KR100835880B1 - 27 Cell-transducing heat shock protein 27 fusion protein - Google Patents

Abstract

A cell transducing heat shock protein 27(HSP27) fusion protein is provided to be penetrated into a cell easily in a concentration dependent manner, thereby inhibiting apoptosis efficiently. A cell transducing HSP27 fusion protein includes an amino acid sequence of SEQ ID : NO. 7 and is characterized in that a protein transport domain consisting of 15-30 amino acids and composed of a hydrophobic domain including at least 5 tryptophans, a hydrophilic domain including at least 4 lysines, and a spacer isolating two domains is covalently bonded to at least one side terminal of a heat shock protein 27. A pharmaceutical composition for preventing and treating nephritis, vasculitis, auto-immune diseases, stroke, myocardial infarction, arrhythmia, angina pectoris, hemochromatosis, diseases caused by radium therapy, progeria, aging related to diseases, sickle cell disease, malaria, pulmonary emphysema, myocardia, auto-immune nephrotic syndrome, betelnet-related oral cavity carcinoma, hyperoxia, Alzheimer's disease, Parkinson's disease, cataract, Lou Gehrig's disease, diabetes, cerebral ischemia, and atopy comprises the HSP27 fusion protein and a pharmaceutically acceptable carrier.

Description

Cell-transducing heat shock protein 27 fusion protein

1 is a schematic diagram of a PEP-1-HSP27 fusion protein expression vector. (A) Structure of PEP-1-HSP27 expression vector based on vector pET-15b. Synthetic PEP-1 oligomers were cloned into NdeI, XhoI restriction sites and human HSP27 cDNA was cloned into XhoI, BamHI restriction sites of pET-15b. (B) Diagram of expressed control HSP27 and PEP-1-HSP27 fusion proteins. Human HSP27 coding frame is shown as 6His and PEP-1 peptide followed by an open box. The resulting vector was named pPEP-1-HSP27. Expression was induced by addition of IPTG.

Figure 2 is a photograph showing the purification of PEP-1-HSP27 fusion protein. The expressed, purified fusion protein was subjected to 12% SDS-polyacrylamide electrophoresis (A) and Western blotting with anti-rabbit polyhistidine antibody (B).

Lanes 1, 2 and 3 are as follows:

Lane 1, non-induced pPEP-1-HSP27;

Lane 2, induced pPEP-1-HSP27;

Lane 3, purified pPEP-1-HSP27.

3 is a photograph showing the introduction of PEP-1-HSP27 fusion protein into cultured astrocyte cells. 1-5 μM of PEP-1-HSP27 fusion protein and control HSP27 were added to the culture medium for 60 minutes each. PEP-1-HSP27 fusion proteins introduced into cells were analyzed by western blotting.

4 shows the effect of the transduced PEP-1-HSP27 fusion protein on the viability of astrocytes. Paraquat (“Pq”, 30 mM) was added to astrocytes treated with 1-5 μM of PEP-1-HSP27 fusion protein for one hour. Cell viability was determined by MTT {3- (4,5-dimethylthiazol-2-yl) -2,5-dipheyltetrazolium bromide; It was evaluated by color analysis using the following abbreviated "MTT"}.

Figure 5 shows the effect on the survival rate of astrocytic cells of the PEP-1-HSP27 and PEP-1-SODs fusion protein introduced at the same time. Paraquat (Pq, 30 mM) was added to astrocytes treated with 3 μM PEP-1-HSP27 and PEP-1-SODs for one hour. Cell viability was evaluated by colorimetric analysis using MTT.

6 shows the effect of PEP-1-HSP27 fusion protein on neuronal viability.

(A) The hippocampus of gerbil mouse brain was stained with cresyl violet 4 days after cerebral ischemic ischemia attack. Negative control (A and B , normal), positive control (C and D, vehicle injected group), injection of 300 μg PEP-1-HSP27 fusion protein after seizure (E and F). Scale bar: a, b and c, 400 μm, d, e and f, 50 μm.

(B) Graph showing neuronal density. Graph bars show mean ± standard deviation of 7 gerbil mice.

