WO2016111420A1 - Cell penetration and enzymatic stabilization functions of α-helix domain in 30kc19 protein, and cargo delivery system using same - Google Patents

Abstract

The present invention relates to a 30Kc19α cell penetrating peptide and a cargo delivery system using the same. Specifically, the 30Kc19α peptide according to the present invention exhibits cell penetration, intracellular stability, or cell penetration and intracellular stability, which are similar or superior to the 30Kc19 entire domain, and thus the 30Kc19α peptide, as a cell penetrating peptide, binds to a cargo, thereby effectively delivering the cargo into cells.

Description

Cell Permeability and Enzyme Stabilization of α-ｈｅｌｉｘ Domain in 30KC19 Protein and a Cargo Delivery System Using the Same

The present invention relates to cell permeability and enzyme stabilization function of the α-helix domain (30Kc19α) in the 30Kc19 protein and cargo delivery system using the same. Specifically, since 30Kc19α 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 30Kc19α can effectively deliver cargo into cells by binding to cargo as a cell permeation peptide. .

In general, cell membranes of living animals, including human cells, do not pass large biomolecules such as proteins or nucleic acids, and large inorganic materials such as magnetic nanoparticles and quantum dots. Some smaller materials are passed through the cell membrane at low efficiency and into the cell, but large substances cannot be delivered into the cell unless the specific receptor for it is on the cell membrane. Therefore, materials used for the treatment and diagnosis of most diseases are difficult to introduce or transfer into cells.

Various methods for introducing or delivering a substance into cells have been developed. In particular, electroporation for delivering nucleic acids, projection using microinjection, and membrane fusion using liposomes are typical. However, the 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. Moreover, unlike nucleic acids, proteins do not have efficient intracellular delivery methods that have been proven to date.

In such a situation, cell-permeable proteins or peptides have recently attracted attention. The most studied of these is the 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. Has been reported to have cell penetrating function [Fawell S. et al., Proc. Natl. Acad. Sci. USA 91, 664-668 (1994). Similar examples include amino acids 267 to 300 of the VP22 protein of HSV-1 (Herpes Simplex Virus type 1) [Elliott G. et al. Cell, 88,223-233 (1997)] and 339 th to 355 th amino acids of Antennapedia (Antp) in Drosophila [Schwarze S.R. et al. Trends Pharmacol Sci. 21, 45-48 (2000). Recently, artificially synthesized positively charged peptides have also been reported to have cell permeability [Laus R. et al. Nature Biotechnol. 18, 1269-1272 (2000). Based on the above facts, studies have been conducted to bind proteins and nucleic acids to the protein transduction domain (PTD) of these cell penetrating proteins and deliver them into cells. Incorporation of green fluorescence protein (GFP) into TAT PTD was observed to penetrate the cell membrane [Park J. et al. J. Gen. Virol., 83, 1173-1181 (2002)], as well as Cre recombinase in TAT [Nicolas J. et al. Biochem. Biophys. Res. Commun. 316, 12-20 (2004)] and Super Oxide Dismutase (SOD) [Eum W. et al. Free Radical Biol. Med. 37, 339-349 (2004)] have been reported to confirm that the activity of this binding protein appears in the cell by binding to and delivered into the cell. However, it has been pointed out that most cell permeation proteins including TAT, which have been studied in the past, are often virus-derived and may exhibit cytotoxicity when introduced in vivo. To overcome this, human-derived cell penetrating proteins or artificially synthesized PTDs have been studied as an alternative. But even this is not the exact study of cytotoxicity.

Techniques for delivering proteins and nucleic acids into living cells using TAT PTD, the most well known PTD, have been disclosed in a variety of prior studies and patents. 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. In addition, protein delivery using TAT is described in various patents. U.S. Patent Publication No. 20060182736 also discloses intracellular delivery of proteins using PTDs of other cell penetrating proteins such as VP22 and Antp in addition to TAT. The prior arts are cell delivery technologies of large molecules such as proteins. However, 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. Although there are studies showing that taking only PTD portion of TAT or VP22 and delivering it to cells, the efficiency of delivery increases and the cytotoxicity is relatively low, but the exact verification is insufficient.

The inventors have identified that the α-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 α-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.

According to one embodiment of the present invention, a cell penetrating peptide is disclosed, comprising an α-helix domain (30Kc19α) of a 30Kc19 protein.

In the cell penetrating peptide according to the present invention, the 30Kc19α 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.

In the cell penetrating peptide according to the present invention, the 30Kc19α is characterized in that it is derived from a silkworm ( Bombyx mori).

