KR20060017348A - Cell-transducing botoxin fusion protein - Google Patents

Abstract

The present invention relates to a botox fusion protein that can be easily penetrated into the dermis or into a cell when applied to the skin surface by forming a covalently bonded botox fusion protein of the botox and a protein transport domain. Protein transport domain of the invention is composed of 15 to 30 amino acids, a hydrophobic domain (hydrophobic domain) containing five or more tryptophan, a hydrophilic domain containing a large number of lysine and a spacer separating the two domains It was composed of a spacer, and compared with the control botoxin, it was smoothly penetrated into cells in a concentration-dependent and time-dependent manner, and lasted more than 36 hours in the cells.

Botoxin, Cell Transduction, Fusion Protein, Skin

Description

Cell-transducing botoxin fusion protein             

Figure 1 shows the expression vector of PEP-1-botoxin fusion protein. (a) Construction of PEP-1-botoxin expression vector based on pET-15b vector. Synthetic PEP-1 oligomers are cloned into NdeI , XhoI restriction sites. Botoxin cDNA was cloned into Xho I and Bam HI restriction sites of pET-15b. (b) Diagram of expressed control botoxin and PEP-1-botoxin fusion proteins. The coding frame of botox is shown as an open box following the 6His and PEP-1 peptides. The resulting vector was named pPEP-1- Botoxin. Expression was induced by addition of IPTG.

Figure 2 relates to the expression, purification and intracellular localization of PEP-1-botoxins. Protein extracts and purified fusion proteins of cells were analyzed by 15% SDS-PAGE (A) (B) and Western blot analysis with anti-rabbit polyhistidine antibody (C). The lanes of A, B and C are as follows:

Lane 1, non-induced pPEP-1-botoxin;

Lane 2, induced pPEP-1-botoxin;

Lane 3, purified pPEP-1-botoxin.

Immunofluorescence and distribution of PEP-1-botoxin fusion proteins introduced when HeLa cells were immobilized with paraformaldehyde (D). HeLa cells were treated with 2μM PEP-1-botoxin fusion protein for 30 minutes, incubated with anti-rabbit polyhistidine (1: 400) and 1 hour with Cy-3 conjugated antibody (1: 1000). Were incubated during.

Control cells themselves (C-left) without PEP-1-botoxin,

Cells treated with PEP-1-botoxin (C-right).

Incorporation of PEP-1-botoxin fusion protein into cultured HeLa cells. (a) 0.25-2 μM PEP-1-botoxin fusion protein and control botoxin were added to the culture medium for 30 minutes. (b) 2 μM of PEP-1-botoxin and control botoxin were added to the culture medium for 10-60 minutes each. Intracellularly introduced PEP-1-botoxin was analyzed by western blotting.

4. Stability of PEP-1-botoxin fusion protein introduced into cultured HeLa cells

Cells infiltrated by 2 μM PEP-1-botoxin were incubated for various time intervals (1-48 h). PEP-1-botoxin fusion proteins introduced into cells were analyzed by western blotting.

5. Histological analysis of animal skin into which PEP-1-botoxin fusion protein was introduced.

50 μg of PEP-1-botoxin fusion protein was applied locally to the shaved area of the skin such as a mouse for 60 minutes. Frozen sections of skin tissue were immunostained with rabbit anti-histidine IgG (1: 400) and stained with biotin-coupled goat antirabbit IgG (1: 200). Sections were visualized with 3,3'-diaminobenzidine and observed under an axioscope microscope.

Botulinum rinum toxin (Botulinum toxin; also mixed with the "beam toxin" or "BTX") is a neurotoxin produced by the anaerobic bacterium Clostridium botulinum rinum (Clostridium botulinum) the bacteria that grow in rotten canned or scavengers) A, It is specified as B, C1, C2, D, E, F and G type. Among them, A, B, and E types are associated with human diseases, and C and D types are associated with diseases of domestic animals and other animals. These seven antigenic forms all have similar structure and molecular weight. A heavy chain having a molecular weight of 100,000 and a light chain of 50,000 are connected by disulfide bonds. The light chain is thought to have zinc dependent endopeptidase activity that separates neuroproteins.

In addition, botoxine paralyzes muscles by blocking the secretion of the neurotransmitter acetylcholine, where motor and muscle meet, and weakens and contracts muscle after synaptic transmission is inhibited by botoxin. The nerve endings thus affected are not degraded and the disturbance of neurotransmitter secretion is irreversible. Therefore, its function is restored by the growth of nerve endings and the formation of new synaptic contacts. Botoxins induce muscle weakness by disrupting the delivery of alpha motor neurons at neuromuscular junctions. Therefore, it can be used for the overactivation state of muscles.

Therefore, if the dose is used as an injection medicine that does not affect the human body at all, the US Food and Drug Administration (FDA) permits it to be used in humans in the early 1980s, using the principle that it can partially paralyze the muscle of the injection site. It has been used as a therapeutic drug for neurological diseases and muscle diseases. Since then, it has been used as a treatment for various muscle diseases such as facial spasms, eyelid spasms, and deaths (斜頸: head distorted and tilted to one side). Since 1990, it has been widely used as an anti-wrinkle medicine. In other words, by using the toxin of Clostridium botulinum, the wrinkles are stretched by paralyzing or worsening the muscles that make wrinkles on the eyes, the forehead, and the forehead.

