WO2016018134A1 - Polypeptide for delivering biomolecules into cells, and use of same - Google Patents

Polypeptide for delivering biomolecules into cells, and use of same Download PDF

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WO2016018134A1
WO2016018134A1 PCT/KR2015/008109 KR2015008109W WO2016018134A1 WO 2016018134 A1 WO2016018134 A1 WO 2016018134A1 KR 2015008109 W KR2015008109 W KR 2015008109W WO 2016018134 A1 WO2016018134 A1 WO 2016018134A1
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polypeptide
domain
cell
cells
seq
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PCT/KR2015/008109
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French (fr)
Korean (ko)
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김학성
유정현
조성민
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한국과학기술원
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    • 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
    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression

Definitions

  • the present invention relates to a polypeptide for delivering a useful biomolecule into a cell, and more particularly, a polypeptide for delivering various molecules into a cell of a eukaryotic cell, a polynucleotide encoding the polypeptide, and a vector comprising the polynucleotide.
  • the present invention relates to a method for producing the polypeptide by culturing the recombinant main cell transformed with the vector and the recombinant host cell.
  • the cell membranes of animal cells do not pass many biomolecules. Although various biomolecules are used in fields such as basic science and medicine, the use of biomolecules inside cells has been limited due to cell membranes.
  • Protein is an important substance that is widely used in basic science and medicine fields, and can have a much more diverse and delicate function than a small molecule substance.
  • proteins do not cross cell membranes, there are significant limitations in their use in the cytoplasm.
  • Protein therapeutics are being actively developed for the effective treatment of diseases including cancer, but most of them are limited to disease targets outside the cell because proteins cannot cross the cell membrane. However, since there are more disease targets in cells, the importance of developing protein therapeutics for them is of great importance. Therefore, in order to develop a protein therapeutic agent having a high therapeutic effect against a wide range of diseases, development of a technology capable of safely delivering a therapeutic protein that targets intracellular disease targets into specific cells is required first.
  • PTD protein transduction domains
  • cell penetrating peptides such as cell penetrating peptides
  • cell penetrating peptides are conjugated to proteins and transferred into cells, or foreign proteins are attached to nanoparticles and transferred into cells, but low efficiency and cell specificity are the biggest. It remains a problem.
  • the present inventors have developed a polypeptide that can safely and efficiently deliver foreign biomolecules into specific cells based on the cell permeation domain of the toxin protein produced by bacteria, and completed the present invention.
  • An object of the present invention is to provide a polypeptide capable of safely and efficiently delivering biomolecules into a specific cell, a polynucleotide encoding the polypeptide, a vector comprising the polynucleotide, a recombinant microorganism into which the vector is introduced, and the recombinant microorganism. It provides a method for producing the polypeptide by culturing.
  • Another object of the present invention is to provide a composition for intracellular delivery containing the polypeptide and a method for intracellular delivery of biomolecules using the composition.
  • FIG. 1 is a schematic diagram of the present invention, which illustrates the polypeptide consisting of three domains and a endoplasmic reticulum residue sequence.
  • FIG. 2 is a schematic diagram illustrating the crystallization structure of a polypeptide that recognizes a Gb3 cell membrane receptor based on the basic concept of FIG. 1 and delivers eGFP into a cell.
  • FIG. 3 is a confocal laser scanning micrograph showing successful delivery of eGFP into Gb3-positive cells using the polypeptide of FIG. 2.
  • FIG. 4 is a schematic diagram illustrating the crystallization structure of a polypeptide that recognizes an EGFR cell membrane receptor based on the basic concept of FIG. 1 and delivers an eGFP into a cell.
  • FIG. 5 is a confocal laser scanning micrograph of eGFP delivered into EGFR positive and negative cells using the polypeptide of FIG. 4.
  • FIG. 6 is a graph illustrating the successful delivery of luciferase by a polypeptide that recognizes a Gb3 cell membrane receptor based on the basic concept of FIG. 1 and delivers luciferase into a cell.
  • FIG. X axis hour
  • Y axis relative activity of intracellular luciferase
  • blue the polypeptide
  • green polypeptide with domain 2 removed
  • red polypeptide with inactive B-subunit bound.
  • a polypeptide is prepared by using domain 2 of a toxin protein produced by P. aeruginosa as a cell permeation domain and binding a receptor recognition domain and a cargo domain to the N and C ends of the cell permeation domain, respectively. It was.
  • the present invention provides a biomolecule into a cell, wherein the cell permeation domain is a basic skeleton, and the receptor recognition domain and the cargo domain are respectively coupled to the N and C ends of the cell permeation domain. It relates to a polypeptide that can be delivered.
  • the "cell permeation domain” refers to a portion that allows the biomolecule to enter the cell through the cell membrane.
  • the domain may use a polypeptide derived from a bacterial toxin protein.
  • a part of Pseudomonas Exotoxin A, a part of Shiga like toxin of E. coli can be used.
  • the "receptor recognition domain” means a portion that recognizes a specific receptor in the cell membrane of the cell to be delivered.
  • the domain may be a variety of molecules that can recognize a specific receptor on the cell membrane, including the polypeptide.
  • natural proteins such as EGF, IL-6, Fc, monoclonal antibodies (scFv), lipids (Repebody), artificial proteins such as DARPin, Monobody, Nanobody, small molecule ligands such as RGD, folate, etc. can be used. have.
  • “cargo domain” refers to a biomolecule to be delivered into the cell.
  • the domain may be conjugated to various C-terminal end of the cell permeation domain in a variety of ways.
  • a nucleotide of a foreign protein can be used to be conjugated to the nucleotide of the polypeptide to produce one binding polypeptide form.
  • the cysteine cysteine
  • maleimide maleimide
  • biomolecules to be delivered may be targets for therapeutic and diagnostic proteins, single-stranded nucleic acids, double-stranded nucleic acids, small molecule drugs, and the like.
  • the polypeptide according to the present invention may be characterized in that the endoplasmic reticulum residue sequence is further connected to the C terminal of the cargo domain (biomolecule) in order to increase the intracellular delivery efficiency of the biomolecule.
  • the endoplasmic reticulum means a sequence that allows the polypeptide to remain in the endoplasmic reticulum of the cell to help foreign molecules penetrate into the cell.
  • the endoplasmic reticulum sequences include sequences such as KDEL and REDLK to help foreign molecules remain in the endoplasmic reticulum.
  • the N-terminal and the receptor recognition domain of the domain 2 may be connected by a linker represented by the amino acid sequence of SEQ ID NO: 4, but is not limited thereto.
  • the C terminal and the cargo domain of the domain 2 may be connected by a linker represented by the amino acid sequence of SEQ ID NO: 5, but is not limited thereto.
  • nucleic acids encoding the respective domains were amplified by PCR and then introduced into E. coli.
  • B-Subunit of cigatoxin was used as the N-terminal receptor recognition domain of domain 2
  • eGFP was used as the C-terminal cargo domain.
  • the expressed polypeptide was purified using Ni-NTA column and gel permeation chromatography (GPC) (SEQ ID NO: 10). 1 ⁇ M of the purified polypeptide was treated in HepG2 (KCTC, No. HC18302) and Vero cell (KCTC, No. AC28810) for 3 hours, and then confirmed by confocal laser scanning microscope. As a result, eGFP was delivered into the cells. It was confirmed that (Fig. 3).
  • hEGF was used as the receptor recognition domain, leaving the cargo domain intact to prove that the receptor recognition domain was interchangeable.
  • the completed gene was inserted into a pET-based vector for expression, and then introduced into E. coli and expressed.
  • the expressed polypeptide was purified using Ni-NTA column and gel permeation chromatography (GPC) (SEQ ID NO: 11). 1 ⁇ M of the purified polypeptide was treated with Hcc827 (ATCC, No. CRL-2868) and Vero cells for 6 hours, and then confirmed by confocal laser scanning. As a result, eGFP was delivered only in Hcc827 cells expressing EGFR. It could be confirmed that (Fig. 5).
  • renilla luciferase was used as the cargo domain while leaving the receptor recognition domain intact to prove that the cargo domain was replaceable.
  • the completed gene was inserted into a pET-based vector for expression, and then introduced into E. coli and expressed.
  • the expressed polypeptide was purified purely using Ni-NTA column and gel permeation chromatography (GPC) (SEQ ID NO: 12).
  • GPC gel permeation chromatography
  • the purified polypeptide was tested using HepG2 cells. As a result of measuring the activity of intracellular luciferase, it was confirmed that a sufficient amount of luciferase was delivered into the cell in 1 hour (FIG. 6).
  • the present invention a polynucleotide encoding the polypeptide;
  • the present invention relates to a recombinant microorganism into which the polynucleotide or the recombinant vector is introduced.
  • the polynucleotide means that thousands or more of the nucleotides, which are a unit in which sugar, phosphate, and base are bonded to each other. Nucleotides form long chains with diester bonds. At this time, the skeleton of sugar and phosphoric acid enters repeatedly, and the order of bases is irregularly arranged between. Irregularities and lengths of base placement produce key information that enters the gene. Polynucleotides are directional, with one end of the chain 5 'end and the other end of the chain necessarily 3' end. At this time, the nature of the terminal 5 ⁇ -phosphate group, 3 ⁇ -hydroxy group is displayed together. Both DNA and RNA are in polynucleotide form. Polynucleotides play an important role in protein biosynthesis.
  • vector refers to a DNA preparation containing a nucleotide sequence of a polynucleotide encoding the target protein operably linked to a suitable regulatory sequence so that the target protein can be expressed 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 the phage vector or cosmid vector
  • pBR, pUC, and pBluescriptII systems are used as plasmid vectors.
  • pGEM-based, pTZ-based, pCL-based and pET-based and the like can be used.
  • the vector usable in the present invention is not particularly limited and known expression vectors can be used.
  • pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vector and the like can be used.
  • pACYC177, pCL, pCC1BAC vectors can be used.
  • a "recombinant microorganism” means that a vector having a polynucleotide encoding at least one target protein is introduced into a host cell, or a polynucleotide encoding at least one target protein is introduced at a microorganism so that the polynucleotide is incorporated into a chromosome.
  • a cell infected with a trait to express a protein and may be any cell, such as eukaryotic cells, prokaryotic cells, etc., but is not particularly limited thereto, bacterial cells such as E.