In the cell penetrating peptide according to the present invention, the 30Kc19α is characterized in that consisting of the first to 88th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 1.

According to another embodiment of the present invention, a cargo delivery system is disclosed comprising a 30Kc19α cell penetrating peptide and a cargo fused to the N-terminus or C-terminus of the cell penetrating peptide.

In the cargo delivery system according to the invention, the cargo is characterized in that the protein, nucleic acids, peptides, lipids, glycolipids, minerals, sugars, contrast agents, drugs or chemicals.

In the cargo delivery system according to the invention, the peptide is characterized in that it is a cytokine, an antibody, an antibody fragment, a therapeutic enzyme or a soluble receptor.

In the cargo delivery system according to the invention, the contrast material is characterized in that the radiopaque contrast material, paramagnetic contrast material, superparamagnetic contrast material or CT contrast material.

According to another embodiment of the present invention, a method for tracking the route of application drug delivery in a subject comprising applying to the subject a 30Kc19α cell penetrating peptide and a contrast agent.

In the method for tracking drug delivery route according to the present invention, the 30Kc19α 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.

According to another embodiment of the present invention, intracellular drug delivery to a subject comprising a composition comprising a 30Kc19α 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.

In the intracellular drug delivery kit according to the present invention, the 30Kc19α is characterized in that consisting of the first to 88th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 1.

According to another embodiment of the present invention, a drug, cosmetic or food composition having an excellent effect of delivering an active ingredient into a cell, including a 30Kc19α cell penetrating peptide, is disclosed.

In the composition according to the present invention, the 30Kc19α is characterized in that consisting of the first to 88th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 1.

30Kc19α according to the present invention 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 α-helix domain and β-sheet domain of 30Kc19 protein.

Figure 2 shows the results of Western blotting analysis of α-helix domain and β-sheet domain.

Figure 3 shows that the α-helix domain acts as a protein trasnduction domain (PTD) in 30Kc19 protein and is produced as a water-soluble protein.

Figure 4 shows the results of SDS-PAGE analysis of 30Kc19α according to Example 1.

5 shows the results of SDS-PAGE analysis of 30Kc19 according to Comparative Example 1. FIG.

Figure 6 shows the results of Western blotting analysis of 30Kc19α protein entered into the cell according to Experimental Example 1 (1).

Figure 7 shows the results of fluorescence image analysis of 30Kc19α into the cell according to Experimental Example 1 (2).

8 shows the results of experiments comparing cell permeability between 30Kc19a and 30Kc19 according to Experimental Example 1 (3).

9 shows the results of spectrophotometer analysis of 30Kc19 and 30Kc19α according to Experimental Example 2. FIG.

Figure 10 shows the results of the spectrofluorometer analysis with the addition concentration and time of 30Kc19α according to Experimental Example 2.

11 shows the results of β-gal analysis of cells according to Experimental Example 3 (1).

12 shows the cytotoxicity evaluation results according to Experimental Example 3 (2).

Unless defined otherwise herein, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art. Various scientific events, including the terms included herein, are well known and available in the art. Although any methods and materials similar or equivalent to those described herein are found to be used in the practice or testing herein, some methods and materials have been described. Depending on the context used by those of ordinary skill in the art, they can be used in a variety of ways, and therefore should not be construed as limiting the invention to particular methods, protocols, and reagents.

As used herein, the singular encompasses the plural objects unless the context clearly dictates otherwise. As used herein, "or" means "and / or" unless stated otherwise. Moreover, the terms “comprising” as well as other forms, such as “having”, “consisting of” and “consisting of” are not limiting.

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 α-helix domain of the 30Kc19 protein (hereinafter referred to as "30Kc19α") 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.

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 α-helix domain (1-88 amino acids of SEQ ID NO: 1) and a β-sheet domain (89-239 amino acids of SEQ ID NO: 1) (Fig. 1). Ongoing studies have shown that the amino acid sequence of the protein transduction domain (PTD), which is a cell permeable part of the 30Kc19 protein, has an excellent effect as a cell-permeable peptide with the α-helix domain of the 30Kc19 protein alone.

The α-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). In addition, the α-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).

According to one embodiment of the invention, a 30Kc19α 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. In addition, 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.

As used herein, the term "Cell Penetrating Peptide (CPP)" refers to a peptide capable of moving cargo into a cell in vitro and / or in vivo. .

As used herein, the term "amino acid" includes D-isomers and modified amino acids as well as 22 standard amino acids that are naturally incorporated into the peptide. In addition, 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. On the other hand, 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. For example, 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.