Botoxins, particularly botoxin type A, are for example strabismus, blepharospasm, cervical dystonia, oromandibular dystonia, and spasmodic dysphonia. It has been used to treat muscle abnormalities, and recently, the function of relieving hyperhidrosis and migraine headaches is also known.

Botoxin-related three-step mechanism has been proposed for the above action. First, it consists of the binding of cell membrane receptors and BTX which have not yet been identified through the heavy chain, and second, internalization of the heavy chain and the light chain, and third, cleavage of the target protein by separation of the light chain and inhibition of acetylcholine secretion.

Botoxin is currently marketed under the trade names DYSPORT (Porton Products Ltd., UK) and BOTOX (Allergan, Inc., Irvine, Calif.).

Commercially available botox products are injected into the muscle under the diagnosis of a neurologist, ophthalmologist, orthopedic surgeon in the form of an injection.

However, injection is inconvenient to use compared to topical skin preparations.

The present invention relates to a botox fusion protein that can be easily penetrated into the dermis or into cells when applied to the skin surface by covalently binding botox to the protein transport domain. It consists of ˜30 amino acids, and consists of a non-hydrophobic domain (hydrophobic domain) containing five or more tryptophan, a hydrophilic domain containing a large number of lysine, and a spacer separating the two domains . Fusion protein in the form covalently bound to the end of the protein ("target protein") that the protein transport domain is intended to transport into the cell is prepared by chemical method or at the genetic level and introduced into the cell or into the skin.

Recently, it has been found that PEP-1 peptides can be used as carriers to transfer heterologous proteins into cells as they are, such as Tat proteins. However, there is almost no experiment using the PEP-1 peptide.

Peptides, polypeptides and proteins, despite their excellent selectivity and potency for certain physiological functions compared to other compounds, are difficult to pass through the cell membrane due to their size and various biochemical properties, and therefore cannot be delivered directly into the cell. Due to its weakness, practical use as an effective therapeutic agent and basic research means is difficult. In general, substances with molecular weights of 600 or more are known to be almost impossible to cross the cell membrane (Schwartze et al., 1999; Van de Waterbeemd et al., 1998).

Many methods have been attempted to deliver proteins, polypeptides and peptides intracellularly. These methods include the following methods. Physical treatment methods (ie microinjection, scrape loading, electroporation and bioolistics), chemically or biologically forming pores in cells Digitinin, pore-forming proteins and ATP treatment, methods using modified proteins or protein carriers (lipidated proteins, immunotoxins and related bioconjugates) (immunotoxins / related bioconjugates) and particle inhalation or fusion methods (liposomes, cell-cell fusion, virus mimics and induced pinocytosis). (Fernandez and Bayley, 1998). The above mentioned methods have the following problems. For example, microinjection is efficient only when studying a single cell or a small number of cells, and immunotoxins usually do not deliver proteins at high concentrations to the cell, as well as proteins with lethal activity. It can also kill target cells when used. In addition, these methods have problems such as intracellular delivery efficiency of proteins, damage to cells, arrival of target proteins, and reproducibility.

In addition, gene therapy is not easy to transport genes, low expression in target cells, short period of protein expression in the cell, very difficult to artificially control the amount of protein expressed in the target cell, etc. It has several problems (Verma, IM and Somia, N. (1997) Nature 389, 239-242).

The protein transduction domain (PTD) of Tat protein, a human immunodeficiency virus protein, has been shown to efficiently pass through cell membranes. Recently, many heterologous proteins have been fused with Tat protein and transported into cells. (Fawell, S. et al. (1994) Proc. Natl. Acad. Sci. USA. 91, 664-668., Schwartze, SR et al. (1999) Science. 285, 1569-1572, Watson, K.). and Edward, RJ (1999) Biochem. Pharmacol. 58, 1521-1528). In addition, our laboratory showed that copper, zinc-superoxide dismutase and catalase fused with Tat protein have biological activity and are efficiently transported into cells (Kwon, HY et al. (2000) FEBS Letter) 485, 163-167, Eum, WS et al. (2002) Mol. Cells. 13, 334-340, Jin, LH et al. (2001) Free Radic. Biol. Med. 31, 1509-1519).

However, it is understood that Tat-fusion proteins are not transported into cells in nature, but are transported into cells in a denatured state and then refolded in the cells to have activity. Thus, the stability and persistence of these Tat fusion proteins do not appear to be more efficient than in their natural state (Schwartze, S.R. et al. (2000) trends in CELL BIOLOGY. 10, 290-295).

Recently, a method of transporting natural heterologous proteins into cells using PEP-1 peptide, which is a more effective method of protein delivery, has been found (Morris, MC et al. (2001) Nat. Biotech. 19, 1173-1176). The PEP-1 peptide consists of 21 amino acids (KETWWETWWTEWSQPKKKRKV) (SEQ ID NO: 3) and has three domains (hydrophobic, spacer, hydrophilic domain). So far, studies using PEP-1 peptides have revealed that the proteins can be naturally transported into cells when administered to cells simultaneously without covalently binding the PEP-1 peptide and foreign proteins. In addition, PEP-1 peptides have several advantages over Tat proteins as protein therapeutics: they efficiently penetrate proteins into cells, are stable in physiological buffers, lack toxicity, and lack serum sensitivity. It has such advantages. However, PEP-1 peptides may be expressed at a constant rate (approximately PEP-1 and protein ≒ 30: 1) with external proteins such as green fluorescent protein (GFP), beta galactosidase (β-Gal), and the like. Since the protein is effectively transported within the cell only by administration, it is necessary to add an expensive PEP-1 peptide.

