • Multimerization forms of hydroxylated EBPPI [G1A4F1] 6-MFP [Mgfp5] 1 double block copolypeptides of 48.0 kDa and 92.0 kDa were identified.
  • FIG. 8 (C) shows EBPPI [G 1 A 4 F 1 ] 6 -MFP [Mgfp5] 1 double block copolypeptides treated with various concentrations of oxidizer of NaIO 4 and copper stained SDS in their hydroxylated form. -PAGE image. Treatment with NaIO 4 results in intermolecular crosslinking of unmodified EBPPI [G 1 A 4 F 1 ] 6 -MFP [Mgfp5] 1 double block copolypeptides due to the formation of di tyrosine residues, resulting in SDS The chemically crosslinked double block copolypeptides remain in the wells during PAGE (lanes (2-3) in FIG. 8 (C)).
  • hydroxylated double block copolypeptides were multiplexed due to the formation of di tyrosine residues at neutral pH as well as the formation of quinones induced by DOPA self oxidation. That is, NaIO 4 induced intermolecular crosslinking through the formation of quinone and di tyrosine residues, and no migration of chemically crosslinked hydroxylated double block copolypeptides occurred during SDS-PAGE.
  • the thermal properties of EBPPI [G 1 A 4 F 1 ] 6 -MFP [Mgfp5] 1 double block copolypeptides range from 10 ° C. to 1 ° C./min heating rate in 10 mM phosphate buffer (pH 5, to prevent oxidation). It observed by measuring 350 nm absorbance in the temperature range of 70 degreeC.
  • FIG. 9 (A) shows EBPPI [G 1 A 4 F 1 ] 6 , 25 ⁇ M of mushroom-derived tyrosinase-catalyzed hydroxylation with or without the control EBPPI [G 1 A 4 F 1 ] Thermal profile of 6- MFP [Mgfp5] 1 double block copolypeptide.
  • Monoblock, EBPPI [G 1 A 4 F 1 ] 6 exhibited complete solubility under T t (approx. 45 ° C.), aqua conditions, and abruptly above LCST due to total aggregation of EBPPI [G 1 A 4 F 1 ] 6 Metastasis was shown.
  • EBPPI [G 1 A 4 F 1 ] 6 -MFP [Mgfp5] 1 double block copolypeptides exhibit different thermal reactivity from the monoblock of EBPPI. Since the MFP block makes EBPPI [G 1 A 4 F 1 ] 6 more hydrophobic, the LCST of EBPPI [G 1 A 4 F 1 ] 6 -MFP [Mgfp5] 1 double block copolypeptides was induced by mushroom tyrosinase. It decreased to 35 ° C. regardless of hydroxylation.
  • LCST behavior was analyzed according to the concentration of block copolypeptide and NaIO 4 oxidant.
  • 250 ⁇ M of the hydroxylated EBPPI [G 1 A 4 F 1 ] 6 -MFP [Mgfp5] 1 double block copolypeptide and 10 mM NaIO 4 treated 25 ⁇ M double block copolypeptide exhibited rapid transition of EBP block.
  • 9 (B) and 9 (C) This is due to the formation of quinones induced by DOPA self oxidation and non-covalent interactions of the MFP blocks as well as di tyrosine residues at neutral pH.
  • the aggregation of high concentrations of MFP block is hydrogen bond, Induced by non-covalent interactions such as stacking, electrostatic and hydrophobic interactions.
  • EBPPI [G 1 A 4 F 1 ] 6 -MFP [Mgfp5] 1 double block copolypeptides lower than EBPPI [G 1 A 4 F 1 ] 6 -MFP [Mgfp5] 1 double block copolypeptides. This is because hydrophobic EBPPI blocks were introduced at both ends of the MFP [Mgfp5] 1 intermediate block.
  • the thermal reactivity of the triple block copolypeptides is similar to the EBPPI [G 1 A 4 F 1 ] 6 -MFP [Mgfp5] 1 double block copolypeptides hydroxylated with 10 mM NaIO 4 as shown in FIG. 9 (C).
  • Triple block copolypeptides aggregated at temperatures higher than the LCST of the EBPPI block. This is because EBPPI blocks in double and triple block copolypeptides can form both chemical crosslinking of MFP through physical crosslinking and quinone formation.
  • Example 8 Bulk scale surface adhesion analysis of (1) a block copolypeptide treated with mushroom derived tyrosinase and (2) a hydroxylated block copolypeptide in a co-expression system
  • Hydroxylated double block and triple block copolypeptides were prepared in mushroom derived tyrosinase treated or bacterial co-expression systems.
  • 10 mM phosphate buffer (pH 5) containing 10 mM and 100 mM NaIO 4 was dissolved in 10, 20 and 30% by weight of EBPPI [G1A4F1] 6-MFP [Mgfp5] 1 double block copolypeptides.
  • the adherent surface was rinsed with acetone, ethanol and water.
  • Each block copolypeptide solution was placed directly on the attachment and mixed with 10 mM NaIO 4 oxidant.
  • the deposits were covered with other deposits to form overlapping regions and cured at 4 ° C. for 1 hour.
  • EBPPI [G 1 A 4 F 1 ] 6 -MFP [Mgfp5] 1 -EBPPI [G 1 A 4 F 1 ] 6 triple block copolypeptide (10% by weight) was treated with 10 mM NaIO 4 , the control EBPPI [G 1 A 3 F 2 ] 12 -EBPP [G 1 A 4 F 1 ] 6 -EBPPI [G 1 A 3 F 2 ] 12 It was confirmed that the surface adhesion is stronger than the triple block copolypeptide. This is because the MFP intermediate block has surface adhesion (FIG. 11).
  • Hydroxylated block copolypeptides were prepared in various weight percents in a bacterial co-expression system. First, 10% by weight of EBPPI [G 1 A 4 F 1 ] 6 -MFP [Mgfp5] 1 double block copolypeptide was dissolved in 10 mM phosphate buffer (pH 5). Each block copolypeptide solution was mixed with 10 mM-100 mM NaIO 4 oxidant prior to placing the attachment. Each block copolypeptide was placed on an attachment and then covered with another attachment to form an overlapped region and cured for 1 hour at 4 ° C.
  • the hydroxylation rate of tyrosine residues was increased in the case of hydroxylated double or triple block copolypeptides in a co-expression system, so that the same block copolypeptides would be hydroxylated in a bacterial co-expression system. Not only does it show strong surface adhesion, but it is also useful for industrial scale-up.
  • the properties of the core-shell structure of EBP-MFP block copolypeptides with or without hydroxyl groups were analyzed by dynamic light scattering (DLS) instruments (Malvern instruments, Worcestershire, UK).
  • the hydrodynamic radius (R h ) of the 12.5 ⁇ M block copolypeptide in 10 mM phosphate buffer (pH5) was measured for 11 min after equilibration at 10 o C and 45 o C for 1 min.
  • a rhodamine 6G fluorescent dye with a final concentration of 0.5 w / v% was used.
  • the fluorescent dyes were each (1) nothing in the PCR tube (# PCR-02-C, Axygen), (2) mixed with EBP double block copolypeptides, and (3) EBPPI hydroxylated via coexpression [G 1 A 4 F 1 ] 6- MFP [Mgfp5] 1 diblock copolypeptide mixed at 10 ° C., and (4) EBPPI [G 1 A 4 F 1 ] hydroxylated via coexpression A mixture of 6- MFP [Mgfp5] 1 double block copolypeptide at 40 ° C. was used.
  • Each block copolypeptide is 25 uM dissolved in 10 mM phosphate buffer (pH5).
  • phosphate buffer pH5

