KR20070095617A - Cell-transducing fusion protein of cyclophilin a - Google Patents

Abstract

A cell-transducing fusion protein of cyclophilin A is provided to be smoothly permeated into cultured cells as well as skin cells of live animals, thereby being applied as a skin topical agent and a cosmetic composition. A cell-transducing fusion protein of cyclophilin A is characterized in that a PEP-1 protein transfer domain is covalently bound to at least one side terminal end of a cyclophilin A protein and it includes an amino acid sequence described as SEQ ID : NO. 7. A skin topical composition comprises the cell-transducing fusion protein of cyclophilin A as an effective ingredient and a pharmaceutically acceptable carrier. A recombinant polynucleotide coding the cell-transducing fusion protein of cyclophilin A includes a nucleotide sequence described as SEQ ID : NO. 6.

Description

Cell-transducing Fusion Protein of Cyclophilin A}

Figure 1 schematically shows the expression vector of the PEP-1-CypA fusion protein. PEP-1-CypA expression vectors were prepared based on the pET-15b vector. Synthetic PEP-1 oligomers were cloned into the NdeI, XhoI restriction sites of the pET-15b vector, and human cyclophilin A ("CypA") cDNA was cloned into the XhoI, BamHI restriction sites of the pET-15b vector. Expression was induced by addition of IPTG.

Figure 2 shows the expression, purification of PEP-1-CypA fusion protein. Cellular protein extracts and purified fusion proteins were analyzed by 12% SDS-PAGE (A) and Western blot analysis with anti-rabbit polyhistidine antibody (B). Lanes 1, 2 and 3 are as follows:

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

Lane 2, induced pPEP-1-CypA;

Lane 3, purified pPEP-1-CypA.

Figure 3 is a photograph showing the cell introduction of PEP-1-CypA fusion protein. (A) 0.5-5 μM PEP-1-CypA fusion protein was added to the culture medium for 60 minutes. (B) 3 μM PEP-1-CypA fusion protein was added to the culture medium for 10-60 minutes.

Figure 4 Western blot analysis of cells pretreated with 3μΜ PEP-1-CypA fusion protein.

5 shows the effect on skin cell viability of PEP-1-cypA fusion proteins introduced into cells. Skin cells treated with PEP-1-cypA for one hour were exposed to LPS. Cell viability was determined by colorimetric analysis using MTT. Each bar is the average obtained from five experiments.

6 is a photograph showing that the cell-introduced PEP-1-CypA fusion protein inhibits LPS-induced COX-2 expression. Skin cells were exposed to LPS in the presence or absence of 0-12 μM PEP-1-CypA fusion protein.

7 is a photograph showing that the cell-introduced PEP-1-CypA fusion protein inhibits LPS-induced COX-2 expression. Skin cells were exposed to LPS with or without 10 μM PEP-1-CypA, cyclosporin (CyS).

[1] Leung DY, Bieber T. (2003) Lancet 361, 151-60.

[2] Novak N, Bieber T, Leung DY. (2003) J. Allergy Clin . Immunol . 112, S128-139.

[3] Boguniewicz M, Leung DY. 10.Atopic dermatitis. (2006) J. Allergy Clin. Immunol . 117, S475-480.

[4] Yao Q, Li M, Yang H, Chai H, Fisher W, Chen C. (2005) World J. Surg. 29, 276-280.

[5] Del Zoppo, GJ, Wagner, S. and Tagaya, M. (1997) Drugs 54, 9-38.

[6] Morris, MC, Depollier, J., Mery, J., Heitz, F. and Divita, G. (2001) Nat . Biotech . 19, 1173-1176.

[7] Sambrook, J., Fritsch, FE, and Maniatis, T. (1989) Molecular cloning , Cold spring harbor laboratory press, Cold spring harbor.

[8] Bradford, MA (1976) Anal . Biochem . 72, 248-254.

Allergy is a phenomenon in which most people's immune mechanisms react abnormally more than usual to this invasive substance without any reaction to most people. In particular, atopic dermatitis, one of the allergic diseases soaring in modern people, is a recurrent chronic inflammatory dermatitis with severe itching, which mainly begins in infants and children, and persists in adulthood or may be caused by adulthood. Atopic dermatitis is characterized by pruritus, chronic recurrent eczema with a batch and characteristic histological pattern, and is characterized by lichenification and epidermal exfoliation in the chronic phase. Some patients with atopic dermatitis may have allergic rhinitis, asthma and respiratory illnesses.