  • coli coli, Streptomyces, Salmonella typhimurium
  • Yeast cells Fungal cells such as Pchia pastoris; Insect cells such as Drozophila and Spodoptera Sf9 cells
  • Animal cells such as CHO, COS, NSO, 293, Bow Melanoma cells, and the like.
  • transformation refers to introducing a vector containing a polynucleotide encoding a target protein into a host cell or integrating a polynucleotide encoding a target protein into a chromosome of the host cell to complete the polynucleotide in the host cell. It means that the encoding protein can be expressed. Transformed polynucleotides include all of them, as long as they can be expressed in the host cell, whether they are inserted into or located outside the chromosome of the host cell. In addition, the polynucleotides include DNA and RNA encoding the target protein.
  • the polynucleotide may be introduced in any form as long as it can be expressed by being introduced into a host cell.
  • the polynucleotide may be introduced into a host cell in the form of an expression cassette, which is a gene construct containing all elements necessary for self-expression.
  • the expression cassette typically includes a promoter, transcription termination signal, ribosomal binding site and translation termination signal operably linked to the polynucleotide.
  • the expression cassette may be in the form of an expression vector capable of self replication.
  • the polynucleotide may be introduced into a host cell in its own form and operably linked to a sequence required for expression in the host cell.
  • the present invention provides a method for producing a polypeptide comprising: (a) culturing the recombinant microorganism to produce a polypeptide; And (b) relates to a method for producing a polypeptide that can deliver the biomolecule into the cell comprising the step of recovering the generated polypeptide.
  • the step of culturing the recombinant microorganism is not particularly limited thereto, but is preferably performed by a known batch culture method, continuous culture method, fed-batch culture method, and the like, and the culture conditions are not particularly limited thereto.
  • the culture conditions are not particularly limited thereto.
  • sodium hydroxide, potassium hydroxide or ammonia or acidic compounds (e.g. phosphoric acid or sulfuric acid) can be used to adjust the appropriate pH (pH 5-9, preferably pH 6-8, most preferably pH 6.8).
  • the incubation temperature can be maintained at 20 to 45 °C, preferably 25 to 40 °C, incubated for about 10 to 160 hours This is preferable.
  • the polypeptide produced by the culture may be secreted into the medium or remain in the cell.
  • the culture medium used may include sugars and carbohydrates (e.g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose), fats and fats (e.g. soybean oil, sunflower seeds) as carbon sources.
  • sugars and carbohydrates e.g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose
  • fats and fats e.g. soybean oil, sunflower seeds
  • fatty acids e.g. palmitic acid, stearic acid and linoleic acid
  • alcohols e.g. glycerol and ethanol
  • organic acids e.g. acetic acid
  • Nitrogen sources include nitrogen-containing organic compounds such as peptone, yeast extract, gravy, malt extract, corn steep liquor, soybean meal and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and Ammonium nitrate) and the like can be used individually or in combination;
  • As a source of phosphorus, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, a corresponding sodium-containing salt, and the like can be used individually or in combination;
  • Other metal salts such as magnesium sulfate or iron sulfate, and essential growth-promoting substances such as amino acids and vitamins.
  • the method for recovering the polypeptide produced in the culturing step of the present invention is a method for recovering the desired polypeptide from the culture medium using a suitable method known in the art according to the culture method, for example, batch, continuous or fed-batch culture method, etc. Can be collected.
  • the core of the present invention is to design a non-toxic toxin from which the toxicity of host cells has been removed to use bacterial toxins as an intracellular delivery medium for foreign proteins, and to design the cell membrane receptors of host cells by using them as a basic skeleton.
  • biomolecule carriers including the "receptor recognition domain” that recognizes it as a target and the “cargo domain” to which foreign proteins are linked.
  • PCR was performed to synthesize a receptor recognition domain and a cargo domain, which are components of the template.
  • PCR conditions were 100 ⁇ M primers, 0.3 ⁇ l, 10X nPfu Buffer 5 ⁇ l, 2 mM dNTP 5 ⁇ l, 2 U nPfu Polymerase and template were added to make the final volume 50 ⁇ l.
  • the primer sets used to synthesize each domain are shown in SEQ ID NOs: 13-20 (Table 1).
  • PCR program is performed once in 95 °C 30 seconds in the first step, the second step (95 °C 30 seconds, 55 °C 30 seconds, 72 °C 30 minutes) is repeated 30 times, finally 72 °C 2 minutes It was performed once.
  • PCR products were cloned into pET21a vector using HindIII and NheI for cargo domain and NdeI and Eco RI for receptor binding domain. All genes were synthesized by E. coli transformation using a vector.
  • Transformation was performed using Origami B (DE3) strain ( E. coli ).
  • the vector was mixed into a competent cell at 4 ° C., heated for 90 seconds in a 42 ° C. constant temperature water bath, and then cooled on ice for 3 minutes. After incubation for 1 hour in a 37 °C shaking culture incubator at 37 °C was plated on a solid medium. The transformed cells were incubated in 500 mL LB medium to OD 0.5 and expressed at 18 ° C. for 24 hours.
  • Toxin proteins produced by bacteria such as intestinal hemorrhagic Escherichia coli or Pseudomonas aeruginosa bind to specific cell membrane receptors of the host and then move to the endoplasmic reticulum through the initial endosomes using the protein delivery system inherent in the host cell and finally penetrate into the cytoplasm Causes cell death.
  • bacteria such as intestinal hemorrhagic Escherichia coli or Pseudomonas aeruginosa
  • bind to specific cell membrane receptors of the host and then move to the endoplasmic reticulum through the initial endosomes using the protein delivery system inherent in the host cell and finally penetrate into the cytoplasm causes cell death.
  • intestinal hemorrhagic Escherichia coli or Pseudomonas aeruginosa bind to specific cell membrane receptors of the host and then move to the endoplasmic reticulum through the initial endosomes using the protein delivery system inherent in the host cell and
  • the system was optimally composed of the "receptor recognition domain", "domain 2", “cargo domain”, and finally the endoplasmic reticulum residual sequence.
  • Various tests of linkers between the receptor recognition domain and domain 2 showed that the use of a linker of about 12 amino acids (SEQ ID NO: 6) was optimal.
  • Tests between domain 2 and the cargo domain were performed in the same manner, and it was found to be optimal to use a linker of about eight amino acids (SEQ ID NO: 7). Since the endoplasmic reticulum sequence was optimally bound to the carboxy terminus of the cargo domain, templates were constructed at the gene level in this order.
  • the template is configured such that the receptor recognition domain and cargo domain can be easily inserted and replaced using restriction enzymes. Inserted into the pET-based vector for the convenience of expression and purification of the template was completed template construction of the basic system (Fig. 1).
  • Example 2 Based on the template constructed in Example 1, a polypeptide for delivering eGFP into cells through Gb3, a cell membrane receptor, was designed and tested.
  • B-subunit of cigatoxin was used as the receptor recognition domain.
  • B-subunit is a part of Shigatoxin and is known to recognize Gb3, a cell membrane receptor of animal cells.
  • genes of B-subunit and eGFP were used as receptor recognition domains and cargo domains, respectively (FIG. 2).
  • Example 2-1 1 ⁇ M of the polypeptide purified in Example 2-1 was treated with HepG2 (KCTC, No. HC18302) and Vero cell (KCTC, No. AC28810) for 3 hours, and then confirmed by confocal laser scanning microscope. It was confirmed that the eGFP was delivered (Fig. 3).
  • Example 3-1 Gene and Polypeptide Synthesis for Intracellular Delivery of eGFP Via EGFR
  • hEGF Human Epidermal Growth Factor
  • Example 3-2 Intracellular Delivery Evaluation of EGFR Mediated eGFP
  • Example 3-1 1 ⁇ M of the polypeptide purified in Example 3-1 was treated with Hcc827 (ATCC, No. CRL-2868) and Vero cells for 6 hours, and then confirmed by confocal laser scanning microscopy in Hcc827 cells expressing EGFR. Only eGFP could be confirmed that delivered (Fig. 5).
  • a polypeptide for delivering luciferase into cells through Gb3, a cell membrane receptor was designed and tested based on the template constructed in Example 1.
  • B-subunit of cigatoxin was used as in Example 2, and renilla luciferase was used as a cargo domain.
  • renilla luciferase was used as a cargo domain.
  • the expressed polypeptide was purified purely using Ni-NTA column and gel permeation chromatography (GPC) (SEQ ID NO: 12).
  • the purified polypeptide was tested using HepG2 cells. As a result of measuring the activity of intracellular luciferase, it was confirmed that a sufficient amount of luciferase was delivered into the cell in 1 hour (FIG. 6).

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Abstract

The present invention relates to polypeptide for delivering useful biomolecules into cells. More particularly, the present invention relates to polypeptide which delivers various molecules into eukaryotic cells, polynucleotide which codes for the polypeptide, a vector which comprises the polynucleotide, a recombinant host cell which has been transformed by means of the vector, and a method for preparing the polypeptide by culturing the recombinant host cell. A polypeptide, according to the present invention, can deliver biomolecules into particular cells with high efficiency and thus is useful for development and preparation of medicine as well as in basic medical and scientific research areas.

Description

생체분자를 세포 내로 전달하는 폴리펩타이드 및 그 용도Polypeptides and their use to deliver biomolecules into cells
본 발명은 유용한 생체분자를 세포 내로 전달하는 폴리펩타이드에 관한 것으로, 보다 구체적으로는 다양한 분자를 진핵세포의 세포 내로 전달해주는 폴리펩타이드, 상기 폴리펩타이드를 코딩하는 폴리뉴클레오타이드, 상기 폴리뉴클레오타이드를 포함하는 벡터, 상기 벡터로 형질전환된 재조합 주세포 및 상기 재조합 숙주세포를 배양하여 상기 폴리펩타이드를 제조하는 방법에 관한 것이다.The present invention relates to a polypeptide for delivering a useful biomolecule into a cell, and more particularly, a polypeptide for delivering various molecules into a cell of a eukaryotic cell, a polynucleotide encoding the polypeptide, and a vector comprising the polynucleotide. The present invention relates to a method for producing the polypeptide by culturing the recombinant main cell transformed with the vector and the recombinant host cell.
