WO2016111420A1 - Fonctions de pénétration cellulaire et de stabilisation enzymatique d'un domaine d'hélice a dans une protéine 30kc19 et système d'administration de cargo l'utilisant - Google Patents

Fonctions de pénétration cellulaire et de stabilisation enzymatique d'un domaine d'hélice a dans une protéine 30kc19 et système d'administration de cargo l'utilisant Download PDF

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WO2016111420A1
WO2016111420A1 PCT/KR2015/004297 KR2015004297W WO2016111420A1 WO 2016111420 A1 WO2016111420 A1 WO 2016111420A1 KR 2015004297 W KR2015004297 W KR 2015004297W WO 2016111420 A1 WO2016111420 A1 WO 2016111420A1
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cell
protein
30kc19α
peptide
penetrating peptide
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Korean (ko)
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박태현
김효주
류지나
박희호
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SNU R&DB Foundation
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Seoul National University R&DB Foundation
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    • 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
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • C07K14/43586—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from silkworms
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/46—Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0002—General or multifunctional contrast agents, e.g. chelated agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/10—Organic compounds
    • A61K49/14—Peptides, e.g. proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22

Definitions

  • the present invention relates to cell permeability and enzyme stabilization function of the ⁇ -helix domain (30Kc19 ⁇ ) in the 30Kc19 protein and cargo delivery system using the same. Specifically, since 30Kc19 ⁇ exhibits cell permeability, intracellular stability, or cell permeability and intracellular stability that is comparable to or superior to that of the 30Kc19 full domain, the 30Kc19 ⁇ can effectively deliver cargo into cells by binding to cargo as a cell permeation peptide. .
  • cell membranes of living animals do not pass large biomolecules such as proteins or nucleic acids, and large inorganic materials such as magnetic nanoparticles and quantum dots.
  • biomolecules such as proteins or nucleic acids
  • inorganic materials such as magnetic nanoparticles and quantum dots.
  • nucleic acid delivery method has a disadvantage in that the nucleic acid can be delivered only to a very limited number of cells, and its efficiency varies greatly depending on the type of cells.
  • proteins do not have efficient intracellular delivery methods that have been proven to date.
  • TAT protein derived from Human Immunodeficiency Virus-1 (HIV-1), which consists of 11 amino acids 47 to 57 in a 86 amino acid TAT protein.
  • HSV-1 Human Immunodeficiency Virus-1
  • Similar examples include amino acids 267 to 300 of the VP22 protein of HSV-1 (Herpes Simplex Virus type 1) [Elliott G. et al.
  • U.S. Patent Publication Nos. 20050112640 and 20050282281 disclose a vector comprising a gene of TAT PTD, by which TAT is expressed by binding to a desired protein.
  • U.S. Patent Publication No. 20080166755 also discloses a technique for binding a protein that assists bone formation with TAT and delivering it to bone producing cells.
  • protein delivery using TAT is described in various patents.
  • 20060182736 also discloses intracellular delivery of proteins using PTDs of other cell penetrating proteins such as VP22 and Antp in addition to TAT.
  • PTDs of other cell penetrating proteins such as VP22 and Antp
  • the prior arts are cell delivery technologies of large molecules such as proteins.
  • TAT and VP22 are derived from viruses that are very harmful to the human body, and full-length TAT is known to induce cell death.
  • TAT and VP22 are derived from viruses that are very harmful to the human body, and full-length TAT is known to induce cell death.
  • the inventors have identified that the ⁇ -helix domain of 30Kc19 protein derived from silkworm ( Bombyx mori ) and known cell permeation function is PTD (protein transduction domain) which is a cell permeable part, and the ⁇ -helix domain of 30Kc19 protein is 30Kc19.
  • the present invention was completed by confirming that there is a cell permeability, intracellular stability, or cell permeability and intracellular stability that is comparable to or even better than that of the 30Kc19 protein.
  • a cell penetrating peptide comprising an ⁇ -helix domain (30Kc19 ⁇ ) of a 30Kc19 protein.
  • the 30Kc19 ⁇ is characterized by having cell permeability, intracellular stability, or cell permeability and intracellular stability that are comparable to or greater than the 30Kc19 full domain.
  • the 30Kc19 ⁇ is characterized in that it is derived from a silkworm ( Bombyx mori).
  • the 30Kc19 ⁇ is characterized in that consisting of the first to 88th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 1.