Although the root cause of atopic dermatitis is not known, environmental factors such as genetic pollution, westernized living environment, pollution, and increased use of various chemical drugs are known as environmental factors. Recent studies have shown that overproduction of the T _H 2 cytokine interleukin 4 (IL-4) increases the concentration of IgE in the serum and abnormally low levels of the T _H 1 cytokine interferon-γ (IFN-γ). When expressed, it inhibits the function of T _H 2 cells. In other words, the balance between these two substances is important. In atopic dermatitis, this balance is broken, resulting in overproduction of IgE. These immunological factors are also known to cause atopic dermatitis.

Cyclophilin is one of the immunophillin families and was originally known as a major cell binding protein for immunosuppressive cyclosporin A ("CsA"), FK506 and the like. Cyclophyllin is divided into CypA, CypB, CypC, CypD, Cyp40, etc. In particular, cyclophilin A (mixed with "CypA" in the description and drawings, claims, etc.) of the present specification is the most abundant protein that binds CsA as a cytoplasmic protein to be.

Cyclophylline is a multifunctional protein involved in peptidyl-prolyl-isomerase activity, protein folding / repair, immune system, tumors, and the like. Many studies have been conducted to use the disease.

There is a great interest in using CypA for therapeutic purposes in in vivo polymers, especially skin diseases. At the moment the most attention is on gene therapy. However, gene therapy is not easy to carry genes, low expression in target cells, short duration of protein expression in cells, and very difficult to artificially control the amount of protein expressed in target cells. There are several problems.

In moving drugs or proteins for treatment into cells, a method of directly delivering a target protein through a cell membrane may be considered. However, proteins are difficult to cross through cell membranes due to their size and biochemical properties. In general, it is known that a substance having a molecular weight of 600 Daltons or more is almost impossible to pass through the cell membrane.

Recently, it has been found that PEP-1 peptides are used to transport natural heterologous proteins into cells as one of protein transport methods. The PEP-1 peptide consists of 21 amino acids (KETWWETWWTEW SQP KKKRKV) and has three domains: hydrophobic domain, spacer and hydrophilic domain. So far, studies using PEP-1 peptides have shown that when PEP-1 peptides and foreign proteins are administered to cells at the same time, the proteins can be transported naturally into the cells. In addition, PEP peptides exhibit several advantages as protein therapeutics over HIV-1 Tat proteins: efficient penetration of proteins into cells and stability in physiological buffers. However, the PEP peptide was found to be effectively transported into the cells only when administered in a constant ratio with an external protein (green fluorescent protein, GFP; β-galactosidase, β-Gal). In addition, it is not yet known whether PEP-1 carries all proteins such as therapeutic proteins into cells.

Accordingly, it is an object of the present invention to provide a fusion protein with enhanced cell permeability of cypA.

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 It is intended to maximize cell permeability by covalently binding the constructed protein transport domain with at least one end of cypA.

Thus, the present inventors fused the PEP-1 peptide carrying the natural protein into the cell to an external protein, human CypA, overexpressed the fusion protein in E. coli, and purified easily and conveniently by affinity chromatography. Purified fusion proteins were effectively transported into cells with biological activity through cultured skin cells and tissue experiments. And it was found that the PEP-1-CypA fusion protein is carried into skin cells and effectively protects skin cell death. The present invention addresses the possibility of PEP-1-CypA fusion protein as an intracellular transporter, protein therapeutics and cosmetics.

In the present invention, PEP-1-CypA which can overexpress and easily purify the PEP-1-CypA fusion protein first Expression vectors were developed. The expression vector is a human CypA cDNA, PEP-1 peptide and six histidines linked in series. Using this expression vector, by over-expression of the PEP-1-CypA fusion protein in E. coli Ni ^{2 +} in the natural state were purified using an affinity chromatography column. The overexpression of the PEP-1-CypA fusion protein was significantly higher, resulting in a higher amount of purified protein. It was confirmed that the PEP-1-CypA fusion protein purified on skin cells cultured by Western blot was delivered to the cells in a concentration and time dependent manner. PEP-1-CypA permeated into cells was maintained for at least 48 hours in the cells and PEP-1-CypA fusion protein was efficiently permeated not only in cultured cells but also in skin tissues of mice. In addition, PEP-1-CypA fusion protein permeated into cells effectively inhibited cell death by immune response.