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Abstract

La présente invention concerne un co-polypeptide à séquences multiples présentant une bonne sensibilité aux stimuli et une bonne adhérence de surface. Lorsque le co-polypeptide à séquences multiples composé d'un peptide à base d'élastine et d'une protéine de pied de moule, de la présente invention, est utilisé, une structure noyau-enveloppe auto-assemblée et un hydrogel, tous deux pouvant être modifiés de manière réversible en fonction de stimuli de température, peuvent être formés, et le co-polypeptide à séquences multiples présente une adhérence de surface remarquablement bonne, ce qui permet de l'utiliser dans le domaine des applications biomédicales.
PCT/KR2018/004029 2017-04-05 2018-04-05 Copolypeptide à séquences multiples, qui est composé d'un peptide à base d'élastine et d'une protéine de pied de moule et qui présente une bonne sensibilité aux stimuli et une bonne adhérence de surface, son procédé de préparation et son utilisation WO2018186702A1 (fr)

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CN114197068B (zh) * 2021-12-31 2023-01-06 清华大学 一种基于生物工程蛋白的高强蛋白纤维及其制备方法
CN117802137A (zh) * 2024-02-19 2024-04-02 上海交通大学 活细胞内原位自聚合水凝胶的构建及提取方法
CN117802137B (zh) * 2024-02-19 2024-09-06 上海交通大学 活细胞内原位自聚合水凝胶的构建及提取方法

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