Accordingly, it is an object of the present invention to provide a botox fusion protein with enhanced cell penatration ability of botox.                         

Another object of the present invention is to develop a formulation that can use botoxin by a simple application method, not by injection method.

In addition, an object of the present invention is a non-hydrophilic domain consisting of 15 to 30 amino acids, including PEP-1, including at least five tryptophan, a hydrophilic domain including a large number of lysine and a spacer separating the two domains By covalently binding the constructed protein transport domain to at least one end of the botoxin to maximize the cell permeability and skin permeability of the botox.

The inventors found that the light chain of the botoxin and heavy chains exhibits a catalytic site in the light chain and covalently binds the protein transport domain PEP-1 to the end of the botox lightchain. Botoxin fusion protein was prepared. The inventors first combined the gene encoding PEP-1 with the 6His expressing nucleotide to the gene encoding the botoxin light chain and overexpressed in E. coli.

The inventors fused (covalently) fused to the botox PEP-1 peptide, which carries a natural botox protein into the cell, overexpressed the fusion protein in E. coli, and metal-chelating affinity. Purification was easy and convenient by chromatography. In addition, it was confirmed through the experiments of cultured HeLa cells and animals that the purified fusion protein has an effective biological activity and transported into the cells.

For this study, we first developed a PEP-BTX expression vector that can overexpress and easily purify the PEP-1-BTX fusion protein. The expression vector is a botox cDNA, PEP peptide (21 amino acids) and six histidines connected in series. Using this expression vector, the PEP-BTX fusion protein was overexpressed in E. coli and purified in a natural state using a Ni ²⁺ -affinity chromatography column. The overexpression of PEP-BTX was significantly higher, resulting in higher amounts of purified protein. It was confirmed that PEP-BTX fusion protein purified on HeLa cells cultured with Western blot was transported to the cells in a concentration and time-dependent manner. PEP-BTX permeated into cells was maintained for at least 36 hours in minimal cells. PEP-BTX penetrated not only the cultured cells but also the skin tissues of mice.

These results indicate that PEP-BTX is well permeated into cells. Therefore, this PEP-BTX fusion protein suggests the possibility of various applications in the field of therapeutics, such as the study of the delivery technology into cells and muscle diseases known to be related to botoxins.

Pharmaceutical compositions containing PEP-1-botoxine fusion protein as an active ingredient can be formulated in injection form or coating form by conventional methods in combination with a carrier that is conventionally acceptable in the pharmaceutical art. Injectable compositions are preferably aqueous isotonic solutions or suspensions, and the compositions mentioned are sterile and / or contain auxiliaries (eg, preservatives, stabilizers, wetting or emulsifier solution promoters, salts or buffers for controlling osmotic pressure). In addition, they may contain other therapeutically valuable substances.

The pharmaceutical preparations thus prepared may be parenterally, ie, subcutaneously, intramuscularly, or topically, as desired. The dosage may range from 1 mg to 10 mg / kg in a daily dosage of 1 to 10 mg / kg. It can be divided into several doses. Dosage levels for a particular patient may vary depending on the patient's weight, age, sex, health condition, time of administration, method of administration, severity of the disease, and the like.

In addition, the coating agent using the botoxin fusion protein of the present invention as an active ingredient can be easily prepared in any form according to a conventional manufacturing method. For example, in preparing a cream coating agent, the PEP-BTX fusion protein of the present invention is contained in a general oil-in-water type (O / W) or water-in-oil type (W / O) cream base, and includes perfume, chelating agent, pigment, and oxidation. Anti-preservatives, preservatives and the like may be used as necessary, and synthetic or natural materials such as proteins, minerals and vitamins may be used in combination for the purpose of improving physical properties.

The present inventors were able to confirm that the PEP-BTX fusion protein penetrates into the skin of the mouse and penetrates smoothly to the dermis layer. Accordingly, it has been found that the botoxin fusion protein can be used as a major component of pharmaceutical compositions and / or coatings (used herein in the same sense as "skin external preparations") compositions.

The present invention provides a method for efficiently delivering a botoxin light chain having a molecular weight of 50,000 Daltons into cells or into the skin. Intracellular delivery of a botoxin protein molecule according to the present invention comprises 15-30 amino acids in a botoxin protein, a hydrophilic domain comprising five or more tryptophans, and a hydrophilic containing four or more lysines. A transport domain consisting of a hydrophilic domain and a spacer separating the two domains is performed by constructing a covalently bonded fusion protein of a cell-permeable transport domain. An example of the transport domain of the present invention includes a PEP-1 peptide consisting of 21 amino acids and consisting of an amino acid sequence such as SEQ ID NO: 3. However, the protein transport domain of the present invention is not limited only to the PEP-1 peptide of SEQ ID NO: 3, and the production of a peptide having a function similar to that of the PEP-1 peptide by partial replacement, addition or lack of the amino acid sequence of PEP-1 Since it is easy for those skilled in the art to which the present invention pertains, a hydrophilic domain consisting of 15 to 30 amino acids, including 5 or more tryptophans, and a hydrophilic domain including 4 or more of lysine (fusion protein using a protein transport domain consisting of a hydrophilic domain and a spacer separating the two domains and a protein transport domain that performs the same or similar protein transport function by substituting a partial amino acid therefrom are also included in the scope of the present invention. Belonging will be self-evident.