These results indicate that the PEP-1-CypA fusion protein permeates well into cells and exhibits well CypA function in cells. Therefore, this PEP-1-CypA fusion protein suggests a variety of applications in the field of skin disease treatment, such as atopic dermatitis.

Pharmaceutical compositions containing the PEP-1-cypA fusion protein as an active ingredient may be formulated in an injectable form or a coating by conventional methods in combination with a carrier which 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 containing the fusion protein of the present invention as an active ingredient can be easily prepared in any form according to a conventional manufacturing method. As an example, in preparing a cream coating agent, the PEP-1-cypA fusion protein of the present invention is contained in a cream base of a general oil-in-water type (O / W) or water-in-oil type (W / O), and contains a perfume, a chelating agent, and a pigment. While antioxidants, preservatives, etc. may be used as necessary, synthetic or natural materials such as proteins, minerals, vitamins, etc. may be used together for the purpose of improving the properties.

In addition, the fusion protein of the present invention can be used as a cosmetic, by adding a pH adjuster, fragrance, emulsifier, preservatives, etc. as needed, in the usual cosmetic preparation method, lotion, gel, water-soluble powder, fat-soluble powder, water-soluble liquid, cream Or formulated with an essence or the like.

The present inventors were able to confirm that the PEP-1-cypA fusion protein penetrates into the skin of the mouse and penetrates smoothly to the dermis layer. Accordingly, it has been found that the PEP-1-cypA fusion protein can be used as a major component of pharmaceutical compositions and / or coatings (as used herein in the same sense as "skin external preparations") compositions.

The present invention provides a method for efficiently delivering cypA intracellularly or into the skin. Intracellular delivery of a cypA protein molecule according to the present invention is composed of 15-30 amino acids in cypA, a hydrophilic domain including 5 or more tryptophans, and a hydrophilic domain including 4 or more of lysine. and a transport domain composed of a spacer separating the two domains and a covalently bonded 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 is also included in the scope of the present invention. Belonging will be self-evident.

Specifically, the present invention relates to a PEP-1-cypA fusion protein, a recombinant nucleotide and vector for producing the fusion protein, a therapeutic composition comprising the fusion protein, a pharmaceutical composition for 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-cypA fusion protein” includes a protein transport domain and cypA, and refers to a covalent complex formed by genetic fusion or chemical bonding of a transport domain and a cargo molecule (ie, cypA in the present invention). it means. In the present specification, it is mixed with "PEP-1-cypA", "cypA fusion protein" or "PEP-cypA".

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.

In addition, the present specification is interchangeable with the expressions "transduction", "transport", "penetration", "transport", "transfer", "transmission", "pass" for "introducing" a protein or peptide into a cell. It was.

At least one side of a cypA protein is a protein transport domain consisting of 15-30 amino acids, 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. It relates to a cell-transducing cypA fusion protein covalently bound to the terminal. 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 between the fusion protein of the invention and the amino acid sequence such as in the range of 85-100%.

In addition, the present invention is characterized in that the cell-introduced cypA 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 cypA protein.

The present invention also relates to an external composition for skin, characterized in that it contains the cell-transducing cypA fusion protein as an active ingredient.

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

The present invention also relates to a recombinant polynucleotide comprising SEQ ID NO: 6, wherein the protein transport domain peptide coding DNA sequence is coupled to cypA cDNA and encodes the cell-inducible 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 transfusion fusion protein, characterized in that comprising 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.

Materials and methods

1) material

Restriction enzymes and T4 DNA ligase were purchased from Promega (USA) and Pfu polymerase was purchased from stratagene (USA). Tat oligonucleotides were synthesized in Gibco BRL custom primer (USA). IPTG was purchased from Duchefa (Netherland). pET-15b and BL21 (DE3) plasmids were purchased from Novagen (USA) and Ni-nitrilo-triacetic acid superflow was purchased from Qiagen (Germany). Human Cyclophilin A (hereinafter, interchangeably with “CypA” in the description and drawings, claims herein) cDNA was isolated from human liver cDNA library by PCR method. All other reagents were used as express products.