동물세포의 세포막은 많은 생체분자들을 통과시키지 않는다. 기초과학과 의약학 등의 분야에서 다양한 생체분자들이 사용되고 있지만, 세포막으로 인해 생체분자의 세포 내부에서의 사용은 많은 제약이 있었다. The cell membranes of animal cells do not pass many biomolecules. Although various biomolecules are used in fields such as basic science and medicine, the use of biomolecules inside cells has been limited due to cell membranes.
특히, 단백질을 세포 내로 전달하는 기술은 생명과학분야에 있어서 굉장히 중요한 기술이다. 단백질은 기초과학, 의약학 분야에 걸쳐서 광범위하게 사용되고 있는 중요한 물질로써, 소분자 물질에 비해 훨씬 다양하고 섬세한 작용을 할 수 있다. 하지만 단백질은 세포막을 통과하지 못하기 때문에 세포질 안에서의 연구에는 사용하기에 커다란 제약이 있었다.In particular, the technology of delivering proteins into cells is a very important technology in the life sciences. Protein is an important substance that is widely used in basic science and medicine fields, and can have a much more diverse and delicate function than a small molecule substance. However, because proteins do not cross cell membranes, there are significant limitations in their use in the cytoplasm.
구체적으로, 세포 내에서 진행되는 생명 현상 및 질병의 원인을 분자 수준에서 규명하기 위해서는 세포 내 단백질의 상호 작용 네트워크와 신호 전달 체계를 이해해야 한다. 다양한 방법이 개발되었지만 세포 내 단백질의 상호 작용이나 신호 전달을 가역적으로 조절하고 세포의 반응을 연구하는 방법이 가장 효과적이다. 따라서, 복잡한 세포 내 신호 전달 네트워크와 메커니즘을 규명하기 위해서는 단백질 상호 작용이나 신호 전달을 강화 또는 억제하는 다양한 외래 단백질을 특정 세포 내로 안전하게 효율적으로 전달하는 기술이 필수적이다.Specifically, in order to identify the cause of the life phenomena and diseases occurring in the cell at the molecular level, it is necessary to understand the interaction networks and signal transduction systems of intracellular proteins. Although a variety of methods have been developed, the most effective method is to reversibly regulate intracellular protein interactions or signal transduction and study cell responses. Therefore, to identify complex intracellular signal transduction networks and mechanisms, a technique for safely and efficiently delivering various foreign proteins into specific cells that enhance or inhibit protein interactions or signal transduction is essential.
외래 단백질을 살아있는 특정 세포 내로 안전하게 전달할 수 있는 기술은 의약분야에도 반드시 필요하다. 암을 비롯한 질병의 효과적인 치료를 위하여 단백질 치료제가 활발히 개발되고 있지만, 단백질이 세포막을 통과할 수 없기 때문에 대부분 세포 밖의 질병 타겟에 한정되고 있다. 그러나, 세포 내에는 보다 많은 질병 타겟이 존재하기 때문에 이를 대상으로 한 단백질 치료제의 개발 중요성이 크게 부각되고 있다. 따라서, 광범위한 질병에 대해 치료 효과가 높은 단백질 치료제를 개발하기 위해서는 세포 내 질병 타켓을 표적하는 치료용 단백질을 특정 세포 안으로 안전하게 전달할 수 있는 기술의 개발이 우선적으로 요구된다. Techniques for the safe delivery of foreign proteins into specific living cells are also essential in the medical field. Protein therapeutics are being actively developed for the effective treatment of diseases including cancer, but most of them are limited to disease targets outside the cell because proteins cannot cross the cell membrane. However, since there are more disease targets in cells, the importance of developing protein therapeutics for them is of great importance. Therefore, in order to develop a protein therapeutic agent having a high therapeutic effect against a wide range of diseases, development of a technology capable of safely delivering a therapeutic protein that targets intracellular disease targets into specific cells is required first.
최근에는 이러한 한계를 극복하기 위하여 PTDs(protein transduction domains)를 사용하는 시도가 많이 이루어지고 있다. PTD는 translocatory protein의 일부분인데, translocatory protein은 독특한 secretory pathway를 통해서 세포 밖으로 나온 후, receptor-independent, non-endocytosis 방식을 통해서 다시금 세포의 cytosol로 들어가게 된다. 또한 이들 단백질은 서로 간의 공통적인 특징을 가지고 있는데, 대부분이 핵 내로 이동하는 성향을 보이며, 아미노산 서열 내에 높은 염기적 성격을 지닌 부분을 가지고 있다. 이러한 높은 염기적 성격을 지닌 부분이 바로 PTD로, translocatory protein이 세포 내로 쉽게 이동하도록 유도하는 부위이다. 최근 인간 면역결핍 바이러스(Human Immunodeficiency Virus type-1) 단백질의 일종인 Tat(Transactivator of transcription) 단백질은 중간부위인 PTD의 특성 때문에 효율적으로 세포막을 통과하여 세포질 내로 쉽게 이동한다는 것이 밝혀졌다.Recently, many attempts have been made to use protein transduction domains (PTDs) to overcome these limitations. PTD is part of the translocatory protein, which exits the cell through a unique secretory pathway and then enters the cell's cytosol through receptor-independent, non-endocytosis. In addition, these proteins have a common characteristic with each other, most of them have a tendency to move into the nucleus, and have a high basic portion in the amino acid sequence. This highly basic moiety is PTD, a site that facilitates translocatory proteins to move into cells. Recently, Tat (Transactivator of transcription) protein, a type of Human Immunodeficiency Virus type-1 protein, has been found to move efficiently through the cell membrane and into the cytoplasm easily due to the characteristics of the intermediate PTD.
이외에도, 종래 PTD를 다른 펩타이드 또는 단백질과 연결시킨 경우, 이러한 융합 단백질의 세포 내 수송이 효율적이라는 것이 밝혀졌고, 그 이후에 PTD를 이용한 다양한 응용이 시도되었다(대한민국 특허등록 제10-0568457호). In addition, when the conventional PTD is linked with other peptides or proteins, it has been found that the intracellular transport of such fusion proteins is efficient, and various applications using PTD have been attempted thereafter (Korean Patent Registration No. 10-0568457).
이처럼, 세포 내로 외래 단백질을 전달하기 위해 많은 연구가 진행되었지만, 세포 막 때문에 큰 진전을 이루지 못하고 있었다. 현재는 주로 세포 침투 펩타이드(cell penetrating peptide) 등을 단백질에 접합시켜 세포 내로 전달하거나 나노 입자에 외래 단백질을 부착하여 세포 내로 전달하는 방법 등이 사용되고 있으나 낮은 효율과 세포에 대한 특이성이 없는 것이 가장 큰 문제점으로 남아 있다. As such, much research has been carried out to deliver foreign proteins into cells, but they have not made great progress because of cell membranes. Currently, cell penetrating peptides, such as cell penetrating peptides, are conjugated to proteins and transferred into cells, or foreign proteins are attached to nanoparticles and transferred into cells, but low efficiency and cell specificity are the biggest. It remains a problem.
이에, 본 발명자들은 박테리아가 생성하는 독소 단백질의 세포투과 도메인을 기반으로 외래 생체분자를 특정 세포 내로 안전하게 고효율로 전달할 수 있는 폴리펩타이드를 개발하고, 본 발명을 완성하였다.Accordingly, the present inventors have developed a polypeptide that can safely and efficiently deliver foreign biomolecules into specific cells based on the cell permeation domain of the toxin protein produced by bacteria, and completed the present invention.
발명의 요약Summary of the Invention
본 발명의 목적은 생체분자를 특정 세포 내로 안전하게 고효율로 전달할 수 있는 폴리펩타이드, 상기 폴리펩타이드를 코딩하는 폴리뉴클레오타이드, 상기 폴리뉴클레오타이드를 포함하는 벡터, 상기 벡터가 도입되어 있는 재조합 미생물 및 상기 재조합 미생물을 배양하여 상기 폴리펩타이드를 제조하는 방법을 제공하는 데 있다. An object of the present invention is to provide a polypeptide capable of safely and efficiently delivering biomolecules into a specific cell, a polynucleotide encoding the polypeptide, a vector comprising the polynucleotide, a recombinant microorganism into which the vector is introduced, and the recombinant microorganism. It provides a method for producing the polypeptide by culturing.
본 발명의 다른 목적은 상기 폴리펩타이드를 함유하는 세포 내 전달용 조성물 및 상기 조성물을 이용한 생체분자의 세포 내 전달방법을 제공하는 데 있다.Another object of the present invention is to provide a composition for intracellular delivery containing the polypeptide and a method for intracellular delivery of biomolecules using the composition.
도 1은 본 발명의 기본 개념도로, 세 도메인과 소포체 잔류 서열로 이루어진 상기 폴리펩타이드를 도식화한 것이다. 1 is a schematic diagram of the present invention, which illustrates the polypeptide consisting of three domains and a endoplasmic reticulum residue sequence.
도 2는 도 1의 기본 개념을 토대로 만든 Gb3 세포막 수용체를 인식하여 eGFP를 세포 내로 전달하는 폴리펩타이드의 결정화 구조 개략도이다. FIG. 2 is a schematic diagram illustrating the crystallization structure of a polypeptide that recognizes a Gb3 cell membrane receptor based on the basic concept of FIG. 1 and delivers eGFP into a cell.
도 3은 도 2의 폴리펩타이드를 이용하여 eGFP를 Gb3 양성 세포 내로 전달에 성공한 공초점레이저주사현미경 사진이다.FIG. 3 is a confocal laser scanning micrograph showing successful delivery of eGFP into Gb3-positive cells using the polypeptide of FIG. 2.
도 4는 도 1의 기본 개념을 토대로 만든 EGFR 세포막 수용체를 인식하여 eGFP를 세포 내로 전달하는 폴리펩타이드의 결정화 구조 개략도이다. 4 is a schematic diagram illustrating the crystallization structure of a polypeptide that recognizes an EGFR cell membrane receptor based on the basic concept of FIG. 1 and delivers an eGFP into a cell.
도 5는 도 4의 폴리펩타이드를 이용하여 eGFP를 EGFR 양성 및 음성 세포 내로 전달한 공초점레이저주사현미경 사진이다. FIG. 5 is a confocal laser scanning micrograph of eGFP delivered into EGFR positive and negative cells using the polypeptide of FIG. 4.