  • a cargo delivery system comprising a 30Kc19 ⁇ cell penetrating peptide and a cargo fused to the N-terminus or C-terminus of the cell penetrating peptide.
  • the cargo is characterized in that the protein, nucleic acids, peptides, lipids, glycolipids, minerals, sugars, contrast agents, drugs or chemicals.
  • the peptide is characterized in that it is a cytokine, an antibody, an antibody fragment, a therapeutic enzyme or a soluble receptor.
  • the contrast material is characterized in that the radiopaque contrast material, paramagnetic contrast material, superparamagnetic contrast material or CT contrast material.
  • a method for tracking the route of application drug delivery in a subject comprising applying to the subject a 30Kc19 ⁇ cell penetrating peptide and a contrast agent.
  • the 30Kc19 ⁇ is characterized in that it consists of the 1st to 88th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 1.
  • intracellular drug delivery to a subject comprising a composition comprising a 30Kc19 ⁇ cell penetrating peptide and a drug and an indication that discloses one or more of the dosage, route of administration, frequency of administration and indications of the composition
  • a kit for use is disclosed.
  • the 30Kc19 ⁇ is characterized in that consisting of the first to 88th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 1.
  • a drug, cosmetic or food composition having an excellent effect of delivering an active ingredient into a cell, including a 30Kc19 ⁇ cell penetrating peptide, is disclosed.
  • the 30Kc19 ⁇ is characterized in that consisting of the first to 88th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 1.
  • 30Kc19 ⁇ exhibits cell permeability, intracellular stability, or cell permeability and intracellular stability that are comparable to or greater than the 30Kc19 full domain, and thus can effectively deliver cargo into cells by binding to cargo as a cell permeation peptide. have.
  • 1 shows the structure of ⁇ -helix domain and ⁇ -sheet domain of 30Kc19 protein.
  • Figure 2 shows the results of Western blotting analysis of ⁇ -helix domain and ⁇ -sheet domain.
  • Figure 3 shows that the ⁇ -helix domain acts as a protein trasnduction domain (PTD) in 30Kc19 protein and is produced as a water-soluble protein.
  • PTD protein trasnduction domain
  • Figure 4 shows the results of SDS-PAGE analysis of 30Kc19 ⁇ according to Example 1.
  • FIG. 5 shows the results of SDS-PAGE analysis of 30Kc19 according to Comparative Example 1.
  • Figure 6 shows the results of Western blotting analysis of 30Kc19 ⁇ protein entered into the cell according to Experimental Example 1 (1).
  • Figure 7 shows the results of fluorescence image analysis of 30Kc19 ⁇ into the cell according to Experimental Example 1 (2).
  • FIG. 9 shows the results of spectrophotometer analysis of 30Kc19 and 30Kc19 ⁇ according to Experimental Example 2.
  • Figure 10 shows the results of the spectrofluorometer analysis with the addition concentration and time of 30Kc19 ⁇ according to Experimental Example 2.
  • the numerical range includes the numerical values defined in the range. All maximum numerical limits given throughout this specification include all lower numerical limits as if the lower numerical limits were clearly written. All minimum numerical limits given throughout this specification include all higher numerical limitations as if the higher numerical limit were clearly written. All numerical limitations given throughout this specification will include all better numerical ranges within the broader numerical range, as the narrower numerical limitations are clearly written.
  • the headings provided herein are not to be construed as limiting the following embodiments, as a reference to the specification in various aspects or as a whole.
  • the present invention is to provide a cell permeation peptide consisting of the ⁇ -helix domain of the 30Kc19 protein (hereinafter referred to as "30Kc19 ⁇ ”) and cargo delivery system using the same.
  • Proteins, nucleic acids, peptides, or viruses have great potential as therapeutic substances, but because of their molecular size, they have not been able to penetrate tissues and cell membranes, so their use is very limited. In addition, even small-sized substances are often unable to penetrate the lipid bilayer of the cell membrane due to their properties and structure. Accordingly, attempts to move proteins, nucleic acids, peptides, or viruses into cells by electroporation or heatshock, etc., but do not damage the cell membrane and at the same time maintaining the activity of the substances intact It was difficult to move the substances into the cell. In the meantime, 30Kc19 protein derived from silkworm ( Bombyx mori ) has been known that can act as a cell-penetrating peptide that can move the large active substances into the cell, the research has been actively conducted.
  • silkworm Bombyx mori
  • the amino acid sequence of 30Kc19 protein derived from silkworm ( Bombyx mori ) is shown in SEQ ID NO: 1.