2) PEP -One- cypA  Fusion Protein Expression Vector Preparation and Transformation

In order to develop a technique for infiltrating a protein with a function into a cell, a fusion protein expression vector capable of delivering a target protein into a cell was prepared, and a human cyclone was readily used to analyze the ability of the PEP-1 peptide to deliver the protein into the cell. Filin A (CypA) protein was selected.

First, PEP-1-CypA To produce a fusion protein, a pET-PEP expression vector containing a PEP-1 peptide (KETWWE TWWTEW SQP KKKRKV) was prepared. Ligation (ligation in pET-15b cut with restriction enzymes XhoⅠ; ( "sub-chain, 5'-TCGAGCACTTTACGTTTTTTTTTCGGCTGACACCATTCGGTCCACCAGGTTTCCCACCAGGTTTCTTTCC-3 'top strand, 5'-TATGAAAGAAACCTGGTGGGAAACCTGGTGGACCGAATGGTCTCAGCCGAAAAAAAAACGTAAAGTGC-3) a NdeⅠ up of two kinds corresponding to PEP-1 peptide nucleotides ) And inserted. Subsequently, two kinds of oligonucleotides were synthesized based on the sequence of the cDNA of human CypA. Forward primer, has a Xho Ⅰ restriction site and a reverse primer 5'-GGATCCTTATC GAGTTGTCCACAGTGAGC-3 '5' -CTCGAGATGGTCAACCCCACCGTGTTCTTC-3 has a restriction site in BamHⅠ.

Polymerase chain reaction (PCR) was carried out in a thermal cycler (Perkin-Elmer, model 9600). The reaction mixture was placed in a 50 μl silicon tube and heated at 94 ° C. for 5 minutes. PCR reactions were performed. After PCR, the reaction was isolated by agarose gel electrophoresis and ligated to TA cloning vector (Invitrogen, Sandiego, USA), and then transformed into competent cells that are easy to clone and from the transformed bacteria. Plasmids were separated by alkaline lysis method. TA vectors containing human CypA cDNA were identified by Xho I and Cleavage with BamH I and insertion into PEP expression vector. E. coli BL21 (DE3) transformed with PEP-1-cypA was selected and colonies were inoculated in 100 ml LB medium and IPTG (0.5 mM) was added to the medium to induce overexpression of recombinant PEP-1-cypA. It was. Overexpressed PEP-1-CypA was confirmed by SDS-PAGE and Western blot analysis.

3) PEP -One- CypA  Expression and Purification of Fusion Proteins

E. coli BL21 (DE3) cells containing PEP-1-CypA cDNA prepared in this laboratory were placed in LB medium containing ampicillin and cultured at 37 ° C. at 200 rpm. When the bacterial concentration in the culture medium was OD ₆₀₀ = 0.5 to 1.0, IPTG was added to the medium to bring the final concentration to 0.5 and 1 mM, followed by further incubation at 30 ° C for 12 hours. The cultured cells were collected by centrifugation, and then 5ml binding buffer (5mM imidazole, 0.5M NaCl, 20mM Tris-HCl, pH 7.9) was added, and pulverized with an ultrasonic grinder. The supernatant was immediately centrifuged Ni ^{2 +} - acrylonitrile triazol setik acid Sepharose super flow load on the columns was washed in binding buffer and 6-fold volume of 10 times the volume of buffer (60mM imidazole, 0.5M NaCl, 20mM Tris-HCl , pH 7.9), followed by elution of the fusion protein with elution buffer (1M imidazole, 0.5M NaCl, 20 mM Tris-HCl, pH 7.9). Subsequently, the fractions containing the fusion protein were collected and PD-10 column chromatography was performed to remove salts contained in the fractions.

Purified protein concentration was determined by Bradford method using bovine serum albumin as standard [8].

4) skin cell culture and PEP -One- CypA Intracellular Permeation of Fusion Proteins

Skin cells (obtained from the Human primary fibroblast cell-Catholic Medical School Dermatology) provide 95% air and 5% CO ₂ at 37 ° C, 20mM HEPES / NaOH (pH 7.4), 5mM NaHCO ₃ , 10% Fetal Bovine Serum (FBS) and Cultured in DMEM (Dulbecco's Modified Eagle's Medium) containing antibiotics (100 μg / ml streptomycin, 100 U / ml penicillin).