Specifically, the present invention relates to a PEP-1 botoxin fusion protein, a recombinant nucleotide and a vector for producing the fusion protein, a treatment comprising the fusion protein, a pharmaceutical composition for the purpose of prevention, a composition for external application for skin, and the like.

Definitions of main terms used in the detailed description of the present invention are as follows.

"PEP-1-botoxin fusion protein" refers to a covalent complex formed by genetic fusion or chemical bonding of a transport molecule and a cargo molecule, comprising a protein transport domain and a botox light chain moiety. In the present specification, it is mixed with "PEP-1-botoxin", "botoxin fusion protein" or "PEP-BTX".

In addition, the "genetic fusion" means a linear, covalent linkage formed through the genetic expression of the DNA sequence encoding the protein.

In addition, "target cell" means a cell to which a cargo molecule is delivered by a transport domain, and a target cell refers to a cell in or outside the body. That is, a target cell is meant to include cells in the body, that is, cells constituting organs or tissues of a living animal or human, or microorganisms found in a living animal or human. In addition, target cells are meant to include extracellular cells, ie cultured animal cells, human cells or microorganisms.

As used herein, the term "protein transport domain" refers to a covalent bond with a peptide or a protein, which may introduce the peptide or protein into a cell without the need for a separate receptor, carrier, or energy. For example, a PEP-1 peptide (SEQ ID NO: 3).

As used herein, “target protein” refers to a molecule that is covalently bound to a PEP-1 protein transport domain and introduced into a cell to exhibit activity.

Also used herein are expressions such as "transduction", "transport", "penetration", "transport", "transfer", "transmission", "pass" for "introducing" a protein or peptide into a cell. Mixed.

According to the present invention, a protein transport domain consisting of 15-30 amino acids and comprising a non-hydrophilic domain including 5 or more tryptophan, a hydrophilic domain including a large number of lysines and a spacer separating the two domains comprises at least one of the botoxin proteins. It relates to a cell-transducing botox fusion protein covalently bonded at one end. In addition, silent changes may result in one or more amino acids in the sequence being substituted with other amino acid (s) of similar polarity that function functionally equivalently. Amino acid substitutions in the sequence may be selected from other members of the class to which the amino acid belongs. For example, hydrophobic amino acid classifications include alanine, valine, leucine, isoleucine, phenylalanine, valine, tryptophan, proline and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. Positive basic amino acids include arginine, lysine and histidine. Negatively charged acidic amino acids include aspartic acid and glutamic acid. Also included within the scope of the invention are fragments or derivatives thereof having the same similar biological activity within a range of homology, such as 85-100%, between the botoxin fusion protein of the invention and the amino acid sequence.

In addition, the present invention is characterized in that the cell-induced botoxin fusion protein has the amino acid sequence of SEQ ID NO: 7.

In addition, the present invention is characterized in that the protein transport domain is covalently bonded to one or both sides of the carboxy terminal and the amino terminal of the botoxin protein.

The present invention also relates to an external composition for skin, comprising the cell-transducing botoxin fusion protein as an active ingredient.

The present invention also relates to a pharmaceutical composition comprising the cell-induced botoxin fusion protein as an active ingredient and comprising a pharmaceutically acceptable carrier.

In addition, the present invention relates to a recombinant polynucleotide comprising SEQ ID NO: 6, which binds a protein transport domain peptide coding DNA sequence to a botox cDNA and encodes the cell-induced botox fusion protein. The scope of the present invention extends not only to recombinant polynucleotides of SEQ ID NO: 6 but also to nucleic acid molecules having sequences by the codon degeneracy of the genetic code.

In addition, the present invention relates to a vector expressing a cell-induced botox fusion protein, characterized in that it comprises the recombinant polynucleotide to express the fusion protein.

Hereinafter, the configuration of the present invention will be described in more detail with reference to specific embodiments. However, the scope of the present invention is not limited by the description of the following examples.

Example  1: PEP-1- Botoxin  Expression and Purification

A PEP-1-botoxin expression vector was prepared to express PEP-1 (KETWWETWWTEWSQPKKKRKV) peptide fused with botoxin.

First, two oligonucleotide top strands 5'-TATGAAAGAAACCTGGTGGGAAACCTGGTGGACCGAATGGTCTCAGCCGAAAAAAAAACGTAAAGTGC-3 'and the bottom strand 5'-TCGAGCACTTTACGTTTTTTTTTCGGCTGA-GACCATTCGGTCCACCAGTC-Phase-synthesized Oligonucleotides were made. Double-chain oligonucleotides were directly linked into the pET-15b vector truncated with Nde I- Xho I.