TABLE 1

According to another embodiment of the present invention, a cargo delivery system is disclosed comprising a 30Kc19α 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.

As used herein, the term "cargo" includes all substances capable of binding into a cell by binding to the 30Kc19α 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.

* As used herein, the term "carrier peptide" refers to a peptide that serves to bind the active ingredient to move the active ingredient to a desired site.

As used herein, the term "drug" is a broad concept that includes substances for alleviating, preventing, treating or diagnosing a disease, wound or particular condition. In the above embodiment, 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.

The term "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. Including, but not limited to, one or more of DNA, cDNA, decoy DNA, RNA, siRNA, miRNA, shRNA, stRNA, snoRNA, snRNA, PNA, antisense oligomer, plasmid and other modified nucleic acids It is not.

As used herein, the term "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. For example, radiopaque contrast materials (for X-ray imaging) include inorganic iodine compounds and organic iodine compounds (e.g., diatrizoat), radiopaque metals and salts thereof (e.g., silver, gold, platinum Etc.) and other radiopaque compounds (eg, calcium salts, barium salts such as barium sulfate, tantalum and tantalum oxide). Paramagnetic contrasting materials (for MR imaging) 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), hydroxybenzyl Ethylene-diamine diacetic acid (HBED). Superparamagnetic contrast agents (for MR imaging) 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. Can be. In addition, the contrast material may include β-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.

As an example, when the cargo according to the present invention is a protein or a peptide, by binding to the DNA expressing the transport peptide to the DNA expressing the transport target and then expressing it, 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. In this case, an expression inducer such as IPTG (isopropyl-1-thio-β-D-galactopyranoside) may be treated to effectively express the fusion protein. Then, 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. Can be. The carrier peptide may bind to a dyeing or fluorescent material, specifically fluorescein isothiocyanate (FITC) or Green Fluorescent Protein (GFP).

According to a specific embodiment of the present invention, a method for tracking a drug delivery route in a subject comprising applying the 30Kc19α 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.

According to a specific embodiment of the invention, for intracellular drug delivery to a subject comprising a composition comprising a 30Kc19α 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.

According to a specific embodiment of the present invention, a drug, cosmetic or food composition having an excellent effect of delivering an active ingredient into a cell, including a 30Kc19α 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 μg / mg to 0.5 mg / mg, more specifically 10 μg / 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.

The 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. For example, 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. In addition, 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. The blending amount of the above components can be easily selected by those skilled in the art within the range that does not impair the object and effect of the present invention, the blending amount is 0.01 to 5% by weight, specifically 0.01 to 3% by weight based on the total weight of the cosmetic composition Can be.

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.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the embodiments of the present invention are provided only to facilitate understanding of the present invention, and the protection scope of the present invention is not limited to the following embodiments.

<Example>

Example 1. Isolation of 30Kc19α Protein

The 30Kc19α 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. At this time, the histidine tag (His6-tag) sequence was included in the carboxyl terminal of 30Kc19α produced to facilitate purification. After culturing the strain in LB medium, 1 mM isopropyl-beta-di-thiogalactopyranoside (IPTG) was added and incubated at 37 ° C. for 4 hours to produce 30Kc19α protein. . Then, the cells collected through centrifugation were sonicated and broken down. The lysate was centrifuged at 12,000 rpm for 30 minutes, and the supernatant was purified using 30 Hiscrap HP column (5 mL, GE Healthcare). Purified 30Kc19α was buffer exchanged with Tris-HCl (pH 8.0) and analyzed by SDS-PAGE (FIG. 4). As can be seen in Figure 4, the band corresponding to 30Kc19α at about 15kDa was observed. The purified proteins were stored at -70 ° C and used for the experiment. Protein quantification was performed using Bovine serum albumin (BSA), a standard protein.

Comparative Example 1. Isolation of 30Kc19 Protein

The same method as in Example 1 was used except that 30Kc19 gene was used instead of 30Kc19α 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.

Experimental Example

Experimental Example 1. Evaluation of cell permeability of 30Kc19α protein (in vitro)

(1) Analysis of Cell Permeability of 30Kc19α

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 Kc19α 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. 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, 30Kc19α delivered to the cells was detected by Western blotting using 30Kc19α antibodies obtained from rabbits. The results are shown in FIG.

As can be seen from FIG. 6, since 30 Kc19α was detected in the cells, it was confirmed that the cells were permeable. In addition, as the concentration increased, the amount of 30Kc19α delivered to the cells also increased, indicating that the cell permeability of 30Kc19α was proportional to the concentration of the external treatment.