To observe intracellular penetration of PEP-1-CypA fusion protein, skin cells were grown for 4-6 hours in 6-well plates and replaced with fresh DMEM culture containing 10% FBS and PEP-1-CypA fusion Proteins were treated in culture. After one hour, cells were treated with trypsin-EDTA (Gibco BRL) and washed thoroughly with phosphate buffered saline (PBS). After crushing the cells, the amount of CypA permeated into the cells was measured by Western blot analysis.

In addition to the skin cells, the same experiment was performed on HaCaT cells (Human Keratocyte cells) obtained from ATCC and B16 cells (Mouse Melanoma cells) obtained from ATCC to confirm that PEP-1-cypA fusion protein permeated into cells. .

5) Weston Blot  analysis

Western blot methods were performed to confirm intracellular penetration of PEP-1-CypA. First, the protein was purified in its natural state. PEP-1-CypA (1-12 μM) fusion protein was treated to the prepared skin cells, and then, one hour later, cells were collected and western blots were performed. Proteins in the cell mill were separated with a 12% SDS polyacrylamide gel and then the proteins in the gel were electrophoresed to a nitrocellulose membrane (Amersham, UK). Protein shifted nitrocellulose membrane was blocked with PBS containing 5% non-dry milk. The membrane was then treated with rabbit anti-histidine polyclonal antibody (Santacruze, USA, 1: 1,000) for one hour. After washing, one hour of reaction with horseradish peroxidase-coupled mouse anti-rabbit IgG antibody (1: 10,000 dilution) was performed. Finally, ECL kit (ECL; Amersham) was used to identify the protein bands in response to human cypA monoantibody.

6) permeated into skin cells PEP -One- CypA  Stability of Fusion Proteins

To measure the intracellular stability of the PEP-1-CypA fusion protein permeated into cells, skin cells were grown in 6-well plates for 4-6 hours and replaced with 1 ml fresh culture medium without FBS and PEP-1 -CypA fusion protein was treated in culture. One hour after treatment cells were treated with trypsin-EDTA and washed well with PBS. Subsequently, the medium containing the FBS was added and the culture was continued, and the cells were collected hourly (1 to 60 hours) to measure the amount and activity of the remaining fusion proteins in the cells by Western blot and activity analysis.

7) Inhibition of immune response

To measure LPS-induced immunosuppressive effects, skin cells were grown in 6-well plates, replaced with 1 ml of fresh culture without FBS, and treated with PEP-1-CypA fusion protein in the culture, followed by LPS. . After administration, expression and cell death of COX-2 were confirmed by Western blot and MTT analysis.

Results

One) PEP -One- CypA  Overexpression and Purification of Fusion Proteins

To overexpress the PEP-1-CypA fusion protein, a PEP-1-CypA expression vector was developed containing human CypA cDNA, PEP peptide (KETWWETWWTEW SQP KKKRKV) and six histidines in succession (FIG. 1).

E. coli cells, which induced overexpression of the fusion protein with IPTG, were disrupted by an ultrasonic grinder at 4 ° C. and centrifuged to separate supernatant proteins by 12% SDS-PAGE. FIG. 2 shows the results of overexpression of PEP-1-CypA and protein blots stained with Coomassie Brilliant Blue (FIG. 2A) and Western blot (FIG. 2B). Lane 2 of FIG. 2A shows the overexpressed fusion protein by treatment with 0.5 mM IPTG and shows that the very high concentration of the fusion protein was overexpressed compared to the control lane 1 (pET-15b vector).

Since the PEP-1-CypA fusion protein contains six histidines at the N-terminus, immobilized metal-chelate affinity chromatography allows the fusion protein to be purified naturally (purity> 95) in a single step. %) Purified (FIG. 2A). Lane 3 in the figure shows the results of staining the purified PEP-1-CypA fusion protein with Coomassie brilliance blue.

Overexpressed and purified PEP-1-CypA fusion protein was once again confirmed by Western blot analysis. As shown in FIG. 2B, the PEP-1-CypA band responding to CypA or 6x histidine antibody was observed at the same position as the protein band of FIG. 2A.