  • the 30Kc19 protein is largely composed of two domains, an ⁇ -helix domain (1-88 amino acids of SEQ ID NO: 1) and a ⁇ -sheet domain (89-239 amino acids of SEQ ID NO: 1) (Fig. 1).
  • PTD protein transduction domain
  • the ⁇ -helix domain of the 30Kc19 protein according to the present invention may have cell permeability that is comparable to that of the 30Kc19 protein or better than that of the 30Kc19 protein (Experimental Example 1).
  • the ⁇ -helix domain of the 30Kc19 protein according to the present invention may have a stability equivalent to that of the 30Kc19 protein or better than that of the 30Kc19 protein (Experimental Examples 2 and 3).
  • a 30Kc19 ⁇ cell penetrating peptide is disclosed.
  • the cell penetrating peptide may be a peptide consisting of the 1st to 88th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 1 or a peptide having a sequence homology of 80% or more with the amino acid sequence.
  • the cell penetrating peptide is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, 97 with a peptide consisting of the first to 88th amino acid sequences of the amino acid sequence represented by SEQ ID NO: 1 Peptides having at least%, at least 98%, or at least 99% sequence homology.
  • the cell penetrating peptide may include a peptide represented by SEQ ID NO: 1 or a peptide in which the sequence and one or more amino acids, three or more amino acids, five or more amino acids, ten or more amino acids, or fifteen or more amino acids are changed. . Since the peptide represented by SEQ ID NO: 1 or a peptide having a sequence homology of 80% or more with the sequence may act as a safe and excellent cell penetrating peptide, it may be combined with cargo to effectively deliver cargo into cells.
  • CPP Cell Penetrating Peptide
  • amino acid includes D-isomers and modified amino acids as well as 22 standard amino acids that are naturally incorporated into the peptide.
  • the peptide may comprise non-standard amino acids that have been post-translational modified.
  • Post-translational modifications include phosphorylation, glycosylation, acylation (eg, acetylation, myristoylation and palmitoylation), alkylation, Carboxylation, hydroxylation, glycation, biotinylation, ubiquitinylation, changes in chemical properties (e.g., beta-elimination deimidization) , Deamidation) and structural changes (eg, formation of disulfide bridges).
  • the peptide may be a wild type peptide identified and isolated from a natural source.
  • the peptide may be an artificial variant, comprising an amino acid sequence in which one or more amino acids are substituted, deleted and / or inserted.
  • Amino acid changes in wild type polypeptides as well as artificial variants include conservative amino acid substitutions that do not significantly affect the folding and / or activity of the protein.
  • the conservative substitutions include basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, valine and methionine), aromatic amino acids (Phenylalanine, tryptophan and tyrosine) and small amino acids (glycine, alanine, serine and threonine). Amino acid substitutions that generally do not alter specific activity are known in the art.
  • the most common exchanges are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu and Asp / Gly.
  • Other examples of conservative substitutions are shown in Table 1 below.
  • a cargo delivery system comprising a 30Kc19 ⁇ cell penetrating peptide and cargo fused to the N-terminus or C-terminus of the cell penetrating peptide.
  • the cell penetrating peptide may be a peptide consisting of the 1st to 88th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 1 or a peptide having a sequence homology of 80% or more with the amino acid sequence.
  • the term "cargo” includes all substances capable of binding into a cell by binding to the 30Kc19 ⁇ cell permeable peptide according to the present invention, for example, any substance that wants to increase cell permeation efficiency, specifically, As an effective substance in drugs, cosmetics or health foods, more specifically, substances that are not easily transported into cells through general routes, and more specifically, proteins, nucleic acids, peptides, minerals, sugars, including nanoparticles, biological It may include, but is not limited to, agents, viruses, contrast agents or other chemicals.
  • carrier peptide refers to a peptide that serves to bind the active ingredient to move the active ingredient to a desired site.
  • the term "drug” is a broad concept that includes substances for alleviating, preventing, treating or diagnosing a disease, wound or particular condition.
  • the drug delivered into the cell by the cell permeable peptide may further comprise a drug carrier such as liposomes, micelles, nanoparticles, magnetic particles or quantum dots.
  • peptide as used herein may include, but is not limited to, hormones, hormone analogs, enzymes, enzyme inhibitors, signal transduction proteins (or peptides), antibodies or vaccines.
  • the nucleic acid may be naturally occurring or artificial DNA or RNA molecules, and may be single stranded or double stranded.