도 6은 도 1의 기본 개념을 토대로 만든 Gb3 세포막 수용체를 인식하여 luciferase를 세포 내로 전달하는 폴리펩타이드가 성공적으로 luciferase를 전달한 것을 시간별로 그린 그래프이다. X축: 시간 (hour), Y축: 세포 내 luciferase의 상대적 활성, 파란색: 상기 폴리펩타이드, 녹색: 도메인 2 가 제거된 폴리펩타이드, 빨간색: 비활성 B-subunit 이 결합된 폴리펩타이드.FIG. 6 is a graph illustrating the successful delivery of luciferase by a polypeptide that recognizes a Gb3 cell membrane receptor based on the basic concept of FIG. 1 and delivers luciferase into a cell. FIG. X axis: hour, Y axis: relative activity of intracellular luciferase, blue: the polypeptide, green: polypeptide with domain 2 removed, red: polypeptide with inactive B-subunit bound.
발명의 상세한 설명 및 바람직한 Detailed description of the invention and preferred 구현예Embodiment
본 발명에서는, 세포막의 특정 수용체를 인식하는 “수용체 인식 도메인”, 박테리아 독소 단백질에서 유래한 “세포투과 도메인”, 전달할 생체분자가 접합되는 “카르고 도메인”, 소포체 잔류 서열이 복합체를 이루어 세포 내로 전달되는 것을 확인하였다. In the present invention, the "receptor recognition domain" that recognizes a specific receptor on the cell membrane, the "cell permeation domain" derived from the bacterial toxin protein, the "cargo domain" to which the biomolecules to be delivered are conjugated, and the vesicle residual sequence are complexed into the cell. Confirmed delivery.
본 발명의 일 실시예에서는 세포투과 도메인으로 녹농균이 생성하는 독소 단백질의 도메인 2를 이용하고, 상기 세포투과 도메인의 N말단 및 C말단에 각각 수용체 인식 도메인 및 카르고 도메인을 결합하여 폴리펩타이드를 제조하였다. In an embodiment of the present invention, a polypeptide is prepared by using domain 2 of a toxin protein produced by P. aeruginosa as a cell permeation domain and binding a receptor recognition domain and a cargo domain to the N and C ends of the cell permeation domain, respectively. It was.
본 발명의 다른 실시예에서는 상기 도메인 2의 구조 및 기능을 분석해 본 결과, 본 시스템은 “수용체 인식 도메인”, “도메인 2”, “카르고 도메인”, 소포체 잔류 서열 순으로 구성되는 것이 최적인 것을 확인할 수 있었다.In another embodiment of the present invention, as a result of analyzing the structure and function of the domain 2, it is optimal that the system consists of the "receptor recognition domain", "domain 2", "cargo domain", the endoplasmic reticulum sequence I could confirm it.
따라서, 본 발명은 일 관점에서, 세포투과 도메인을 기본 골격으로, 상기 세포투과 도메인의 N말단 및 C말단에 수용체 인식 도메인 및 카르고 도메인이 각각 결합되어 있는 것을 특징으로 하는, 생체분자를 세포 내로 전달할 수 있는 폴리펩타이드에 관한 것이다. Accordingly, in one aspect, the present invention provides a biomolecule into a cell, wherein the cell permeation domain is a basic skeleton, and the receptor recognition domain and the cargo domain are respectively coupled to the N and C ends of the cell permeation domain. It relates to a polypeptide that can be delivered.
본 발명에서, “세포투과 도메인”은 생체분자가 세포막을 통과하여 세포 내로 들어갈 수 있게 하는 부분을 의미한다. 상기 도메인은 박테리아의 독소 단백질에서 유래한 폴리펩타이드를 사용할 수 있다. 예를 들어, 녹농균의 외독소(Pseudomonas Exotoxin A)의 일부, 장출혈성 대장균의 시가톡신(Shiga like toxin)의 일부 등이 사용될 수 있다.In the present invention, the "cell permeation domain" refers to a portion that allows the biomolecule to enter the cell through the cell membrane. The domain may use a polypeptide derived from a bacterial toxin protein. For example, a part of Pseudomonas Exotoxin A, a part of Shiga like toxin of E. coli can be used.
본 발명에서, “수용체 인식 도메인” 은 전달하고자 하는 세포의 세포막에 있는 특정 수용체를 인식하는 부분을 의미한다. 상기 도메인에는 폴리펩타이드를 포함하여 세포막에 있는 특정 수용체를 인식할 수 있는 다양한 분자가 이용될 수 있다. 예를 들어, EGF, IL-6, Fc 등의 자연계 단백질, 단일체인항체(scFv), 리피바디(Repebody), DARPin, Monobody, Nanobody 등의 인공단백질, RGD, folate 등의 소분자 리간드 등을 사용할 수 있다.In the present invention, the "receptor recognition domain" means a portion that recognizes a specific receptor in the cell membrane of the cell to be delivered. The domain may be a variety of molecules that can recognize a specific receptor on the cell membrane, including the polypeptide. For example, natural proteins such as EGF, IL-6, Fc, monoclonal antibodies (scFv), lipids (Repebody), artificial proteins such as DARPin, Monobody, Nanobody, small molecule ligands such as RGD, folate, etc. can be used. have.
본 발명에서, “카르고 도메인”은 세포 내로 전달하고자 하는 생체분자를 의미한다. 상기 도메인은 다양한 생체분자가 다양한 방법으로 세포투과 도메인의 C말단에 접합될 수 있다. 예를 들어, 외래 단백질의 뉴클레오타이드가 상기 폴리펩타이드의 뉴클레오타이드에 접합되어 하나의 결합 폴리펩타이드 형태로 생산되도록 사용할 수 있다. 다른 예로, 시스테인(cysteine)을 삽입한 후 생체분자에 말레이미드(maleimide)를 결합하여 시스테인과 말레이미드 간의 접합반응을 이용하여 생체분자를 접합시킬 수 있다. 전달하고자 하는 생체분자의 예로는 치료 및 진단용 단백질, 단일나선 핵산, 이중나선 핵산, 소분자 약제 등이 대상이 될 수 있다.In the present invention, "cargo domain" refers to a biomolecule to be delivered into the cell. The domain may be conjugated to various C-terminal end of the cell permeation domain in a variety of ways. For example, a nucleotide of a foreign protein can be used to be conjugated to the nucleotide of the polypeptide to produce one binding polypeptide form. As another example, after the cysteine (cysteine) is inserted and the maleimide (maleimide) to the biomolecule can be conjugated to the biomolecule using a conjugation reaction between the cysteine and maleimide. Examples of biomolecules to be delivered may be targets for therapeutic and diagnostic proteins, single-stranded nucleic acids, double-stranded nucleic acids, small molecule drugs, and the like.
한편, 본 발명에 따른 폴리펩타이드는 생체분자의 세포 내 전달 효율을 높이기 위하여, 상기 카르고 도메인(생체분자)의 C말단에 소포체 잔류 서열이 추가로 연결되어 있는 것을 특징으로 할 수 있다.On the other hand, the polypeptide according to the present invention may be characterized in that the endoplasmic reticulum residue sequence is further connected to the C terminal of the cargo domain (biomolecule) in order to increase the intracellular delivery efficiency of the biomolecule.
본 발명에서, 소포체 잔류 서열은 상기 폴리펩타이드가 세포의 소포체에 잔류하게 하여 외래분자가 세포 내로 투과되는 데 도움을 주는 서열을 의미한다. 상기 소포체 잔류 서열은 KDEL, REDLK 등의 서열을 포함하여 외래분자가 소포체에 잔류할 수 있도록 도움을 준다. In the present invention, the endoplasmic reticulum means a sequence that allows the polypeptide to remain in the endoplasmic reticulum of the cell to help foreign molecules penetrate into the cell. The endoplasmic reticulum sequences include sequences such as KDEL and REDLK to help foreign molecules remain in the endoplasmic reticulum.
본 발명에서는, 수용체 인식 도메인과 도메인 2의 사이에 들어갈 링커를 여러 가지로 테스트 한 결과, 약 12개 아미노산 가량의 링커(서열번호 6)를 사용하는 것이 최적인 것을 확인할 수 있었다. 또한 도메인 2와 카르고 도메인의 사이 역시 같은 방법으로 테스트 한 결과, 약 8개 아미노산 가량의 링커(서열번호 7)를 사용하는 것이 최적인 것을 확인할 수 있었다. In the present invention, as a result of various tests of the linker to be inserted between the receptor recognition domain and the domain 2, it was confirmed that it is optimal to use a linker of about 12 amino acids (SEQ ID NO: 6). In addition, between the domain 2 and the cargo domain was tested in the same way, it was confirmed that it is optimal to use a linker (SEQ ID NO: 7) of about 8 amino acids.
본 발명에 있어서, 상기 도메인 2의 N말단과 수용체 인식 도메인은 서열번호 4의 아미노산 서열로 표시된 링커에 의해 연결된 것을 특징으로 할 수 있으나, 이에 국한되는 것은 아니다. In the present invention, the N-terminal and the receptor recognition domain of the domain 2 may be connected by a linker represented by the amino acid sequence of SEQ ID NO: 4, but is not limited thereto.
본 발명에 있어서, 상기 도메인 2의 C말단과 카르고 도메인은 서열번호 5의 아미노산 서열로 표시되는 링커에 의해 연결된 것을 특징으로 할 수 있으나, 이에 국한되는 것은 아니다. In the present invention, the C terminal and the cargo domain of the domain 2 may be connected by a linker represented by the amino acid sequence of SEQ ID NO: 5, but is not limited thereto.
본 발명에서는 template의 구성요소인 수용체 인식 도메인 및 카르고 도메인을 합성하기 위해 상기 각각의 도메인을 코딩하는 핵산을 PCR을 통해 증폭한 다음, 대장균에 도입하였다.In the present invention, to synthesize the receptor recognition domain and the cargo domain, which is a component of the template, nucleic acids encoding the respective domains were amplified by PCR and then introduced into E. coli.