Next, two initiators were synthesized based on the cDNA sequence of botoxin. Sense initiator 5′-ACACTCGAGATGCAATTTGTTAATAAACAATTTAATT-3 ′ contains the Xho I restriction enzyme cleavage site and antisense initiator 5′-TGTGGATCCTGACTTATTGTATCCTTTATCTAATGATT-3 ′ has the Bam HI restriction enzyme cleavage site. The polymerase chain reaction (PCR) was carried out and the polymerase chain reaction product was cut into Xho I and Bam HI, inducted (Invitek, Berlin, German), using T4 DNA ligase (Takaka, Otsu, Shiga, Japan). Were linked to TA-cloning vector and pPEP-1 vector and cloned into E. coli DH5 cells.

Plasmids were transformed into E. coli BL21 cells to produce PEP-1-botoxin fusion proteins. The transformed bacterial cells were incubated at 37 ° C. in 100 ml of LB medium until the D ₆₀₀ value was 0.5-1.0, and cultured for 16 hours by introducing 0.5 mM IPTG at 30 ° C. The harvested cells were lysed at 4 ° C. in binding buffer (5 mM imidazole, 500 mM NaCl, 20 mM Tris-HCl, pH 7.9) at 4 ° C. and the recombinant PEP-1-botoxins formed were purified. Briefly, isolated cell extracts were loaded undenatured on a Ni ²⁺ -nitrilotriacetic acid Sepharose affinity column (Qiagen, Valencia, CA, USA). The column was washed with 10-fold binding buffer and 6-fold wash buffer (60 mM imidazole, 500 mM NaCl, and 20 mM Tris-HCl, pH 7.9) before the fusion protein was absorbed in Ilution buffer (1 M imidazole, 500 mM NaCl, 20 mM Tris-). HCl, pH 7.9). Fractions containing the fusion protein were collected and salts removed using a PD-10 column (Amersham, Braunschweig, Germany). Protein concentration was predicted by the Bradford procedure using bovine serum albumin as the standard.

Example  2: PEP-1- into Mammalian Cells Botoxin  Introduction of fusion proteins

HeLa cells were cultured in 6 well plates for 4-6 hours to introduce PEP-1-botoxin fusion proteins. The culture medium was then replaced with 1 ml of fresh medium. HeLa cells were treated with various concentrations of PEP-1-botoxin for 30 minutes and then the cells were treated with trypsin-EDTA (Gibco, Grand Island, NY, USA) and washed with phosphate buffered saline (PBS). The cells were harvested to prepare cell extracts for Western blot analysis.

Intracellular stability of the transduced PEP-1-botoxin fusion protein was confirmed as follows: HeLa cells were treated with 2 μM native PEP-1-botoxin for 1 hour and then washed. The cells were transferred to fresh culture medium to remove unintroduced PEP-1-botoxin. Cells were then incubated for 48 more hours and cell extracts were prepared for Western blot analysis.

Example  3: immunofluorescence and fluorescence analysis ( Immunofluorescecnce  and fluorescence analysis)

Immunofluorescence assay was performed using conjugated Cy-3 antibodies. In brief, HeLa cells were cultured on glass coverslips and treated with PEP-1-botoxin fusion protein. After incubation at 37 ° C. for 30 minutes, the cells were washed twice with trypsin-EDTA and PBS and fixed at room temperature for 10 minutes with 0.5 ml of PBS containing 4% paraformaldehyde. Cells were washed with PBS and then incubated with polyhistidine antibody and incubated with PBS containing Cy-3 antibody (1: 1000) for one hour. The distribution of fluorescence was analyzed by fluorescence microscopy (Carl Zeiss, EL-Einsatz, German).

Example  4: PEP-1- on Mouse Skin Botoxin  Introduction of fusion protein

About 30 g of male ICR mouse weight was used to study the introduction of PEP-1-botoxin fusion protein into mouse skin. Animals used in this experiment were treated according to the Principles of Laboratory Animal Care (NIH publication No. 86-23). Animals are anesthetized with nitrogen and oxygen containing 3% isoflurance and 50 g of control botox and PEP-1-botoxine fusion proteins are applied to the shaved area of the animal skin at various time intervals. It was. Frozen tissue pieces were then prepared and fixed for 4 minutes with 4% paraformaldehyde. To eliminate nonspecific immune responses, free-floating sections were incubated for one hour at room temperature with 0.3% Triton X-100 and PBS containing 10% normal goat serum. It was then incubated with rabbit antihistidine IgG (1: 500) for 24 hours at room temperature. After washing three times with PBS for 10 minutes, the sections were incubated with biotin-bound goat antirabbit IgG (dilution 1: 200; Vector Laboratories, Burlingame, CA, USA) for one hour. It was then visualized with 3,3-diaminobenzidine (40 mg DAB / 0.045% H ₂ O ₂ in 100 ml PBS) on gelatin coated slides. Immune responses were observed under an Axisoscope microscope (Carl Zeiss, Gottingen, Germany).

result

PEP-1- with Biological Activity Botoxin  Fusion protein generation

Botoxin cDNA was subcloned into pET-15b plasmid to make PEP-1-botoxin vector, a cell permeable expression vector. The formed PEP-1-botoxin expression vector contains the cDNA sequence encoding botox, and contains the PEP-1 protein (21 amino acids) and six histidine residues at the amino terminus. In addition, the inventors constructed a botoxin expression vector (p botoxin) to produce a control botoxin protein without PEP-1 introducing peptide (FIG. 1).