2) with skin cells PEP -One- CypA Permeation of Fusion Proteins

It was observed how the permeation of skin cells of purified PEP-1-CypA fusion protein in nature changed with time and concentration. As shown in FIG. 3, Western blot analysis confirmed that natural PEP-1-CypA was permeated into skin cells in a time and concentration-dependent manner. 3A shows the results when 3 μM PEP-1-CypA was treated for 10 to 120 minutes, and FIG. 3B was treated with 0.25-3 μM PEP-1-CypA in the cell culture solution for 1 hour. When the degree of intracellular penetration of PEP-1-CypA was observed for each concentration, it was confirmed that the amount of PEP-1-CypA permeated into cells was increased in a concentration-dependent manner. In addition, when observed by time it was confirmed that the fusion protein permeated within 10 minutes of administration, it was confirmed that the amount of PEP-1-CypA permeated into the cell increases in proportion to the treatment time. These results indicate that the PEP-1-CypA fusion protein is time and concentration dependent intracellular permeation occurs.

PEP-1-CypA penetrated into skin cells must remain stable in cells for a significant period of time before they can be effectively applied to protein treatment. As shown in FIG. 4, PEP-1-CypA permeated into cells was degraded with time, and the amount of protein gradually decreased, but it was found to exist in cells for at least 48 hours. Therefore, PEP-1-CypA permeated into cells is useful for protein treatment because it maintains stability for at least 48 hours.

3) PEP -One- CypA Biological Activity of Fusion Proteins

Fusion proteins penetrated into skin cells must maintain their inherent activity before they can be applied to protein therapy. Therefore, the degree of biological activity of the fusion protein penetrated into the cell is a very important problem. Therefore, in order to examine the biological activity of the purified PEP-1-CypA fusion protein, COX-2 expression and cell death induced by LPS were confirmed using MTT analysis (FIGS. 5 and 6). It was confirmed that the purified PEP-1-CypA fusion protein inhibited cell death and COX-2 expression by LPS with time and concentration. When administered in combination with the immunosuppressive cyclosporin (CyS) it can be seen that it is more effective (Fig. 7).

The present invention provides a technique for the direct penetration of human cyclophilin A into cells at the protein level in an effective manner.

In addition, the cypA 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 an external preparation for skin and cosmetics.

It is thought that PEP-1-CypA fusion protein can be effectively used to treat skin diseases by intracellular administration of CypA, which plays a major role in restoring skin inflammation of atopic dermatitis.

Based on the present invention, since PEP-1-CypA can be delivered directly into a cell to protect skin cells and tissues from atopic dermatitis, the present invention can be widely used more effectively, such as in the functional cosmetics industry.