본 발명에서는, 도메인 2의 N말단의 수용체 인식 도메인으로 시가톡신의 B-Subunit을 사용하고, C말단의 카르고 도메인으로 eGFP를 사용하였다. 상기에서 완성된 유전자 구조체의 발현을 위해 pET계 벡터로 삽입한 다음, 대장균에 도입한 후 발현하였다. 발현된 폴리펩타이드는 Ni-NTA 컬럼과 겔침투크로마토그래피(Gel permeation chromatography, GPC)를 이용하여 순수하게 정제하였다(서열번호 10). 상기에서 정제된 폴리펩타이드 1 μM을 HepG2(KCTC, No. HC18302)와 Vero cell(KCTC, No. AC28810)에 3시간씩 처리한 다음, 공초점레이저주사현미경으로 확인한 결과, 세포 내로 eGFP가 전달된 것을 확인할 수 있었다(도 3).In the present invention, B-Subunit of cigatoxin was used as the N-terminal receptor recognition domain of domain 2, and eGFP was used as the C-terminal cargo domain. Inserted into the pET-based vector for expression of the completed gene construct, and then introduced into E. coli and expressed. The expressed polypeptide was purified using Ni-NTA column and gel permeation chromatography (GPC) (SEQ ID NO: 10). 1 μM of the purified polypeptide was treated in HepG2 (KCTC, No. HC18302) and Vero cell (KCTC, No. AC28810) for 3 hours, and then confirmed by confocal laser scanning microscope. As a result, eGFP was delivered into the cells. It was confirmed that (Fig. 3).
본 발명의 다른 실시예에서는, 수용체 인식 도메인이 교체 가능한 것을 증명하기 위해 카르고 도메인은 그대로 두고, 수용체 인식 도메인으로 hEGF를 사용하였다. 상기에서 완성된 유전자는 발현을 위해 pET계 벡터로 삽입한 다음, 대장균에 도입한 후 발현하였다. 발현된 폴리펩타이드는 Ni-NTA 컬럼과 겔침투크로마토그래피(Gel permeation chromatography, GPC)를 이용하여 순수하게 정제되었다(서열번호 11). 상기에서 정제된 폴리펩타이드 1 μM을 Hcc827(ATCC, No. CRL-2868)과 Vero cell에 6시간씩 처리한 다음, 공초점레이저주사현미경으로 확인한 결과, EGFR을 발현하는 Hcc827 세포 내에서만 eGFP가 전달된 것을 확인할 수 있었다(도 5).In another embodiment of the present invention, hEGF was used as the receptor recognition domain, leaving the cargo domain intact to prove that the receptor recognition domain was interchangeable. The completed gene was inserted into a pET-based vector for expression, and then introduced into E. coli and expressed. The expressed polypeptide was purified using Ni-NTA column and gel permeation chromatography (GPC) (SEQ ID NO: 11). 1 μM of the purified polypeptide was treated with Hcc827 (ATCC, No. CRL-2868) and Vero cells for 6 hours, and then confirmed by confocal laser scanning. As a result, eGFP was delivered only in Hcc827 cells expressing EGFR. It could be confirmed that (Fig. 5).
본 발명의 또 다른 실시예에서는, 카르고 도메인이 교체 가능한 것을 증명하기 위해 수용체 인식 도메인은 그대로 두고, 카르고 도메인으로 renilla luciferase를 사용하였다. 상기에서 완성된 유전자는 발현을 위해 pET계 벡터로 삽입한 다음, 대장균에 도입한 후 발현하였다. 발현된 폴리펩타이드는 Ni-NTA 컬럼과 겔침투크로마토그래피(Gel permeation chromatography, GPC)를 이용하여 순수하게 정제되었다(서열번호 12). 상기에서 정제된 폴리펩타이드는 HepG2 cell을 이용하여 테스트 해 보았다. 세포 내 luciferase의 활성을 측정해 본 결과, 1시간 만에 세포 내로 충분한 양의 luciferase가 전달 된 것을 확인할 수 있었다(도 6).In another embodiment of the present invention, renilla luciferase was used as the cargo domain while leaving the receptor recognition domain intact to prove that the cargo domain was replaceable. The completed gene was inserted into a pET-based vector for expression, and then introduced into E. coli and expressed. The expressed polypeptide was purified purely using Ni-NTA column and gel permeation chromatography (GPC) (SEQ ID NO: 12). The purified polypeptide was tested using HepG2 cells. As a result of measuring the activity of intracellular luciferase, it was confirmed that a sufficient amount of luciferase was delivered into the cell in 1 hour (FIG. 6).
본 발명은 다른 관점에서, 상기 폴리펩타이드를 코딩하는 폴리뉴클레오타이드; 상기 폴리뉴클레오타이드를 포함하는 재조합 벡터; 상기 폴리뉴클레오타이드 또는 상기 재조합 벡터가 도입되어 있는 재조합 미생물에 관한 것이다. In another aspect, the present invention, a polynucleotide encoding the polypeptide; A recombinant vector comprising the polynucleotide; The present invention relates to a recombinant microorganism into which the polynucleotide or the recombinant vector is introduced.
본 발명에서, 폴리뉴클레오타이드는 당과 인산기와 염기가 결합한 단위체인 뉴클레오타이드가 수천 개 이상 결합한 것을 의미한다. 뉴클레오타이드는 인산이에스테르 결합으로 긴 사슬을 형성한다. 이때 당과 인산의 골격이 반복적으로 들어가고, 사이사이로 염기의 순서가 불규칙하게 배치된다. 염기 배치의 불규칙성과 길이에 따라 유전자에 들어가는 핵심 정보가 형성된다. 폴리뉴클레오타이드는 한쪽 사슬의 끝이 5`말단이며 다른 쪽 사슬 끝은 반드시 3`말단으로 되어 방향성을 가진다. 이때 말단의 성격을 5`-인산기, 3`-히드록시기 등으로 함께 표시해준다. DNA와 RNA 모두 폴리뉴클레오타이드 형태로 되어 있다. 폴리뉴클레오타이드는 단백질 생합성에 중요한 역할을 한다.In the present invention, the polynucleotide means that thousands or more of the nucleotides, which are a unit in which sugar, phosphate, and base are bonded to each other. Nucleotides form long chains with diester bonds. At this time, the skeleton of sugar and phosphoric acid enters repeatedly, and the order of bases is irregularly arranged between. Irregularities and lengths of base placement produce key information that enters the gene. Polynucleotides are directional, with one end of the chain 5 'end and the other end of the chain necessarily 3' end. At this time, the nature of the terminal 5`-phosphate group, 3`-hydroxy group is displayed together. Both DNA and RNA are in polynucleotide form. Polynucleotides play an important role in protein biosynthesis.
본 발명에서, "벡터"는 적당한 숙주세포에서 목적 단백질을 발현할 수 있도록 적합한 조절 서열에 작동 가능하게 연결된 상기 목적 단백질을 암호화하는 폴리뉴클레오타이드의 염기서열을 함유하는 DNA 제조물을 의미한다. 상기 조절 서열은 전사를 개시할 수 있는 프로모터, 그러한 전사를 조절하기 위한 임의의 오퍼레이터 서열, 적합한 mRNA 리보좀 결합 부위를 코딩하는 서열 및 전사와 해독의 종결을 조절하는 서열을 포함할 수 있으며 목적에 따라 다양하게 제조될 수 있다. 벡터의 프로모터는 구성적 또는 유도성 일 수 있다. 벡터는 적당한 숙주 내로 형질전환 된 후, 숙주 게놈과 무관하게 복제되거나 기능할 수 있으며, 게놈 그 자체에 통합될 수 있다. In the present invention, "vector" refers to a DNA preparation containing a nucleotide sequence of a polynucleotide encoding the target protein operably linked to a suitable regulatory sequence so that the target protein can be expressed 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.
본 발명에서 사용되는 벡터는 숙주세포 중에서 복제 가능한 것이면 특별히 한정되지 않으며 당 업계에 알려진 임의의 벡터를 이용할 수 있다. 통상 사용되는 벡터의 예로는 천연 상태이거나 재조합 된 상태의 플라스미드, 파지미드, 코스미드, 바이러스 및 박테리오파지를 들 수 있다. 예를 들어, 파지 벡터 또는 코스미드 벡터로서 pWE15, M13, λMBL3, λMBL4, λIXII, λASHII, λAPII, λt10, λt11, Charon4A, 및 Charon21A 등을 사용할 수 있으며, 플라스미드 벡터로서 pBR계, pUC계, pBluescriptII계, pGEM계, pTZ계, pCL계 및 pET계 등을 사용할 수 있다. 본 발명에서 사용 가능한 벡터는 특별히 제한되는 것이 아니며 공지된 발현 벡터를 사용할 수 있다. 바람직하게는 pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC 벡터 등을 사용할 수 있다. 가장 바람직하게는 pACYC177, pCL, pCC1BAC 벡터를 사용할 수 있다.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. For example, pWE15, M13, λMBL3, λMBL4, λIXII, λASHII, λAPII, λt10, λt11, Charon4A, and Charon21A can be used as the phage vector or cosmid vector, and pBR, pUC, and pBluescriptII systems are used as plasmid vectors. , pGEM-based, pTZ-based, pCL-based and pET-based and the like can be used. The vector usable in the present invention is not particularly limited and known expression vectors can be used. Preferably, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vector and the like can be used. Most preferably, pACYC177, pCL, pCC1BAC vectors can be used.
본 발명에서, “재조합 미생물”은 하나 이상의 목적 단백질을 암호화하는 폴리뉴클레오타이드를 갖는 벡터가 숙주세포에 도입되거나, 하나 이상의 목적 단백질을 암호화하는 폴리뉴클레오타이드가 미생물에 도입되어 폴리뉴클레오타이드가 염색체에 통합되어 목적 단백질을 발현시키도록 형질이 감염된 세포를 의미하며, 진핵세포, 원핵세포 등의 모든 세포가 될 수 있는데, 특별히 이에 제한되지 않으나, 대장균, 스트렙토미세스, 살모넬라 티피뮤리움 등의 박테리아 세포; 효모 세포; 피치아 파스토리스 등의 균류 세포; 드로조필라, 스포도프테라 Sf9 세포 등의 곤충 세포; CHO, COS, NSO, 293, 보우 멜라노마 세포 등의 동물 세포가 될 수 있다. In the present invention, a "recombinant microorganism" means that a vector having a polynucleotide encoding at least one target protein is introduced into a host cell, or a polynucleotide encoding at least one target protein is introduced at a microorganism so that the polynucleotide is incorporated into a chromosome. Means a cell infected with a trait to express a protein, and may be any cell, such as eukaryotic cells, prokaryotic cells, etc., but is not particularly limited thereto, bacterial cells such as E. coli, Streptomyces, Salmonella typhimurium; Yeast cells; Fungal cells such as Pchia pastoris; Insect cells such as Drozophila and Spodoptera Sf9 cells; Animal cells such as CHO, COS, NSO, 293, Bow Melanoma cells, and the like.