[1] Bowling, AC, Schulz, JB, Brown, RHJr. and Beal, MF (1993) J. Neurochem . 61, 2322-2325.

[2] Robberecht, W., Sapp P., Viaene MK, Rosen D., McKenna-Yasek D., Haines J., Horvitz R., Theys P. and Brown R. Jr. (1994) J. Neurochem . 62, 384-387.

[3] Rosen DR, Bowling AC, Patterson D., Usdin TB, Sapp P., Mezey E., McKenna-Yasek D., O'Regan J., Rahmani Z., Ferrante RJ, Brownstein MJ, Kowall NW, Beal MF, Horvitz HR and Brown RHJr. (1994) Hum . Mol . Genet . 3, 981-987.

[4] Floyd, RA (1990) FASEB J. 4, 2587-2597

[5] Halliwell B. and Gutteridge J.M.C. (1999) Free radicals in biology and medicine, Oxford University Press, Oxford.

[6] Latchman, DS (2005) Int . J. Hyperthermai . 21, 393-402.

[7] Franklin, TB, Krueger-Naug, AM, Clarke, DB, Arrigo, AP and Currie, RW (2005) Int . J. Hyperthermai . 21, 379-392.

[8] Verma, IM and Somia, N. (1997) Nature 389, 239-242.

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

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

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

[12] McCord, JM and Fridovich, I. (1969) J. Biol . Chem . 244, 6049-6055.

The present invention relates to an HSP27 fusion protein that can be easily penetrated into cells by forming a covalently bonded HSP27 fusion protein with human heat shock protein 27 (hereinafter referred to as "HSP27") 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 is composed of a (spacer), and compared with the control group smoothly penetrates in a concentration-dependent manner, and effectively inhibited cell death.

Amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder known as Lou Gehrig 'disease, has not yet been identified for its cause, but the family-type amyotrophic lateral sclerosis About 20% of mutations were observed in the gene of Cu, Zn-superoxide dismutase (mixed with "SOD" or "Cu, Zn-SOD"). The cause is expected to be related to Cu, Zn-SOD. There are more than 100 types of Cu, Zn-SOD mutants discovered to date, and mutants that have been found to be unique according to race have also been reported (10, 11). Cu, Zn-SOD is superoxide ^anion is well known as an important antioxidant in the living body, which catalyzes a reaction to convert to H ₂ O ₂ and O ₂ (· O _2). Early studies of familial muscular dystrophy lateral sclerosis showed that the activity of Cu, Zn-SOD in blood or brain tissues of patients was about 50% less than normal [1-3]. Therefore, it is believed that the decrease of the activity of the mutant SOD prevents free radicals from being removed in vivo, leading to oxidative damage of cells, and thus familial muscular atrophic lateral sclerosis.

Reactive oxygen species (ROS) are inevitably produced as by-products of various intracellular metabolism in all living organisms that use oxygen to obtain energy. These reactive oxygen species damage biopolymers such as intracellular proteins, nucleic acids and fats and are deeply involved in the progress of various diseases in the human body. In particular, it is involved in carcinogenesis, stroke, arthritis, arteriosclerosis, radiation damage and inflammatory reactions, and plays an important role in promoting aging in the normal aging process [4, 5].

Table 1 summarizes the diseases associated with reactive oxygen species or Cu, Zn-superoxide dismutase.

category Concrete example Inflammatory / immune damage Glomerulonephritis, vasculitis, autoimmune diseases, etc. Ischemia Stroke, myocardial infarction, arrhythmia, angina, etc. Drug and Toxic Induction Reactions skip Iron Overload: Tissue and Plasma Idiopathic hemochromatosis, etc. Radiation-related damage Nuclear exposure, radiotherapy, etc. Aging Progesteritis, disease-related aging Red blood cells Sickle cell disease, malaria, etc. Bronchus Emphysema Heart and cardiovascular system Cardiomyopathy, etc. kidney Autoimmune nephrotic syndrome Camouflage Betecnet-Related Oral Cancer, etc. Brain / nerve system / nerve muscle abnormalities Hyperbaric Oxygen, Alzheimer's Disease, Parkinson's Disease, etc. Eye Cataracts, etc. skin Disease due to UV irradiation

* Source: Free radicals in biology and medicine, Oxford University Press, Oxford pp 618 ~ 619.