PEP-1- Botoxin  Expression and Purification of Fusion Proteins

After expression induction, PEP-1-botoxin and the control botoxin fusion protein were purified. That is, the fusion protein was expressed on E. coli and the isolated cell extract was loaded on a Ni 2+ -nitriloacetic acid Sepharose affinity column under conditions that were not denatured. The fusion protein containing fractions were mixed to remove salt using PD-10 column. Obtained from E. coli Crude cell extracts and purified PEP-1-botoxin fusion proteins were electrophoresis with 15% SDS-PAGE. The results of expression and purification are shown in Figures 2A and 2B. The bands in lanes 2 and 3 show that the protein is highly expressed and is a major component of the total water soluble protein. In addition, the yields of the purified fusion protein were about 45 mg / L. Expressed and purified proteins were confirmed by Western blot analysis using anti-rabbit polyhistidine antibody. PEP-1-botoxin was identified in the corresponding band of FIG. 2C.

HeLa  PEP-1- into Cells Botoxin  Introduction of fusion proteins transduction )

To study the ability of PEP-1-botoxin fusion proteins to be introduced into cells, PEP-1-botoxin fusion proteins were conjugated with Cy-3 fluorescein (1: 1000) and immunofluorescence microscopy. (FIG. 2D). As shown in FIG. 2D, the native PEP-1-botoxin fusion protein was successfully introduced into the cell, whereas the control botoxin was not introduced into the cell. These results indicate that the PEP-1 peptide has similar functions and mechanisms to the HIV-1 Tat protein, homeodomain of Antennapedia, and the herpes simplex virus VP22 protein.

In order to measure the cell transduction ability of PEP-1-botoxin fusion protein, PEP-1-botoxin fusion protein cell transduction was analyzed according to the amount change. PEP-1-botoxin fusion proteins of various concentrations (0.25-2 μM) were added to HeLa cells in culture medium and left for 30 minutes, and the amount of the introduced protein was measured by Western blotting (FIG. 3A). The amount of protein introduced into HeLa cells increased in concentration-dependently as the amount of fusion protein in the culture medium increased. Cell introduction of purified PEP-1-botoxin fusion protein in the undenatured state was performed by adding the fusion protein to HeLa culture medium for various time (10-60 min) at 2 μM concentration, and the protein introduced by Western blotting. The levels were analyzed. Intracellular concentrations of PEP-1-botoxin introduced into cells were identified within 10 minutes and gradually increased up to 60 minutes (FIG. 3B).

Intracellular stability of the PEP-1-botoxin fusion protein introduced into HeLa cells is shown in FIG. 4. PEP-1-botoxin fusion protein was added to HeLa cell culture medium at various concentrations of 2 μM for various times, and the level of the protein introduced was analyzed by Western blotting. As shown in FIG. 4, intracellular levels of PEP-1-botoxin fusion protein introduced into cells were first detected after one hour and then decreased continuously during the observation period. However, the transduced botoxin was maintained at marked levels in HELa cells for 36 hours.

PEP-1- to Mouse Skin Botoxin  Penetration of PEP-1-Botoxin fusion protein into mice skin

We assessed the ability of PEP-1-botoxin fusion proteins to introduce cells into mouse skin. The fusion protein was sprayed onto mouse skin for one hour, and the penetration of the fusion protein was analyzed by immunohistochemical method. As shown in FIG. 5, the cell transduction signal was remarkably detected in the dermis of the skin treated with PEP-1-botoxin fusion protein. Unlike the PEP-1-botoxin fusion protein, the control botoxin could not be introduced into the skin. These results indicate that PEP-1-botoxin fusion protein can not only be introduced into cultured cells but also penetrate into live animal skin.

The present invention has made it possible to use botoxin, which was used only as an injection formulation, as a skin coating form.

In addition, the present invention is to be smoothly transduced into cells and skin cells by co-combining botoxin with the protein transport domain in a native form (native form).

In addition, the botoxin fusion protein of the present invention can be easily penetrated into not only cultured cells but also skin cells of living animals, and thus can be applied as a skin external preparation.