본 발명에서, "형질전환"은 표적 단백질을 암호화하는 폴리뉴클레오타이드를 포함하는 벡터를 숙주세포 내에 도입하거나 표적 단백질을 암호화하는 폴리뉴클레오타이드를 숙주세포의 염색체에 통합 완성시켜 숙주세포 내에서 상기 폴리뉴클레오타이드가 암호화하는 단백질이 발현할 수 있도록 하는 것을 의미한다. 형질전환된 폴리뉴클레오타이드는 숙주세포 내에 발현될 수 있기만 한다면, 숙주세포의 염색체 내에 삽입되어 위치하거나 염색체 외에 위치하든지 상관없이 이들 모두를 포함한다. 또한, 상기 폴리뉴클레오타이드는 표적 단백질을 암호화하는 DNA 및 RNA를 포함한다. 상기 폴리뉴클레오타이드는 숙주세포 내로 도입되어 발현될 수 있는 것이면, 어떠한 형태로 도입되는 것이든 상관없다. 예를 들면, 상기 폴리뉴클레오타이드는 자체적으로 발현되는데 필요한 모든 요소를 포함하는 유전자 구조체인 발현 카세트(expression cassette)의 형태로 숙주세포에 도입될 수 있다. 상기 발현 카세트는 통상 상기 폴리뉴클레오타이드에 작동 가능하게 연결되어 있는 프로모터(promoter), 전사 종결 신호, 리보좀 결합부위 및 번역 종결신호를 포함한다. 상기 발현 카세트는 자체 복제가 가능한 발현 벡터 형태일 수 있다. 또한, 상기 폴리뉴클레오타이드는 그 자체의 형태로 숙주세포에 도입되어, 숙주세포에서 발현에 필요한 서열과 작동 가능하게 연결되어 있는 것일 수도 있다.In the present invention, "transformation" refers to introducing a vector containing a polynucleotide encoding a target protein into a host cell or integrating a polynucleotide encoding a target protein into a chromosome of the host cell to complete the polynucleotide in the host cell. It means that the encoding protein can be expressed. Transformed polynucleotides include all of them, as long as they can be expressed in the host cell, whether they are inserted into or located outside the chromosome of the host cell. In addition, the polynucleotides include DNA and RNA encoding the target protein. The polynucleotide may be introduced in any form as long as it can be expressed by being introduced into a host cell. For example, the polynucleotide may be introduced into a host cell in the form of an expression cassette, which is a gene construct containing all elements necessary for self-expression. The expression cassette typically includes a promoter, transcription termination signal, ribosomal binding site and translation termination signal operably linked to the polynucleotide. The expression cassette may be in the form of an expression vector capable of self replication. In addition, the polynucleotide may be introduced into a host cell in its own form and operably linked to a sequence required for expression in the host cell.
본 발명은 또 다른 관점에서, (a) 상기 재조합 미생물을 배양하여 폴리펩타이드를 생성하는 단계; 및 (b) 상기 생성된 폴리펩타이드를 회수하는 단계를 포함하는 생체분자를 세포 내로 전달할 수 있는 폴리펩타이드 제조방법에 관한 것이다.In another aspect, the present invention provides a method for producing a polypeptide comprising: (a) culturing the recombinant microorganism to produce a polypeptide; And (b) relates to a method for producing a polypeptide that can deliver the biomolecule into the cell comprising the step of recovering the generated polypeptide.
본 발명에서, 재조합 미생물을 배양하는 단계는 특별히 이에 제한되지 않으나, 공지된 회분식 배양방법, 연속식 배양방법, 유가식 배양방법 등에 의해 수행됨이 바람직하고, 배양조건은 특별히 이에 제한되지 않으나, 염기성 화합물(예: 수산화나트륨, 수산화칼륨 또는 암모니아) 또는 산성 화합물(예: 인산 또는 황산)을 사용하여 적정 pH(pH 5 내지 9, 바람직하게는 pH 6 내지 8, 가장 바람직하게는 pH 6.8)를 조절할 수 있고, 산소 또는 산소-함유 가스 혼합물을 배양물에 도입시켜 호기성 조건을 유지할 수 있으며, 배양온도는 20 내지 45℃, 바람직하게는 25 내지 40℃를 유지할 수 있고, 약 10 내지 160 시간 동안 배양함이 바람직하다. 상기 배양에 의하여 생산된 상기 폴리펩타이드는 배지 중으로 분비되거나 세포 내에 잔류할 수 있다. In the present invention, the step of culturing the recombinant microorganism is not particularly limited thereto, but is preferably performed by a known batch culture method, continuous culture method, fed-batch culture method, and the like, and the culture conditions are not particularly limited thereto. (E.g. sodium hydroxide, potassium hydroxide or ammonia) or acidic compounds (e.g. phosphoric acid or sulfuric acid) can be used to adjust the appropriate pH (pH 5-9, preferably pH 6-8, most preferably pH 6.8). And introducing an oxygen or oxygen-containing gas mixture into the culture to maintain aerobic conditions, the incubation temperature can be maintained at 20 to 45 ℃, preferably 25 to 40 ℃, incubated for about 10 to 160 hours This is preferable. The polypeptide produced by the culture may be secreted into the medium or remain in the cell.
아울러, 사용되는 배양용 배지는 탄소 공급원으로는 당 및 탄수화물(예: 글루코오스, 슈크로오스, 락토오스, 프럭토오스, 말토오스, 몰라세, 전분 및 셀룰로오스), 유지 및 지방(예: 대두유, 해바라기씨유, 땅콩유 및 코코넛유), 지방산(예:팔미트산, 스테아르산 및 리놀레산), 알콜(예: 글리세롤 및 에탄올) 및 유기산(예:아세트산) 등을 개별적으로 사용하거나 또는 혼합하여 사용할 수 있고; 질소 공급원으로는 질소-함유 유기 화합물(예: 펩톤, 효모 추출액, 육즙, 맥아 추출액, 옥수수 침지액, 대두 박분 및 우레아), 또는 무기 화합물(예: 황산암모늄, 염화암모늄, 인산암모늄, 탄산암모늄 및 질산암모늄) 등을 개별적으로 사용하거나 또는 혼합하여 사용할 수 있으며; 인 공급원으로서 인산 이수소칼륨, 인산수소이칼륨, 이에 상응하는 나트륨 함유 염 등을 개별적으로 사용하거나 또는 혼합하여 사용할 수 있고; 기타 금속염(예: 황산마그네슘 또는 황산철), 아미노산 및 비타민과 같은 필수성장-촉진 물질을 포함할 수 있다. In addition, the culture medium used may include sugars and carbohydrates (e.g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose), fats and fats (e.g. soybean oil, sunflower seeds) as carbon sources. Oils, peanut oils and coconut oils), fatty acids (e.g. palmitic acid, stearic acid and linoleic acid), alcohols (e.g. glycerol and ethanol) and organic acids (e.g. acetic acid), etc. may be used individually or in combination. ; Nitrogen sources include nitrogen-containing organic compounds such as peptone, yeast extract, gravy, malt extract, corn steep liquor, soybean meal and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and Ammonium nitrate) and the like can be used individually or in combination; As a source of phosphorus, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, a corresponding sodium-containing salt, and the like can be used individually or in combination; Other metal salts such as magnesium sulfate or iron sulfate, and essential growth-promoting substances such as amino acids and vitamins.
본 발명의 상기 배양 단계에서 생산된 폴리펩타이드를 회수하는 방법은 배양방법, 예를 들어 회분식, 연속식 또는 유가식 배양 방법 등에 따라 당해 분야에 공지된 적합한 방법을 이용하여 배양액으로부터 목적하는 폴리펩타이드를 수집할 수 있다.The method for recovering the polypeptide produced in the culturing step of the present invention is a method for recovering the desired polypeptide from the culture medium using a suitable method known in the art according to the culture method, for example, batch, continuous or fed-batch culture method, etc. Can be collected.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.
실시예Example
실시예 1: 단백질 구조 분석을 통한 기본 시스템 설계Example 1: Basic System Design Through Protein Structure Analysis
본 발명에서는 외래 생체분자를 세포 내로 전달하는 기술을 개발하기 위하여 박테리아가 생성하는 독소 단백질의 특성을 기반으로 기본 시스템을 설계하여 외래 생체분자를 특정 세포 내로 안전하게 고효율로 전달할 수 있는 기술을 개발하고자 하였다. In the present invention, in order to develop a technology for delivering foreign biomolecules into cells, a basic system was designed based on the characteristics of toxin proteins produced by bacteria, and thus, a technique for safely and efficiently delivering foreign biomolecules into specific cells was proposed. .
본 발명의 핵심은 박테리아의 독소를 외래 단백질의 세포 내 전달 매개체로 이용하기 위해 숙주세포에 대한 독성이 제거된 비 독성 독소를 새롭게 구조 기반으로 설계하고 이를 기본 골격으로 하여 숙주세포의 세포막 수용체를 특이적으로 인식하는 “수용체 인식 도메인”, 그리고 외래 단백질이 연결되는 “카르고(cargo) 도메인” 을 포함한 새로운 생체분자 전달체를 개발하는 것이다.The core of the present invention is to design a non-toxic toxin from which the toxicity of host cells has been removed to use bacterial toxins as an intracellular delivery medium for foreign proteins, and to design the cell membrane receptors of host cells by using them as a basic skeleton. The development of new biomolecule carriers, including the "receptor recognition domain" that recognizes it as a target and the "cargo domain" to which foreign proteins are linked.
실시예 1-1: 도메인의 합성 방법Example 1-1 Synthesis of Domains
본 발명에서는 template의 구성요소인 수용체 인식 도메인 및 카르고 도메인을 합성하기 위해 PCR을 수행하였다.In the present invention, PCR was performed to synthesize a receptor recognition domain and a cargo domain, which are components of the template.