Heat shock protein ("HSP") is known as molecular chaperone and anti-apoptotic protein and is expressed in most cells and is very well preserved. Heat shock proteins are also divided into five groups based on amino acid sequence and molecular weight. 100-110 kDa family, 83-90 kDa group, 66-78 kDa group, 60 kDa group, 15-30 kDa group. HSP27 belongs to the small heat shock protein family and is expressed in mammalian tissues including muscle and nervous tissues, and is abundant in motor neurons and sensory neurons [6, 7].

HSP27 is a protein that performs various functions in cells and is an important enzyme in cells that prevents damage to cells from harmful oxygen damage. There is a great interest in using HSP27 for therapeutic purposes in various diseases [3]. At the moment the most attention is on gene therapy. However, gene therapy is not easy to transport 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. It has a problem [8].

In moving drugs or proteins for treatment into cells, one can think of direct delivery of target proteins across the cell membrane. 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, as a method of transporting proteins, a technique for transporting natural heterologous proteins into cells using PEP-1 peptides has been discovered [9]. The PEP-1 peptide consists of 21 amino acids (KETWWETWWTEW SQP KKKRKV) and has three domains: a hydrophobic domain, a spacer, and a hydrophilic domain. So far, studies using PEP-1 peptides have shown that when PEP-1 peptides and foreign proteins are simultaneously administered to cells, the proteins can be transported into the cells in their natural state. In addition, PEP-1 peptides have several advantages as protein therapeutics such as permeability of proteins into cells, stable in physiological buffers, and non-toxic, compared to Tat proteins.

Therefore, in the present invention, the PEP-1 peptide carrying the natural protein into the cell was fused to human protein HSP27, the fusion protein was overexpressed in Escherichia coli, and the metal-chelating affinity chromatography was performed. It was confirmed by cell and animal experiments that the purified fusion protein was easily and conveniently purified, and that the purified fusion protein was effectively transported into cells with biological activity. And it was confirmed that PEP-1-HSP27 fusion protein increases the activity of superoxide dismutase (SOD). Therefore, the present invention is considered to be a very breakthrough technology in the PEP-1-HSP27 fusion protein in intracellular transport, protein therapeutics and applicability.

The present invention utilizes PEP-1 peptide to heat shock protein 27 to increase the activity of superoxide dismutase known to play an important role in defense against atrophic lateral sclerosis known as Lou Gehrig's disease and harmful oxygen. It was performed to see if the "combined with" HSP27 "can be transported into the cell and whether the PEP-1-HSP27 fusion protein carried into the cell exhibited the function of the protein in the cell.

We firstly overexpress and easily purify the PEP-1-HSP27 fusion protein PEP-1-HSP27 Expression vectors were developed. This expression vector is a human HSP27 cDNA, PEP-1 peptide (21 amino acids) and six histidines are connected in series. Using this expression vector, by over-expression of the PEP-1-HSP27 fusion protein in E. coli Ni ^{2 +} in the natural state were purified using an affinity chromatography column. The overexpression of PEP-1-HSP27 was quite high, resulting in a high amount of purified protein. It was confirmed that PEP-1-HSP27 fusion protein purified on skin cells cultured by Western blot was carried to the cells in a concentration-dependent manner. It was found that PEP-1-HSP27 fusion protein permeated into cells effectively inhibits damage caused by harmful oxygen and greatly increases the activity of SOD.

These results indicate that the PEP-1-HSP27 fusion protein is well permeable into cells and exhibits well HSP27 function in cells. Therefore, this PEP-1-HSP27 fusion protein suggests the possibility of various applications in the treatment of brain diseases and various diseases, such as increased activity of SOD with respect to harmful oxygen.

Pharmaceutical compositions containing the PEP-1-rpS3 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-HSP27 fusion protein of the present invention is contained in a creamy oil-in-water (O / W) or water-in-oil (W / O) cream base, which contains perfumes, chelating agents, and pigments. 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 an essence or the like.