<110> CHOI, JIN HEE          BIOBUD Co., Ltd. <120> Cell transducing botoxin fusion protein <130> wjtj-0408-pepp-btx <160> 7 <170> KopatentIn 1.71 <210> 1 <211> 68 <212> DNA <213> Artificial Sequence <220> <223> top strand oligonucleotide coding PEP-1 <400> 1 tatgaaagaa acctggtggg aaacctggtg gaccgaatgg tctcagccga aaaaaaaacg 60 taaagtgc 68 <210> 2 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> bottom strand oligonucleotide coding PEP-1 <400> 2 tcgagcactt tacgtttttt tttcggctga gaccattcgg tccaccaggt ttcccaccag 60 gtttctttcc 70 <210> 3 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> protein transducing domain called PEP-1 <400> 3 Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys   1 5 10 15 Lys Lys Arg Lys Val              20 <210> 4 <211> 37 <212> DNA <213> Clostridium botulinum <400> 4 acactcgaga tgcaatttgt taataaacaa tttaatt 37 <210> 5 <211> 38 <212> DNA <213> Clostridium botulinum <400> 5 tgtggatcct gacttattgt atcctttatc taatgatt 38 <210> 6 <211> 1365 <212> DNA <213> Artificial Sequence <220> <223> recombinant polynucleotide coding PEP-1-botoxin fusion protein <220> <221> CDS (222) (10) .. (1353) <400> 6 acactcgag atg ccc ttt gtt aat aaa caa ttt aat tat aaa gat cct gta 51            Met Pro Phe Val Asn Lys Gln Phe Asn Tyr Lys Asp Pro Val              1 5 10 aat ggt gtt gat att gct tat ata aaa att cca aat gca gga caa atg 99 Asn Gly Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Ala Gly Gln Met  15 20 25 30 caa gta aaa gct ttt aaa att cat aat aaa ata tgg gtt att cca gaa 147 Gln Val Lys Ala Phe Lys Ile His Asn Lys Ile Trp Val Ile Pro Glu                  35 40 45 aga gat aca ttt aca aat cct gaa gaa gga gat tta aat cca cca cca 195 Arg Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro Pro              50 55 60 gaa gca aaa caa gtt cca gtt tca tat tat gat tca aca tat tta agt 243 Glu Ala Lys Gln Val Pro Val Ser Tyr Tyr Asp Ser Thr Tyr Leu Ser          65 70 75 acc gat aat gaa aaa gat aat tat tta aag gga gtt acc aaa tta ttt 291 Thr Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val Thr Lys Leu Phe      80 85 90 gag aga att tat tca act gat ctt gga aga atg ttg tta aca tca ata 339 Glu Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu Thr Ser Ile  95 100 105 110 gta agg gga ata cca ttt tgg ggt gga agt aca ata gat aca gaa tta 387 Val Arg Gly Ile Pro Phe Trp Gly Gly Ser Thr Ile Asp Thr Glu Leu                 115 120 125 aaa gtt att gat act aat tgt att aat gtg ata caa cca gat ggt agt 435 Lys Val Ile Asp Thr Asn Cys Ile Asn Val Ile Gln Pro Asp Gly Ser             130 135 140 tat aga tca gaa gaa ctt aat cta gta ata ata gga ccc tca gct gat 483 Tyr Arg Ser Glu Glu Leu Asn Leu Val Ile Ile Gly Pro Ser Ala Asp         145 150 155 att ata cag ttt gaa tgt aaa agc ttt gga cat gaa gtt ttg aat ctt 531 Ile Ile Gln Phe Glu Cys Lys Ser Phe Gly His Glu Val Leu Asn Leu     160 165 170 acg cga aat ggt tat ggc tct act caa tac att aga ttt agc cca gat 579 Thr Arg Asn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro Asp 175 180 185 190 ttt aca ttt ggt ttt gag gag tca ctt gaa gtt gat aca aat cct ctt 627 Phe Thr Phe Gly Phe Glu Glu Ser Leu Glu Val Asp Thr Asn Pro Leu                 195 200 205 tta ggt gca ggc aaa ttt gct aca gat cca gca gta aca tta gca cat 675 Leu Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala Val Thr Leu Ala His             210 215 220 gaa ctt ata cat gct gga cat aga tta tat gga ata gca att aat cca 723 Glu Leu Ile His Ala Gly His Arg Leu Tyr Gly Ile Ala Ile Asn Pro         225 230 235 aat agg gtt ttt aaa gta aat act aat gcc tat tat gaa atg agt ggg 771 Asn Arg Val Phe Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met Ser Gly     240 245 250 tta gaa gta agc ttt gag gaa ctt aga aca ttt ggg gga cat gat gca 819 Leu Glu Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly His Asp Ala 255 260 265 270 aag ttt ata gat agt tta cag gaa aac gaa ttt cgt cta tat tat tat 867 Lys Phe Ile Asp Ser Leu Gln Glu Asn Glu Phe Arg Leu Tyr Tyr Tyr                 275 280 285 aat aag ttt aaa gat ata gca agt aca ctt aat aaa gct aaa tca ata 915 Asn Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn Lys Ala Lys Ser Ile             290 295 300 gta ggt act act gct tca tta cag tat atg aaa aat gtt ttt aaa gag 963 Val Gly Thr Thr Ala Ser Leu Gln Tyr Met Lys Asn Val Phe Lys Glu         305 310 315 aaa tat ctc cta tct gaa gat aca tct gga aaa ttt tcg gta gat aaa 1011 Lys Tyr Leu Leu Ser Glu Asp Thr Ser Gly Lys Phe Ser Val Asp Lys     320 325 330 tta aaa ttt gat aag tta tac aaa atg aaa aca gag att tac aca gag 1059 Leu Lys Phe Asp Lys Leu Tyr Lys Met Lys Thr Glu Ile Tyr Thr Glu 335 340 345 350 gat gat aat ttt gtt aag ttt ttt aaa gta ctt aac aga aaa aca tat 1107 Asp Asp Asn Phe Val Lys Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr                 355 360 365 ttg aat ttt gat aaa gcc gta ttt aag ata aat ata gta cct aag gta 1155 Leu Asn Phe Asp Lys Ala Val Phe Lys Ile Asn Ile Val Pro Lys Val             370 375 380 aat tac aca ata tat gat gga ttt aat tta aga aat aca aat tta gca 1203 Asn Tyr Thr Ile Tyr Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala         385 390 395 gca aac ttt aat ggt caa aat aca gaa att aat aat atg aat ttt act 1251 Ala Asn Phe Asn Gly Gln Asn Thr Glu Ile Asn Asn Met Asn Phe Thr     400 405 410 aaa cta aaa aat ttt act gga ttg ttt gaa ttt tat aag ttg cta tgt 1299 Lys Leu Lys Asn Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys 415 420 425 430 gta aga ggg ata ata act tct aaa act aaa tca tta gat gaa gga tac 1347 Val Arg Gly Ile Ile Thr Ser Lys Thr Lys Ser Leu Asp Glu Gly Tyr                 435 440 445 aat aag tcaggat ccaca 1365 Asn lys <210> 7 <211> 448 <212> PRT <213> Artificial Sequence <400> 7 Met Pro Phe Val Asn Lys Gln Phe Asn Tyr Lys Asp Pro Val Asn Gly   1 5 10 15 Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Ala Gly Gln Met Gln Val              20 25 30 Lys Ala Phe Lys Ile His Asn Lys Ile Trp Val Ile Pro Glu Arg Asp          35 40 45 Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro Pro Glu Ala      50 55 60 Lys Gln Val Pro Val Ser Tyr Tyr Asp Ser Thr Tyr Leu Ser Thr Asp  65 70 75 80 Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val Thr Lys Leu Phe Glu Arg                  85 90 95 Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu Thr Ser Ile Val Arg             100 105 110 Gly Ile Pro Phe Trp Gly Gly Ser Thr Ile Asp Thr Glu Leu Lys Val         115 120 125 Ile Asp Thr Asn Cys Ile Asn Val Ile Gln Pro Asp Gly Ser Tyr Arg     130 135 140 Ser Glu Glu Leu Asn Leu Val Ile Gly Pro Ser Ala Asp Ile Ile 145 150 155 160 Gln Phe Glu Cys Lys Ser Phe Gly His Glu Val Leu Asn Leu Thr Arg                 165 170 175 Asn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro Asp Phe Thr             180 185 190 Phe Gly Phe Glu Glu Ser Leu Glu Val Asp Thr Asn Pro Leu Leu Gly         195 200 205 Ala Gly Lys Phe Ala Thr Asp Pro Ala Val Thr Leu Ala His Glu Leu     210 215 220 Ile His Ala Gly His Arg Leu Tyr Gly Ile Ala Ile Asn Pro Asn Arg 225 230 235 240 Val Phe Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met Ser Gly Leu Glu                 245 250 255 Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly His Asp Ala Lys Phe             260 265 270 Ile Asp Ser Leu Gln Glu Asn Glu Phe Arg Leu Tyr Tyr Tyr Asn Lys         275 280 285 Phe Lys Asp Ile Ala Ser Thr Leu Asn Lys Ala Lys Ser Ile Val Gly     290 295 300 Thr Thr Ala Ser Leu Gln Tyr Met Lys Asn Val Phe Lys Glu Lys Tyr 305 310 315 320 Leu Leu Ser Glu Asp Thr Ser Gly Lys Phe Ser Val Asp Lys Leu Lys                 325 330 335 Phe Asp Lys Leu Tyr Lys Met Lys Thr Glu Ile Tyr Thr Glu Asp Asp             340 345 350 Asn Phe Val Lys Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr Leu Asn         355 360 365 Phe Asp Lys Ala Val Phe Lys Ile Asn Ile Val Pro Lys Val Asn Tyr     370 375 380 Thr Ile Tyr Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala Ala Asn 385 390 395 400 Phe Asn Gly Gln Asn Thr Glu Ile Asn Asn Met Asn Phe Thr Lys Leu                 405 410 415 Lys Asn Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys Val Arg             420 425 430 Gly Ile Ile Thr Ser Lys Thr Lys Ser Leu Asp Glu Gly Tyr Asn Lys         435 440 445  