  • the nucleic acid molecule may be one or more, which may be nucleic acid molecules of the same type (eg, having the same nucleotide sequence), or may be nucleic acid molecules of other types.
  • RNA RNA
  • siRNA miRNA
  • shRNA stRNA
  • snoRNA snRNA
  • PNA antisense oligomer
  • plasmid plasmid and other modified nucleic acids It is not.
  • contrast material means any material used for the imaging of structures or fluids in vivo in medical imaging.
  • the contrast material may include a radiopaque contrast agent, a paramagnetic contrast agent, a superparamagnetic contrast agent, a computed tomography (CT) contrast medium, or other contrast materials. It is not limited to this.
  • radiopaque contrast materials for X-ray imaging
  • inorganic iodine compounds and organic iodine compounds e.g., diatrizoat
  • radiopaque metals and salts thereof e.g., silver, gold, platinum Etc.
  • other radiopaque compounds e.g, calcium salts, barium salts such as barium sulfate, tantalum and tantalum oxide.
  • Paramagnetic contrasting materials include gadolinium diethylene triaminepentaacetic acid (Gd-DTPA) and derivatives thereof, and other gadolinium, manganese, iron, dysprosium, copper, and europium (europium), erbium, chromium, nickel and cobalt complexes such as 1,4,7,10-tetraazacyclododecane-N, N ', N ", N"'-tetraacetic acid ( DOTA), ethylenediaminetetraacetic acid (EDTA), 1,4,7,10-tetraazacyclododecane-N, -N ', N "-triacetic acid (D03A), 1,4,7-triazacyclononane -N, N ', N "-triacetic acid (NOTA), 1,4,8,10-tetraazacyclotetradecane-N, N', N", N "'-tetraacetic acid (TETA), hydroxybenzy
  • Superparamagnetic contrast agents may include magnetite, super-paramagnetic iron oxide (SPIO), ultrasmall superparamagnetic iron oxide (USPIO) and monocrystalline iron oxide (monocrystailine). Can be.
  • Other suitable contrast agents include iodinated and non-iodinated, ionic and nonionic CT contrast agents, and contrast agents such as spin-labels, or other diagnostically effective agents.
  • the contrast material may include ⁇ -galactosidase, green fluorescent protein, blue fluorescent protein or luciferase. When expressed in a cell, it may comprise a marker gene encoding a protein that is readily detectable.
  • Various labels may be used, such as radionuclides, fluorescents, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, and the like.
  • the cargo according to the present invention is a protein or a peptide
  • the transport peptide and the transport peptide in the form of a fusion protein of the transport target and the peptide The subject can be combined.
  • Specific examples of the binding by the fusion protein are as follows: In preparing a primer for producing the fusion protein, the nucleotide encoding the carrier peptide is attached to the nucleotide expressing the moving target, and then the obtained nucleotide is converted into a restriction enzyme.
  • the pET vector is inserted into an example vector, and BL-21 (DE3) is introduced into an example cell and transformed.
  • an expression inducer such as IPTG (isopropyl-1-thio- ⁇ -D-galactopyranoside) may be treated to effectively express the fusion protein.
  • IPTG isopropyl-1-thio- ⁇ -D-galactopyranoside
  • the fusion protein expressed through a method such as His tag purification (purification) is purified, and then dialyzed using PBS, and concentrated in a kit, for example, by centrifugation for 5 to 20 minutes at 2,000 to 4,000 rpm.
  • the carrier peptide may bind to a dyeing or fluorescent material, specifically fluorescein isothiocyanate (FITC) or Green Fluorescent Protein (GFP).
  • FITC fluorescein isothiocyanate
  • GFP Green Fluorescent Protein
  • a method for tracking a drug delivery route in a subject comprising applying the 30Kc19 ⁇ cell penetrating peptide and a contrast agent to the subject is disclosed.
  • the cell penetrating peptide may be a peptide consisting of the 1st to 88th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 1 or a peptide having a sequence homology of 80% or more with the amino acid sequence.
  • composition Kits for intracellular drug delivery to a subject comprising a composition comprising a 30Kc19 ⁇ cell penetrating peptide and a drug and an indication that discloses one or more of the dosage, route of administration, frequency of administration and indications of the composition Kits are disclosed.
  • a drug, cosmetic or food composition having an excellent effect of delivering an active ingredient into a cell, including a 30Kc19 ⁇ cell penetrating peptide is disclosed.