The present inventors were able to confirm that the PEP-1-HSP27 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-rpS3 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 HSP27 into cells, including nerve cells and skin cells. Intracellular delivery of the HSP27 protein molecule according to the present invention is composed of 15-30 amino acids in HSP27, 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-HSP27 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-HSP27 fusion protein" includes a protein transport domain and HSP27, and refers to a covalent complex formed by genetic fusion or chemical bonding of a transport domain and a cargo molecule (ie, HSP27 in the present invention). it means. In this specification, it is mixed with "PEP-1-HSP27", "HSP27 fusion protein" or "PEP-HSP27".

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.

The present invention provides a heat shock protein 27 comprising a protein transport domain consisting of 15-30 amino acids, a non-hydrophilic domain including 5 or more tryptophan, a hydrophilic domain including 4 or more lysines, and a spacer separating the two domains. It relates to a cell-transducing heat shock protein 27 (HSP27) fusion protein covalently bound to at least one end of. 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 HSP27 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 HSP27 protein.

In addition, the present invention relates to a skin external preparation composition for the treatment and prevention of skin aging, atopy, and skin irritation by UV irradiation, comprising the cell-transducing HSP27 fusion protein as an active ingredient.

The present invention also relates to a cosmetic composition comprising the cell-transducing HSP27 fusion protein as an active ingredient.

In addition, the present invention is characterized in that the glomerulonephritis, vasculitis, autoimmune diseases, stroke, myocardial infarction, arrhythmia, angina pectoris, characterized in that the cell-derived HSP27 fusion protein as an active ingredient, comprising a pharmaceutically acceptable carrier Idiopathic hemochromatosis, disease caused by radiation therapy, premature ejaculation, disease-related aging, sickle cell disease, malaria, emphysema, cardiomyopathy, autoimmune nephrotic syndrome, Betelnet-related oral cancer, hyperbaric oxygen, Alzheimer's disease, Parkinson's disease, cataracts, Lou Gehrig It relates to a pharmaceutical composition for the treatment and prevention of diseases, diabetes, cerebral ischemia, atopic disease.

The present invention also relates to recombinant polynucleotides including SEQ ID NO: 6, wherein the protein transport domain peptide coding DNA sequence is coupled to HSP27 cDNA to encode the cell-introduced 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-introduced HSP27 fusion protein, characterized in that it comprises the recombinant polynucleotide to express the HSP27 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.

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 sepharose superflow was purchased from Qiagen (Germany). Human heat shock protein 27 (HSP27) cDNA was isolated from human liver cDNA library by PCR method. All other reagents were used as express products.

Example  One: Recombination PEP -One- HSP27  Fusion protein of  Expression vector preparation and transformation

In order to develop a technique for infiltrating a protein having a function into a cell, a fusion protein expression vector capable of delivering a target protein into a cell was prepared, and human heat shock was performed to easily analyze the ability of the PEP-1 peptide to deliver the protein into the cell. Protein 27 was selected.

First, PEP-1-HSP27 To produce a fusion protein, a pET-PEP expression vector containing a PEP-1 peptide (KETWWE TWWTEW SQP KKKRKV; 21 amino acids) was prepared. Up of the two types for the PEP-1 peptide nucleotides; the (top strand, 5'-TATGAAAGAAACCTGGTGGGAAACCTGGTGGACCGAATGGTCTCAGCCGAAAAAAAAACGTAAAGTGC-3 'Sub-chain, 5'-TCGAGCACTTTACGTTTTTTTTTCGGCTGACACCATTCGGTCC ACCAGGTTTCCCACCAGGTTTCTTTCC-3' ) Nde Ⅰ - in pET-15b cut with restriction enzymes Xho Ⅰ Inserted by ligation. Subsequently, two kinds of oligonucleotides were synthesized based on the sequence of the cDNA of human HSP27. The forward primer has a Xho I restriction site with 5'-CTCGAGATGACCGAGCGCCGCGTCCCCTTC-3 'and the reverse primer has a Bam HI restriction site with 5'-GGATCCTTACTT GGCGGCAGTCTCATCGGA-3'.