PCR 조건은 100 μM primer를 각각 0.3 ㎕, 10X nPfu Buffer 5 ㎕, 2 mM dNTP 5 ㎕, 2 U nPfu Polymerase와 template를 넣어 최종 부피가 50 ㎕가 되도록 하였다. 각각의 도메인을 합성하기 위해 사용한 primer set는 서열번호 13 내지 20과 같다(표 1). PCR program은 첫 번째 단계에서 95℃ 30초를 1회 수행 후, 두 번째 단계 (95℃ 30초, 55℃ 30초, 72℃ 30분)를 30회 반복 수행하고, 마지막으로 72℃ 2분을 한차례 수행하였다. PCR product는 카르고 도메인의 경우 HindIII와 NheI으로, 수용체 결합 도메인의 경우 NdeI과 EcoRI을 이용하여 pET21a vector에 cloning하였다. 모든 유전자는 벡터를 이용하여 대장균 형질전환을 통해 합성하였다.The PCR conditions were 100 μM primers, 0.3 μl, 10X nPfu Buffer 5 μl, 2 mM dNTP 5 μl, 2 U nPfu Polymerase and template were added to make the final volume 50 μl. The primer sets used to synthesize each domain are shown in SEQ ID NOs: 13-20 (Table 1). PCR program is performed once in 95 ℃ 30 seconds in the first step, the second step (95 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 30 minutes) is repeated 30 times, finally 72 ℃ 2 minutes It was performed once. PCR products were cloned into pET21a vector using HindIII and NheI for cargo domain and NdeI and Eco RI for receptor binding domain. All genes were synthesized by E. coli transformation using a vector.
Figure PCTKR2015008109-appb-T000001
Figure PCTKR2015008109-appb-T000001
형질전환은 Origami B(DE3) strain(E. coli)을 이용하였다. 4℃ competent cell에 벡터를 섞고, 42℃ 항온수조에서 90초간 가열한 후, 얼음 위로 옮겨 3분간 냉각하여 실시하였다. 37℃ 진탕배양기에서 1시간 동안 배양한 후 고형배지에 도말하여 37℃에서 배양하였다. 형질전환된 cell을 500 mL LB배지에 OD 0.5까지 배양한 후 18℃에서 24시간 발현하였다.Transformation was performed using Origami B (DE3) strain ( E. coli ). The vector was mixed into a competent cell at 4 ° C., heated for 90 seconds in a 42 ° C. constant temperature water bath, and then cooled on ice for 3 minutes. After incubation for 1 hour in a 37 ℃ shaking culture incubator at 37 ℃ was plated on a solid medium. The transformed cells were incubated in 500 mL LB medium to OD 0.5 and expressed at 18 ° C. for 24 hours.
실시예 1-2: 세포투과 도메인 개발을 위한 박테리아 유래 독소 단백질 구조 및 기능 분석Example 1-2 Bacterial Derived Toxin Protein Structure and Function Analysis for Cell Permeation Domain Development
장출혈성 대장균이나 녹농균 등의 박테리아가 생성하는 독소 단백질들은 숙주의 특정 세포막 수용체와 결합한 후 숙주세포가 본래 갖고 있는 단백질 전달 시스템을 이용하여 초기 엔도솜을 거쳐 소포체로 이동한 후에 최종적으로 세포질로 침투하여 세포의 사멸을 일으킨다. 본 연구에서는 위와 같이 오랜 시간 동안 진화해온 박테리아 독소 단백질의 특성에 착안하여 외래 생체분자를 세포 내로 효율적으로 전달하는 새로운 기술을 개발하고자 하였다. Toxin proteins produced by bacteria such as intestinal hemorrhagic Escherichia coli or Pseudomonas aeruginosa bind to specific cell membrane receptors of the host and then move to the endoplasmic reticulum through the initial endosomes using the protein delivery system inherent in the host cell and finally penetrate into the cytoplasm Causes cell death. In this study, we focused on the characteristics of bacterial toxin proteins that have evolved for a long time as described above, and developed a new technique for efficiently delivering foreign biomolecules into cells.
녹농균이 생성하는 독소 단백질의 구조를 분석한 결과, 4가지의 도메인으로 나눌 수 있었으며, 그 중 도메인 2의 독소 단백질이 세포막을 투과시키는 기능을 갖고 있음을 알게 되었다. 도메인 2의 유전자를 합성한 후, 대장균을 형질전환하여 발현테스트를 한 결과, 성공적으로 발현 되는 것을 확인할 수 있었다. As a result of analyzing the structure of the toxin protein produced by Pseudomonas aeruginosa, it was found that the toxin protein of the Pseudomonas aeruginosa was divided into four domains. After synthesizing the gene of domain 2, E. coli was transformed and tested for expression, and it was confirmed that the expression was successful.
실시예 1-3: 수용체 인식 도메인 및 카르고 도메인 연결을 위한 연결체 및 소포체 잔류 서열 결합Example 1-3 Binding and Vesicle Residue Sequence Binding for Receptor Recognition Domain and Cargo Domain Linkage
상기 도메인 2의 구조 및 기능을 분석해 본 결과, 본 시스템은 “수용체 인식 도메인”, “도메인 2”, “카르고 도메인”, 마지막으로 소포체 잔류 서열 순으로 구성되는 것이 최적으로 판단되었다. 수용체 인식 도메인과 도메인 2 사이에 들어갈 링커를 여러 가지로 테스트 한 결과, 약 12개 아미노산 가량의 링커(서열번호 6)를 사용하는 것이 최적인 것으로 확인되었다. 도메인 2와 카르고 도메인 사이 역시 같은 방법으로 테스트 한 결과, 약 8개 아미노산 가량의 링커(서열번호 7)를 사용하는 것이 최적인 것으로 확인되었다. 소포체 잔류 서열은 카르고 도메인의 카르복시 말단에 결합하는 것이 최적으로 판단되었기에 상기 순서로 유전자 수준으로 template 를 구축하였다. As a result of analyzing the structure and function of the domain 2, the system was optimally composed of the "receptor recognition domain", "domain 2", "cargo domain", and finally the endoplasmic reticulum residual sequence. Various tests of linkers between the receptor recognition domain and domain 2 showed that the use of a linker of about 12 amino acids (SEQ ID NO: 6) was optimal. Tests between domain 2 and the cargo domain were performed in the same manner, and it was found to be optimal to use a linker of about eight amino acids (SEQ ID NO: 7). Since the endoplasmic reticulum sequence was optimally bound to the carboxy terminus of the cargo domain, templates were constructed at the gene level in this order.
상기 template는 수용체 인식 도메인과 카르고 도메인이 제한효소 등을 이용하여 손쉽게 삽입되고 교체될 수 있도록 구성하였다. 상기 template의 발현과 정제의 편의성을 위하여 pET계 벡터에 삽입하여 기본 시스템의 template 구축을 완료하였다(도 1).The template is configured such that the receptor recognition domain and cargo domain can be easily inserted and replaced using restriction enzymes. Inserted into the pET-based vector for the convenience of expression and purification of the template was completed template construction of the basic system (Fig. 1).
실시예 2: Gb3를 매개로 한 eGFP의 세포 내 전달Example 2 Intracellular Delivery of eGFP Via Gb3
실시예 1에서 구축된 template를 기반으로 세포막 수용체인 Gb3를 매개로 eGFP를 세포 내로 전달하는 폴리펩타이드를 설계 및 실험하였다.Based on the template constructed in Example 1, a polypeptide for delivering eGFP into cells through Gb3, a cell membrane receptor, was designed and tested.
실시예 2-1: Gb3를 매개로 한 eGFP의 세포 내 전달을 위한 유전자 및 폴리펩타이드 합성Example 2-1 Gene and Polypeptide Synthesis for Intracellular Delivery of Gb3-Mediated eGFP
수용체 인식 도메인으로 시가톡신의 B-subunit을 사용하였다. B-subunit은 시가톡신의 일부로써, 동물세포의 세포막 수용체인 Gb3를 인식하는 것으로 알려져 있다. 그리하여 B-subunit과 eGFP의 유전자를 각각 수용체 인식 도메인과 카르고 도메인으로 사용하였다(도 2). B-subunit of cigatoxin was used as the receptor recognition domain. B-subunit is a part of Shigatoxin and is known to recognize Gb3, a cell membrane receptor of animal cells. Thus, genes of B-subunit and eGFP were used as receptor recognition domains and cargo domains, respectively (FIG. 2).
상기에서 완성된 유전자 구조체의 발현을 위해 pET계 벡터로 삽입한 다음, 대장균에 도입한 후 발현하였다. 발현된 폴리펩타이드는 Ni-NTA 컬럼과 겔침투크로마토그래피(Gel permeation chromatography, GPC)를 이용하여 순수하게 정제하였다(서열번호 10).Inserted into the pET-based vector for expression of the completed gene construct, and then introduced into E. coli and expressed. The expressed polypeptide was purified using Ni-NTA column and gel permeation chromatography (GPC) (SEQ ID NO: 10).
실시예 2-2: Gb3를 매개로 한 eGFP의 세포 내 전달 평가 Example 2-2 Evaluation of Intracellular Delivery of GG3 Mediated eGFP
실시예 2-1에서 정제된 폴리펩타이드 1 μM을 HepG2(KCTC, No. HC18302)와 Vero cell(KCTC, No. AC28810)에 3시간씩 처리한 다음, 공초점레이저주사현미경으로 확인한 결과, 세포 내로 eGFP가 전달된 것을 확인할 수 있었다(도 3).1 μM of the polypeptide purified in Example 2-1 was treated with HepG2 (KCTC, No. HC18302) and Vero cell (KCTC, No. AC28810) for 3 hours, and then confirmed by confocal laser scanning microscope. It was confirmed that the eGFP was delivered (Fig. 3).
실시예 3: EGFR를 매개로 한 eGFP의 세포 내 전달 Example 3: Intracellular Delivery of eGFP Via EGFR
수용체 인식 도메인이 교체 가능함을 증명하기 위하여, 실시예 1에서 구축된 template를 기반으로 세포막 수용체인 EGFR를 매개로 eGFP를 세포 내로 전달하는 폴리펩타이드를 설계 및 실험하였다.In order to prove that the receptor recognition domain is interchangeable, based on the template constructed in Example 1, a polypeptide that delivers eGFP into cells through EGFR, a cell membrane receptor, was designed and tested.
실시예 3-1: EGFR를 매개로 한 eGFP의 세포 내 전달을 위한 유전자 및 폴리펩타이드 합성Example 3-1 Gene and Polypeptide Synthesis for Intracellular Delivery of eGFP Via EGFR
수용체 인식 도메인으로 human Epidermal Growth Factor(hEGF)를 사용하였다. hEGF는 동물세포의 세포막 수용체인 EGFR을 인식하는 사람 유래 호르몬이다. 그리하여 hEGF와 eGFP의 유전자를 각각 수용체 인식 도메인과 카르고 도메인으로 사용하였다(도 4). Human Epidermal Growth Factor (hEGF) was used as the receptor recognition domain. hEGF is a human-derived hormone that recognizes EGFR, the cell membrane receptor for animal cells. Thus, genes of hEGF and eGFP were used as receptor recognition domains and cargo domains, respectively (FIG. 4).