  • the composition according to the present invention is 0.2 mg / ml to 2 mg / ml of a peptide consisting of the 1 to 88 amino acid sequence of the amino acid sequence represented by SEQ ID NO: 1 or a peptide having a sequence homology of 80% or more with the amino acid sequence, Or 0.3 mg / ml to 1.5 mg / ml, or 0.4 mg / ml to 1.0 mg / ml. Specifically, it may include 1 ⁇ g / mg to 0.5 mg / mg, more specifically 10 ⁇ g / mg to 0.1 mg / mg. When included in the above range is not only suitable for showing the intended effect of the present invention, it can satisfy both the stability and safety of the composition, it may be appropriate to include in the above range in terms of cost-effectiveness.
  • composition according to the invention can be applied to all animals, including humans, dogs, chickens, pigs, cattle, sheep, guinea pigs or monkeys.
  • the pharmaceutical composition may be administered orally, rectally, transdermal, intravenous, intramuscular, intraperitoneal, intramedullary, intradural or subcutaneous.
  • Formulations for oral administration may be, but are not limited to, tablets, pills, soft or hard capsules, granules, powders, solutions or emulsions.
  • Formulations for parenteral administration may be, but are not limited to, injections, drops, lotions, ointments, gels, creams, suspensions, emulsions, suppositories, patches or sprays.
  • the pharmaceutical composition may include additives such as diluents, excipients, lubricants, binders, disintegrants, buffers, dispersants, surfactants, colorants, flavoring or sweetening agents as necessary.
  • Cosmetic compositions according to the invention may be provided in all formulations suitable for topical application.
  • it may be provided in the form of a solution, an emulsion obtained by dispersing an oil phase in an aqueous phase, an emulsion obtained by dispersing an aqueous phase in an oil phase, a suspension, a solid, a gel, a powder, a paste, a foam, or an aerosol.
  • Such formulations may be prepared according to conventional methods in the art.
  • the cosmetic composition may include other ingredients that can give a synergistic effect to the main effect, preferably within a range that does not impair the main effect.
  • the cosmetic composition may further include a moisturizer, an emulsifier, a surfactant, a UV absorber, a preservative, a fungicide, an antioxidant, a pH adjuster, an organic or inorganic pigment, a perfume, a cooling agent or a restriction agent.
  • a moisturizer an emulsifier, a surfactant, a UV absorber, a preservative, a fungicide, an antioxidant, a pH adjuster, an organic or inorganic pigment, a perfume, a cooling agent or a restriction agent.
  • the formulation of the food composition according to the present invention is not particularly limited, but may be, for example, formulated into tablets, granules, powders, solutions, solid preparations, and the like. Each formulation may be appropriately selected and blended by those skilled in the art according to the formulation or purpose of use, in addition to the active ingredient, and may be synergistic when applied simultaneously with other raw materials.
  • the 30Kc19 ⁇ gene from Bombyx mori was inserted at the site between EcoR I and Xho I of pET23a vector (Novagen).
  • the plasmid thus obtained was transformed into E. coli BL21 (DE3), and only the E. coli clone into which the plasmid was inserted was selected using ampicillin selection.
  • the histidine tag (His6-tag) sequence was included in the carboxyl terminal of 30Kc19 ⁇ produced to facilitate purification.
  • IPTG isopropyl-beta-di-thiogalactopyranoside
  • Example 2 The same method as in Example 1 was used except that 30Kc19 gene was used instead of 30Kc19 ⁇ gene (FIG. 5). As a result of analyzing the 30Kc19 by SDS-PAGE, a band corresponding to 30Kc19 was observed at about 35kDa as can be seen from FIG. 5.
  • HeLa cells and CHO cells were cultured in DMEM medium (Hyclone) with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin streptomycin. When the cells occupied about 70% of the bottom surface, they were replaced with fresh medium added with 30 Kc19 ⁇ at concentrations of 0 mg / ml, 0.5 mg / ml and 1.0 mg / ml and incubated at 37 ° C. for 4 hours. To identify 30Kc19 delivered to the cells, cells were first washed three times with PBS and then removed from the bottom of the plate using 1% trypsin solution. Cells detached by centrifugation were obtained and disrupted with RIPA buffer.
  • DMEM medium Hyclone
  • FBS fetal bovine serum
  • the supernatant obtained through centrifugation was subjected to 12% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, the gel was transferred to nitrocellulose membrane and then blocked with 5% skim milk. Then, 30Kc19 ⁇ delivered to the cells was detected by Western blotting using 30Kc19 ⁇ antibodies obtained from rabbits. The results are shown in FIG.