Polymerase chain reaction (PCR) was carried out in a thermocycler (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 reactants were separated by agarose gel electrophoresis and linked to a TA cloning vector (Invitrogen, Sandiego, USA), transformed into competent cells, and alkaline elution of the plasmid from the transformed bacteria. Separated by law [10]. The TA vector containing the human HSP27 cDNA Xho Ⅰ and It was cut with Bam HI and inserted into the PEP expression vector. E. coli BL21 (DE3) transformed with PEP-1-HSP27 was selected, and colonies were inoculated in 100 ml LB medium and IPTG (0.5 mM) was added to the medium to overexpress the recombinant PEP-1-HSP27. Induced. Overexpressed PEP-1-HSP27 was confirmed by SDS-PAGE and Western blotting.

Example  2: recombination PEP -One- HSP27  Fusion protein field  Expression and Purification

E. coli BL21 (DE3) cells (PEP-1-HSP27) containing human HSP27 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 = 0.5-1.0, IPTG was added to the medium to bring the final concentration to 0.5 mM, and then further incubated 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 by an ultrasonic grinder. The supernatant was immediately centrifuged Ni ^{2 +} - nitrile three super acid Sepharose flow ^{(Ni 2 + -nitrilotriacetic acid sepharose super} flow) load to the column, washed with buffer, binding buffer and 6 times the volume of 10-fold volume (60mM already in The fusion protein was washed with dozol, 0.5M NaCl, 20mM Tris-HCl, pH 7.9) followed by elution buffer (1M imidazole, 0.5M NaCl, 20mM Tris-HCl, pH 7.9). Fractions containing the fused protein were collected and the salt contained in the fractions was removed using a PD-10 column.

Purified protein concentration was measured by Bradford method using bovine serum albumin as a standard [11].

Example  3: cell culture and PEP -One- HSP27 Intracellular Permeation of Fusion Proteins

Astrocytes and skin cells were obtained from ATCC, supply 95% air and 5% CO2 at 37 ° C, 20 mM HEPES / NaOH (pH 7.4), 5 mM NaHCO ₃ , 10% fetal bovine serum And Dulbecco's Modified Eagle's Medium (DMEM) medium containing antibiotics (100 μg / ml streptomycin, 100 U / ml penicillin).

To observe intracellular penetration of PEP-1-HSP27 fusion protein, astrocytes and skin cells were grown in 6-well plates for 4-6 hours and replaced with fresh DMEM culture containing 10% FBS, and recombinant PEP- 1-HSP27 (3 μM) was treated in the 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 HSP27 permeated into the cells was measured by Western blot analysis.

Example  4: Weston Blot  analysis( Wesern blot analysis )

Western blot methods were performed to confirm intracellular penetration of PEP-1-HSP27. One hour after the cells were treated with the fusion protein of PEP-1-HSP27 (3 μM), only cells were collected and western blots were performed. Proteins in the cell mill were separated using 12% SDS polyacrylamide gel electrophoresis, and the proteins in the gels were then electrophoresed to a nitrocellulose membrane (Amersham, UK). The protein-shifted nitrocellulose membrane was blocked with PBS containing 5% non-dry milk. The membrane was then treated with a 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, the ECL kit (ECL; Amersham) was used to identify protein bands that respond to human HSP27 monoantibody.

Example  5: Superoxide Dismutase ( SOD ) Enzyme activity measurement

The activity of SOD was measured by spectrophotometric observation of the degree of inhibition of ferricytochrome c reduction by xanthine / xanthine oxidase reaction according to McCord and Fridovich's method (1969) [12]. .

Standard assays were performed in 50 mM calcium phosphate buffer (pH 7.8) containing 3 ml of 0.1 mM EDTA at 25 ° C. The reaction mixture contained 10 μM pericytochrome c, 50 μM xanthine and a sufficient amount of xanthine oxidase and the reduction of pericytochrome c was measured at 550 nm. The amount of superoxide dismutase that reduced cytochrome c by 50% under the above conditions was defined as 1 unit.