Claims (8)

A protein transport domain consisting of 15-30 amino acids and comprising a non-hydrophilic domain containing 5 or more tryptophan, a hydrophilic domain containing a large number of lysines, and a spacer separating the two domains is provided at least at one end of the botoxin protein. Covalently bound cell-transducing botox fusion protein. The cell-induced botox fusion protein according to claim 1, wherein the cell-induced botox fusion protein has an amino acid sequence represented by SEQ ID NO. [Claim 3] The cell-inducible botoxin fusion protein according to claim 1 or 2, wherein a protein transport domain is covalently bonded to one or both sides of the carboxy terminal and the amino terminal of the botoxin protein. An external composition for skin, comprising the cell-transducing botoxin fusion protein of claim 1 or 2 as an active ingredient. A pharmaceutical composition comprising the cell-induced botoxin fusion protein of claim 1 or 2 as an active ingredient and a pharmaceutically acceptable carrier. A recombinant polynucleotide encoding a protein transport domain peptide coding DNA sequence to a botox cDNA encoding the cell-induced botox fusion protein of claim 1. The recombinant polynucleotide of claim 6, wherein the recombinant polynucleotide has a nucleotide sequence of SEQ ID NO. 6. A vector expressing a cell-introducing botox fusion protein, comprising the recombinant polynucleotide of claim 6 to express the fusion protein of claim 1.

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