  • 30Kc19 ⁇ protein was labeled with green fluorescent Alexa Fluor® 488 (Invitrogen). Fluorescence bound 30Kc19 ⁇ protein was added to HeLa cells, incubated at 37 ° C. for 4 hours, and washed three times with PBS. Nuclei stained with Hoechst on the washed cells and the cells were observed using a confocal microscope (Olympus, Japan) in the live cell image in the living state. The results of analyzing the intracellular location according to the concentration of 30Kc19 ⁇ protein (0 mg / ml, 0.2 mg / ml and 0.4 mg / ml) through fluorescence images are shown in FIG. 7. The results of staining the nuclei at each concentration and the combined image of 30Kc19 ⁇ and nuclei were shown.
  • the enzyme stability of 30Kc19 ⁇ protein was evaluated using Green Fluorescent Protein (GFP).
  • GFP Green Fluorescent Protein
  • the GFP gene was inserted into the EcoR 1 and Xho 1 sites of the pET23a vector (Invitrogen) to obtain a vector.
  • E. coli BL21 (DE3) strain transformed using the plasmid was incubated in LB medium, followed by incubation at 27 ° C. for 6 hours by adding 1 mM IPTG to produce GFP protein. The cells collected by centrifugation were then sonicated. This was obtained by purification using His Trp HP column (5ml, GE Healthcare).
  • GFP was incubated at 37 ° C. for 24 hours in the presence or absence of 400 ug / ml of 30Kc19 ⁇ protein and 30Kc19 protein cultured in Experimental Example 1, and the activity of GFP was measured. The results are shown in FIG.
  • Beta galactosidase was transfected into lipofectamine3000 in HeLa cells, and after 24 hours, 30Kc19 ⁇ protein was treated with 0 ⁇ g / ml, 40 ⁇ g / ml, 80 ⁇ g / ml, 120 ⁇ g / ml and 200 ⁇ g / ml, respectively.
  • ⁇ -gal assay was performed after 24 hours, and the results are shown in FIG. 10.
  • the 30Kc19 ⁇ protein was stable in the cell.
  • the 30Kc19a protein is 80 ⁇ g / ml concentration showed the highest stability.

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  • Tropical Medicine & Parasitology (AREA)
  • Radiology & Medical Imaging (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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  • Engineering & Computer Science (AREA)
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  • Peptides Or Proteins (AREA)
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Abstract

La présente invention concerne un peptide de pénétration cellulaire 30Kc19α et un système d'administration de cargo l'utilisant. Plus particulièrement, le peptide 30Kc19α selon la présente invention présente une pénétration cellulaire, une stabilité intracellulaire ou une pénétration cellulaire et une stabilité intracellulaire qui sont similaires ou supérieures au domaine entier de la 30Kc19 et, par conséquent, le peptide 30Kc19α, en tant que peptide de pénétration cellulaire, se lie à un cargo, ce qui permet d'administrer efficacement le cargo dans des cellules.
PCT/KR2015/004297 2015-01-07 2015-04-29 Fonctions de pénétration cellulaire et de stabilisation enzymatique d'un domaine d'hélice a dans une protéine 30kc19 et système d'administration de cargo l'utilisant Ceased WO2016111420A1 (fr)

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KR1020150001973A KR101626343B1 (ko) 2015-01-07 2015-01-07 30Kc19 단백질 내 α－ｈｅｌｉｘ 도메인의 세포 투과성과 효소 안정화 기능 및 이를 이용한 카고 전달 시스템
KR10-2015-0001973 2015-01-07

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WO2021194257A1 (fr) * 2020-03-25 2021-09-30 (주) 업테라 Dimère peptidique perméable aux cellules, son procédé de préparation et système de distribution de cargo l'utilisant
KR102195740B1 (ko) 2020-03-25 2020-12-28 서울대학교산학협력단 세포 투과성 펩티드 이량체, 이의 제조방법 및 이를 이용한 카고 전달 시스템
US11358992B2 (en) 2020-04-17 2022-06-14 Uppthera Cell-penetrating cereblon recombinant fusion protein and use thereof
KR102185390B1 (ko) 2020-04-17 2020-12-01 (주) 업테라 세포 투과성 세레블론 재조합 융합 단백질 및 이의 용도

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CN110372780A (zh) * 2019-07-08 2019-10-25 吉林大学 抗肿瘤多肽及其在抗肿瘤领域的应用

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