Example  6: immunohistostaining ( Immunohistochemistry )

Immunohistoscopic staining was performed to confirm that the PEP-1-HSP27 fusion protein penetrated into the brain tissue to protect neurons. Experimental animals used male gerbil mice. The animals were anesthetized with 3% isoflurance in a nitrogen-oxygen mixed gas and brain tissues were made into frozen tissue sections.

Prior to immunohistostaining, frozen tissue sections were dried at room temperature for 30 minutes and then completely dried at 37 ° C. for at least 2 hours before starting the experiment. Tissue sections were buffered with 0.01 M PBS (PBS) for 10 minutes and washed again with distilled water for 10 minutes. After washing with PBS twice for 7 minutes, and then reacted for 20 minutes in a PBS solution containing 0.5% hydrogen peroxide to remove endogenous peroxidase in the tissue. After washing twice with PBS for 7 minutes each, the tissues were reacted with 5% normal goat serum in PBS for 30 minutes to prevent non-specific reactions. Subsequently, immunohistostaining was performed using a conventional streptavidin-biotin peroxidase method.

The primary antibody was a rabbit anti-mouse histidine (Santa cruz, USA) antibody. All antibodies used for immunohistochemistry were diluted in 0.1 M PBS (pH 7.4) containing 2% normal goat serum and 0.1% Triton X-100. The skin tissue was diluted 1: 500 primary antibody and reacted at 4 ° C. for 48 hours. Thereafter, the biotin-bound rabbit anti-mouse IgG (Vector, USA) with the secondary antibody was diluted 1: 200 and reacted at room temperature for 2 hours, followed by the peroxidase-linked streptavidin (Vector, diluted 1: 200). USA) at room temperature for 2 hours. After the reaction of each step was washed 4-5 times with PBS solution.

Antigen-reacted tissues were developed in Tris buffer solution (pH 7.4) containing 0.03% hydrogen peroxide and 0.05% DAB (3,3'-diaminobenzidine tetrachloride; Sigma, USA) for 1-5 minutes. After completion of the color reaction, the tissue was encapsulated using a crystal mount (Biomeda, USA), which is a water-soluble encapsulant, and then photographed after a microscope observation (Axioscope microscope; Carl Zeiss, Germany).

Result 1: recombination PEP -One- HSP27  Fusion protein of  Overexpression and Refining

To overexpress the PEP-1-HSP27 fusion protein, a PEP-1-HSP27 expression vector was developed containing human HSP27 cDNA, PEP peptide (KETWWETWWTEW SQP KKKRKV; 21 amino acids) 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 PEP-1-HSP27 overexpression and purified protein stained with Coomassie Brilliant Blue (FIG. 2A) and Western blot (FIG. 2B). Figure 2A shows well overexpression of the fusion protein induced by treatment with 0.5mM IPTG.

Since the PEP-1-HSP27 fusion protein contains six histidines at the N-terminus, immobilized metal-chelate affinity chromatography allows the fusion protein to be pure in nature (purity> 95). %) Purified (FIG. 2A). Purified PEP-1-HSP27 fusion protein was confirmed by staining with Coomassie Brilliant Blue.

Overexpressed and purified PEP-1-HSP27 fusion protein was once again confirmed by Western blot analysis. As shown in FIG. 2B, the PEP-1-HSP27 fusion protein band in response to HSP27 or 6x histidine antibody was observed at the same position as the protein band of FIG. 2A.

Result 2: recombination PEP -One- HSP27 Neuronal Permeation of Fusion Proteins

It was observed how the permeation of the skin cells of the purified PEP-1-HSP27 fusion protein in nature changed with concentration. As shown in FIG. 3, Western blotting confirmed that natural PEP-1-HSP27 penetrated into skin cells in a concentration-dependent manner. Figure 3 shows the results when 1-5μM PEP-1-HSP27 fusion protein was treated in cell culture for 1 hour. When the degree of intracellular penetration of PEP-1-HSP27 was observed for each concentration, it was confirmed that the amount of PEP-1-HSP27 permeated into cells increased in a concentration-dependent manner. These results indicate that the PEP-1-HSP27 fusion protein permeates into cells in a concentration dependent manner.