(2) Analysis of location of 30Kc19α in cells

30Kc19α protein was labeled with green fluorescent Alexa Fluor® 488 (Invitrogen). Fluorescence bound 30Kc19α 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 30Kc19α 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 30Kc19α and nuclei were shown.

As can be seen from Figure 7, it was confirmed that the higher the concentration of 30Kc19α, the greater the amount of protein is located in the cytoplasm.

(3) Comparison of 30Kc19α and 30Kc19 Cell Permeability

In order to compare the cell permeability between 30Kc19α and 30Kc19, HeLa cells and CHO cells were treated with 30Kc19a and 30Kc19 at the same concentration, and after 24 hours, cells were lysed using RIPA buffer to confirm the concentration of protein in the cytoplasm. The results are shown in FIG.

As can be seen from Figure 8, it was confirmed that more 30Kc19α enters the cytoplasm than 30Kc19.

Experimental Example 2. Evaluation of enzyme stability of 30Kc19α protein (in vitro)

The enzyme stability of 30Kc19α protein was evaluated using Green Fluorescent Protein (GFP). 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 30Kc19α protein and 30Kc19 protein cultured in Experimental Example 1, and the activity of GFP was measured. The results are shown in FIG.

In addition, the concentration of 30Kc19α and the degree of fluorescence of GFP over time were measured and shown in FIG. 10.

As can be seen from FIG. 9, in the absence of 30Kc19α or 30Kc19 protein, GFP activity decreased by about 30% or more after 24 hours, but when 400 μg / ml of 30Kc19α or 30Kc19 protein was added, the GFP activity was maintained at 50% or more. Confirmed. It was also confirmed from FIG. 10 that the higher the concentration of 30Kc19α, the higher the GFP activity was maintained.

Experimental Example 3. Evaluation of intracellular stability of 30Kc19α protein (in vivo)

(1) Intracellular stability evaluation

Beta galactosidase was transfected into lipofectamine3000 in HeLa cells, and after 24 hours, 30Kc19α protein was treated with 0 ㎍ / ml, 40 ㎍ / ml, 80 ㎍ / ml, 120 ㎍ / ml and 200 ㎍ / ml, respectively. Β-gal assay was performed after 24 hours, and the results are shown in FIG. 10.

As can be seen from FIG. 11, it was confirmed that the 30Kc19α protein was stable in the cell. In particular, when the 30Kc19a protein is 80 μg / ml concentration showed the highest stability.

(2) Cytotoxicity Assessment

After absorbing 30Kc19α protein in CHO cells and incubated for 24 hours, the absorbance was measured by CCK-8 assay. The results are shown in FIG.

As can be seen from Figure 12, it was confirmed that the cells treated with 30Kc19α protein showed similar viability to the control cells not treated with the protein. From this it was demonstrated that the 30Kc19α protein according to the present invention does not cause toxicity in the cell and can be injected intracellularly.

Claims (10)

1. A cell penetrating peptide comprising an α-helix domain (30Kc19α) of a 30Kc19 protein.
2. The method of claim 1,

   Wherein said 30Kc19α has cell permeability, intracellular stability, or cell permeability and intracellular stability that is comparable to or greater than the 30Kc19 full domain.
3. The method of claim 1,

   The 30Kc19α is a cell penetrating peptide derived from Bombyx mori .
4. The method of claim 1,

   The 30Kc19α is a cell penetrating peptide consisting of the first to 88th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 1.
5. A cell penetrating peptide according to any one of claims 1 to 4; And

   Cargo fused to the N-terminus or C-terminus of the cell penetrating peptide

   Cargo delivery system comprising a.
6. The method of claim 5,

   Wherein the cargo is a protein, nucleic acid, peptide, lipid, glycolipid, mineral, sugar, contrast, drug or chemical.
7. The method of claim 6,

   Wherein said peptide is a cytokine, an antibody, an antibody fragment, a therapeutic enzyme or a soluble receptor.
8. The method of claim 6,

   Said contrast material is radiopaque contrast material, paramagnetic contrast material, superparamagnetic contrast material or CT contrast material, cargo delivery system.
9. A method for tracking drug delivery to a subject, comprising applying the cell penetrating peptide and the contrast medium according to any one of claims 1 to 4 to the subject.
10. A cell of a subject comprising the cell penetrating peptide according to any one of claims 1 to 4, a composition comprising a drug, and instructions which disclose one or more of the dosage, route of administration, frequency of administration and indications of the composition. Kit for my drug delivery.

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