상기에서 완성된 유전자 구조체의 발현을 위해 pET계 벡터로 삽입한 다음, 대장균에 도입한 후 발현하였다. 발현된 폴리펩타이드는 Ni-NTA 컬럼과 겔침투크로마토그래피(Gel permeation chromatography, GPC)를 이용하여 순수하게 정제되었다(서열번호 11).Inserted into the pET-based vector for expression of the completed gene construct, and then introduced into E. coli and expressed. The expressed polypeptide was purified using Ni-NTA column and gel permeation chromatography (GPC) (SEQ ID NO: 11).
실시예 3-2: EGFR를 매개로 한 eGFP의 세포 내 전달 평가 Example 3-2: Intracellular Delivery Evaluation of EGFR Mediated eGFP
실시예 3-1에서 정제된 폴리펩타이드 1 μM을 Hcc827(ATCC, No. CRL-2868)과 Vero cell에 6시간씩 처리한 다음, 공초점레이저주사현미경으로 확인한 결과, EGFR을 발현하는 Hcc827 세포 내에서만 eGFP가 전달된 것을 확인할 수 있었다(도 5).1 μM of the polypeptide purified in Example 3-1 was treated with Hcc827 (ATCC, No. CRL-2868) and Vero cells for 6 hours, and then confirmed by confocal laser scanning microscopy in Hcc827 cells expressing EGFR. Only eGFP could be confirmed that delivered (Fig. 5).
실시예 4: Gb3를 매개로 한 luciferase의 세포 내 전달Example 4: Intracellular Delivery of Gfer3 Mediated Luciferase
본 발명이 다양한 단백질의 세포 내 전달이 가능함을 증명하기 위하여, 실시예 1에서 구축된 template를 기반으로 세포막 수용체인 Gb3를 매개로 luciferase를 세포 내로 전달하는 폴리펩타이드를 설계 및 실험하였다.In order to prove that the present invention is capable of intracellular delivery of various proteins, a polypeptide for delivering luciferase into cells through Gb3, a cell membrane receptor, was designed and tested based on the template constructed in Example 1.
수용체 인식 도메인으로 실시예 2와 같이 시가톡신의 B-subunit을 사용하였고, renilla luciferase를 카르고 도메인으로 사용하였다. 완성된 유전자 구조체의 발현을 위해 pET계 벡터로 삽입한 다음, 대장균에 도입한 후 발현하였다. 발현된 폴리펩타이드는 Ni-NTA 컬럼과 겔침투크로마토그래피(Gel permeation chromatography, GPC)를 이용하여 순수하게 정제되었다(서열번호 12).As a receptor recognition domain, B-subunit of cigatoxin was used as in Example 2, and renilla luciferase was used as a cargo domain. For expression of the completed gene construct was inserted into the pET-based vector, and then introduced into E. coli and expressed. The expressed polypeptide was purified purely using Ni-NTA column and gel permeation chromatography (GPC) (SEQ ID NO: 12).
상기에서 정제된 폴리펩타이드는 HepG2 cell을 이용하여 테스트 해 보았다. 세포 내 luciferase의 활성을 측정한 결과, 1시간 만에 세포 내로 충분한 양의 luciferase가 전달된 것을 확인할 수 있었다(도 6). The purified polypeptide was tested using HepG2 cells. As a result of measuring the activity of intracellular luciferase, it was confirmed that a sufficient amount of luciferase was delivered into the cell in 1 hour (FIG. 6).
이상으로, 본 발명 내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.As described above, specific parts of the present disclosure have been described in detail, and for those skilled in the art, these specific descriptions are merely preferred embodiments, and the scope of the present disclosure is not limited thereto. Will be obvious. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
전자파일 첨부하였음.Electronic file attached.

Claims (16)

  1. 세포투과 도메인을 기본 골격으로, 상기 세포투과 도메인의 N말단 및 C말단에 수용체 인식 도메인 및 카르고 도메인이 각각 결합되어 있는 것을 특징으로 하는, 생체분자를 세포 내로 전달할 수 있는 폴리펩타이드.A polypeptide capable of delivering a biomolecule into a cell, characterized in that the receptor recognition domain and the cargo domain are respectively coupled to the N- and C-terminals of the cell-permeable domain based on the cell-permeable domain.
  2. 제1항에 있어서, 상기 세포투과 도메인은 녹농균 유래 독소 단백질의 일부 또는 장출혈성 대장균의 시가톡신(Shiga like toxin)인 것을 특징으로 하는, 생체분자를 세포 내로 전달할 수 있는 폴리펩타이드.The polypeptide of claim 1, wherein the cell permeation domain is a part of P. aeruginosa-derived toxin protein or Shiga like toxin of E. coli.
  3. 제2항에 있어서, 상기 녹농균 유래 독소 단백질의 일부는 서열번호 1의 아미노산 서열로 표시되는 도메인 2인 것을 특징으로 하는, 생체분자를 세포 내로 전달할 수 있는 폴리펩타이드.The polypeptide of claim 2, wherein a part of the Pseudomonas aeruginosa-derived toxin protein is domain 2 represented by the amino acid sequence of SEQ ID NO: 1. 4.
  4. 제1항에 있어서, 상기 수용체 인식 도메인은 EGF, IL-6, Fc, 단일체인항체(scFv), 리피바디(Repebody), DARPin, Monobody, Nanobody, RGD 및 folate로 구성된 군에서 선택되는 것을 특징으로 하는, 생체분자를 세포 내로 전달할 수 있는 폴리펩타이드. The method of claim 1, wherein the receptor recognition domain is selected from the group consisting of EGF, IL-6, Fc, monoclonal antibody (scFv), repeat body (Repebody), DARPin, Monobody, Nanobody, RGD and folate A polypeptide capable of delivering a biomolecule into a cell.
  5. 제4항에 있어서, 상기 수용체 인식 도메인은 서열번호 2 또는 3으로 표시되는 것을 특징으로 하는, 생체분자를 세포 내로 전달할 수 있는 폴리펩타이드.5. The polypeptide of claim 4, wherein the receptor recognition domain is represented by SEQ ID NO: 2 or 3. 6.
  6. 제1항에 있어서, 상기 카르고 도메인은 치료용 단백질, 단일나선 핵산, 이중나선 핵산 및 소분자 약제로 구성된 군에서 선택되는 것을 특징으로 하는, 생체분자를 세포 내로 전달할 수 있는 폴리펩타이드.The polypeptide of claim 1, wherein the cargo domain is selected from the group consisting of therapeutic proteins, single-stranded nucleic acids, double-stranded nucleic acids, and small molecule pharmaceuticals.
  7. 제1항에 있어서, 상기 도메인 2의 N말단과 수용체 인식 도메인은 서열번호 6의 아미노산 서열로 표시되는 링커에 의해 연결되는 것을 특징으로 하는, 생체분자를 세포 내로 전달할 수 있는 폴리펩타이드.The polypeptide of claim 1, wherein the N-terminal of the domain 2 and the receptor recognition domain are linked by a linker represented by the amino acid sequence of SEQ ID NO: 6.
  8. 제1항에 있어서, 상기 도메인 2의 C말단과 카르고 도메인은 서열번호 7의 아미노산 서열로 표시되는 링커에 의해 연결되는 것을 특징으로 하는, 생체분자를 세포 내로 전달할 수 있는 폴리펩타이드.The polypeptide of claim 1, wherein the C terminus and the cargo domain of the domain 2 are linked by a linker represented by the amino acid sequence of SEQ ID NO. 7.
  9. 제1항에 있어서, 상기 카르고 도메인의 C말단에 소포체 잔류서열이 추가로 연결되어 있는 것을 특징으로 하는, 생체분자를 세포 내로 전달할 수 있는 폴리펩타이드.The polypeptide of claim 1, wherein the endoplasmic reticulum sequence is further linked to the C terminus of the cargo domain.
  10. 제9항에 있어서, 상기 소포체 잔류서열은 서열번호 8 또는 9로 표시되는 것을 특징으로 하는, 생체분자를 세포 내로 전달할 수 있는 폴리펩타이드.10. The polypeptide of claim 9, wherein the endoplasmic reticulum residue sequence is represented by SEQ ID NO: 8 or 9.
  11. 제1항 내지 제10항 중 어느 한 항의 폴리펩타이드를 코딩하는 폴리뉴클레오타이드.A polynucleotide encoding the polypeptide of any one of claims 1 to 10.
  12. 제11항의 폴리뉴클레오타이드를 포함하는 재조합 벡터.Recombinant vector comprising the polynucleotide of claim 11.
  13. 제11항의 폴리뉴클레오타이드가 도입되어 있는 재조합 미생물.A recombinant microorganism into which the polynucleotide of claim 11 is introduced.
  14. 제12항의 재조합 벡터가 도입되어 있는 재조합 미생물.A recombinant microorganism into which the recombinant vector of claim 12 is introduced.
  15. 다음의 단계를 포함하는, 생체분자를 세포 내로 전달할 수 있는 폴리펩타이드의 제조방법:A method for preparing a polypeptide capable of delivering a biomolecule into a cell, comprising the following steps:
    (a) 제13항의 재조합 미생물을 배양하여 제1항의 폴리펩타이드를 생성하는 단계; 및(a) culturing the recombinant microorganism of claim 13 to produce the polypeptide of claim 1; And
    (b) 상기 생성된 폴리펩타이드를 회수하는 단계.(b) recovering the generated polypeptide.
  16. 다음의 단계를 포함하는, 생체분자를 세포 내로 전달할 수 있는 폴리펩타이드의 제조방법:A method for preparing a polypeptide capable of delivering a biomolecule into a cell, comprising the following steps:
    (a) 제14항의 재조합 미생물을 배양하여 제1항의 폴리펩타이드를 생성하는 단계; 및(a) culturing the recombinant microorganism of claim 14 to produce the polypeptide of claim 1; And
    (b) 상기 생성된 폴리펩타이드를 회수하는 단계.(b) recovering the generated polypeptide.
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