Result 3: PEP -One- HSP27 Biological Functions of Fusion Proteins

Fusion proteins penetrated into neurons 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 function of the purified PEP-1-HSP27 fusion protein, cell survival rate induced by paraquat was confirmed by MTT analysis (FIG. 4). After treatment of purified PEP-1-HSP27 fusion protein with (1-5μM) concentration with paraquat (30mM) in neurons, cell viability was measured and the fusion protein protected cell death by paraquat by concentration. Confirmed.

In addition, by measuring the SOD activity by reacting with superoxide dismutase (SOD) mutants, it was found that the activity of the SOD mutants was increased (data not shown), and suppressed cell death more than the SOD mutant. It was confirmed that (Fig. 5). Increased viability of cells infiltrated with PEP-1-HSP27 fusion protein indicates that the fusion protein effectively protects against cell death by paraqaut.

Result 4: PEP -One- CCS Protective effect of fusion protein in animal experiments

In order to confirm the cell death protective effect of the fusion protein in the cerebral ischemia animal model, 300 ㎍ of the fusion protein was intraperitoneally injected 30 minutes before the induction of cerebral ischemia and confirmed by tissue immunostaining 4 days later. As shown in the figure, it was found that the PEP-1-HSP27 fusion protein almost completely protects neurons (FIG. 6). These results indicate that the fusion protein effectively penetrates into cells and protects neuronal cell death.

The present invention provides a technique for the direct penetration of human heat shock protein 27 (HSP27) into the cell at the protein level in an effective manner.

Harmful oxygen damages cellular biopolymers and is reportedly deeply involved in the progression of over 100 human diseases (10–12). Therefore, the PEP-1-HSP27 fusion protein of the present invention may be effectively used for protein treatment of diseases by increasing the activity of superoxide dismutase, which plays a major role in protecting damage caused by harmful oxygen. It is feed.

Based on the present invention, PEP-1-HSP27 fusion protein can be delivered directly into cells to protect cells and tissues from harmful oxygen harmful to our human body, which is why we have degenerative diseases such as Lou Gehrig's disease, diabetes, cerebral ischemia, Parkinson's, atopy It can be usefully used for the treatment and prevention of, and can be widely used in the functional cosmetics industry.

Attach sequence list electronic file  

Claims (8)

At least one side of the heat shock protein 27 is a protein transport domain consisting of 15-30 amino acids, a non-hydrophilic domain containing 5 or more tryptophan, a hydrophilic domain containing 4 or more lysines, and a spacer separating the two domains. A cell-transducing heat shock protein 27 (HSP27) fusion protein having the amino acid sequence of SEQ ID NO: 7 covalently bound to a terminus. delete delete A skin external composition for treating and preventing skin aging, atopy and dermatitis caused by UV irradiation, comprising the cell-introduced thermal shock protein 27 fusion protein of claim 1 as an active ingredient. A cosmetic composition comprising the cell introducing thermal shock protein 27 fusion protein of claim 1 as an active ingredient. Glomerulonephritis, vasculitis, autoimmune disease, stroke, myocardial infarction, arrhythmia, angina pectoris, idiopathic, characterized in that the cell-induced thermal shock protein 27 fusion protein of claim 1 as an active ingredient and comprises a pharmaceutically acceptable carrier Hemochromatosis, Radiation Therapy, Premature Ejaculation, Disease-Related Aging, Sickle Cell Disease, Malaria, Emphysema, Cardiomyopathy, Autoimmune Nephrotic Syndrome, Betelnet-Related Oral Cancer, Hyperbaric Oxygen, Alzheimer's Disease, Parkinson's Disease, Cataracts, Lou Gehrig's Disease , Pharmaceutical composition for the treatment and prevention of diabetes mellitus, cerebral ischemia, atopic disease. The recombinant polynucleotide of SEQ ID NO: 6, wherein the protein transport domain peptide coding DNA sequence is coupled to the heat shock protein 27 cDNA encoding the cell-introduced heat shock protein 27 fusion protein of claim 1. A vector expressing the cell-introduced thermal shock protein 27 fusion protein, characterized in that it comprises the recombinant polynucleotide of claim 7 to express the fusion protein of claim 1.

Images