<110> Hallym University <120> Cell-transducing cypA fusion protein <130> sychoi-cypA <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 caccattcgg 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> 30 <212> DNA <213> Artificial Sequence <220> <223> forwaed primer of human cyclophilin A <400> 4 ctcgagatgg tcaaccccac cgtgttcttc 30 <210> 5 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of human cyclophilin A <400> 5 ggatccttat cgagttgtcc acagtgagc 29 <210> 6 <211> 573 <212> DNA <213> Artificial Sequence <220> <223> recombinant polynucleotide coding PEP-1-cypA fusion protein <220> <221> CDS (222) (1) .. (564) <400> 6 atg aaa gaa acc tgg tgg gaa acc tgg tgg acc gaa tct cag ccg aaa 48 Met Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Ser Gln Pro Lys   1 5 10 15 aaa aaa cgt aaa gtg ctc gag atg gtc aac ccc acc gtg ttc ttc gac 96 Lys Lys Arg Lys Val Leu Glu Met Val Asn Pro Thr Val Phe Phe Asp              20 25 30 att gcc gtc gac ggc gag ccc ttg ggc cgc gtc tcc ttt gag ctg ttt 144 Ile Ala Val Asp Gly Glu Pro Leu Gly Arg Val Ser Phe Glu Leu Phe          35 40 45 gca gac aag gtc cca aag aca gca gaa aat ttt cgt gct ctg agc act 192 Ala Asp Lys Val Pro Lys Thr Ala Glu Asn Phe Arg Ala Leu Ser Thr      50 55 60 gga gag aaa gga ttt ggt tat aag ggt tcc tgc ttt cac aga att att 240 Gly Glu Lys Gly Phe Gly Tyr Lys Gly Ser Cys Phe His Arg Ile Ile  65 70 75 80 cca ggg ttt atg tgt cag ggt ggt gac ttc aca cgc cat aat ggc act 288 Pro Gly Phe Met Cys Gln Gly Gly Asp Phe Thr Arg His Asn Gly Thr                  85 90 95 ggt ggc aag tcc atc tat ggg gag aaa ttt gaa gat gag aac ttc acc 336 Gly Gly Lys Ser Ile Tyr Gly Glu Lys Phe Glu Asp Glu Asn Phe Thr             100 105 110 cta aag cat acg ggt cct ggc atc ttg tcc atg gca aat gct gga ccc 384 Leu Lys His Thr Gly Pro Gly Ile Leu Ser Met Ala Asn Ala Gly Pro         115 120 125 aac aca aat ggt tcc cag ttt ttc atc tgc act gcc aag act gag tgg 432 Asn Thr Asn Gly Ser Gln Phe Phe Ile Cys Thr Ala Lys Thr Glu Trp     130 135 140 ttg gat ggc aag cat gtg gtg ttt ggc aaa gtg aaa gaa ggc atg aat 480 Leu Asp Gly Lys His Val Val Phe Gly Lys Val Lys Glu Gly Met Asn 145 150 155 160 att gtg gag gcc atg gag cgc ttt ggg tcc agg aat ggc aag acc agc 528 Ile Val Glu Ala Met Glu Arg Phe Gly Ser Arg Asn Gly Lys Thr Ser                 165 170 175 aag aag atc acc att gct gac tgt gga caa ctc gaa taagga tcc 573 Lys Lys Ile Thr Ile Ala Asp Cys Gly Gln Leu Glu             180 185 <210> 7 <211> 188 <212> PRT <213> Artificial Sequence <400> 7 Met Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Ser Gln Pro Lys   1 5 10 15 Lys Lys Arg Lys Val Leu Glu Met Val Asn Pro Thr Val Phe Phe Asp              20 25 30 Ile Ala Val Asp Gly Glu Pro Leu Gly Arg Val Ser Phe Glu Leu Phe          35 40 45 Ala Asp Lys Val Pro Lys Thr Ala Glu Asn Phe Arg Ala Leu Ser Thr      50 55 60 Gly Glu Lys Gly Phe Gly Tyr Lys Gly Ser Cys Phe His Arg Ile Ile  65 70 75 80 Pro Gly Phe Met Cys Gln Gly Gly Asp Phe Thr Arg His Asn Gly Thr                  85 90 95 Gly Gly Lys Ser Ile Tyr Gly Glu Lys Phe Glu Asp Glu Asn Phe Thr             100 105 110 Leu Lys His Thr Gly Pro Gly Ile Leu Ser Met Ala Asn Ala Gly Pro         115 120 125 Asn Thr Asn Gly Ser Gln Phe Phe Ile Cys Thr Ala Lys Thr Glu Trp     130 135 140 Leu Asp Gly Lys His Val Val Phe Gly Lys Val Lys Glu Gly Met Asn 145 150 155 160 Ile Val Glu Ala Met Glu Arg Phe Gly Ser Arg Asn Gly Lys Thr Ser                 165 170 175 Lys Lys Ile Thr Ile Ala Asp Cys Gly Gln Leu Glu             180 185

Claims (8)

A cell-transducing cyclophilin A fusion protein, wherein the PEP-1 protein transport domain is covalently bound to at least one end of the cyclophilin A protein. The cell introducing cyclophilin A fusion protein according to claim 1, wherein the cell introducing cyclophilin A fusion protein has an amino acid sequence represented by SEQ ID NO: 7. 6. According to claim 1 or 2, wherein the cell transduction cyclophilin A fusion protein, characterized in that the protein transport domain is covalently bonded to one or both of the carboxy terminal and the amino terminal of the cyclophilin A protein. The topical skin composition according to claim 1 or 2, comprising the cell-introduced cyclophilin A fusion protein as an active ingredient. A pharmaceutical composition comprising the cell introducing cyclophilin A fusion protein of claim 1 or 2 as an active ingredient and comprising a pharmaceutically acceptable carrier. A recombinant polynucleotide encoding a protein transport domain peptide coding DNA sequence to cyclophylline A cDNA encoding the cell-introduced cyclophylline A fusion protein of claim 1. The recombinant polynucleotide of claim 6, wherein the recombinant polynucleotide has a nucleotide sequence represented by SEQ ID NO: 6. 7. A vector for expressing a cell-introducing cyclophilin A fusion protein, characterized in that it comprises the recombinant polynucleotide of claim 6 to express the fusion protein of claim 1.

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