KR101611658B1 - A Peptide for Efficiently Introducing Proteins into Cells and Remaining them - Google Patents

Abstract

The present invention relates to a method for introducing intracellular proteins with high efficiency, and more particularly, to a method for introducing intracellular proteins with high efficiency using a specific short mitochondrial target peptide, sMTS, and to provide intracellular introduction and maintenance efficiency of biofunctional substances such as proteins, nucleic acids, And to a fused composition used therefor.

Description

[0001] The present invention relates to peptides for the efficient introduction and maintenance of intracellular proteins {A Peptide for Efficiently Introducing Proteins into Cells and Remaining them}

The present invention relates to a method for introducing intracellular proteins with high efficiency, and more particularly, to a method for introducing intracellular proteins with high efficiency using a specific short mitochondrial target peptide sMTS, And a fusant composition used therefor and their use.

In general, hydrophilic or large molecular mass substances can not enter the cell by the barrier of cell membrane. Cell membranes prevent macromolecules such as peptides, proteins, and nucleic acids from entering the cell and, even if they enter the cell through a physiological mechanism called endocytosis by cell membrane receptors, they are fused with the cell's lysosomal compartment As a result, many restrictions are imposed on the treatment and prevention of diseases using the macromolecules. In the case of anticancer agents, it is necessary to overcome obstacles such as multidrug resistance in order to deliver the drug into cells. In order to prevent drug degradation, many methods of delivering various macromolecules and drug - containing carriers into cells directly without passing through the endocytosis process have been proposed. These methods include microinjection, electroporation, etc., which can damage cell membranes. Other methods include using pH-sensitive liposomes and cell permeable materials. However, even if a drug is delivered into cells through such a method, there is a problem that it must be moved to a specific organ in order to exhibit its effect.

One of the new alternatives is that the cell penetrating peptide (CPP) or protein transduction domain (PTD) has been used as a therapeutic protein, which has been difficult to use as a drug due to its low cell membrane permeability and fast in vivo half- It is getting a lot of attention because it can increase the utilization value of macromolecules such as genes. It has already been reported that cell-permeable peptides can chemically bind to various substances as well as proteins, allowing them to permeate into cells (Maarja and Ulo, Current Opinion in Pharmacology, 6: 509-514, 2006).

Such membrane permeable peptides are typically peptides composed of the 47th amino acid to the 57th amino acid of Tat protein (Schwarze SR et al., Science, 285: 1569-1572, 1999), a transcription factor of human immunodeficiency virus, (Joliot A. et al., Proc Natl Acad Sci, 88: 1864-1868, 1991), which is composed of the 339th amino acid to the 355th amino acid of the antennapedia protein.

Much research has been done on the in vivo transport of macromolecules by these peptides. (Schwarze SR et al., Science, 285: 1569-1572, 1999), topical application of cyclosporin A by an arginine oligomer, by in vivo delivery of beta-galactosidase by Tat peptide (Jonathan B. Rothbard. Et al., Nature Medicine, 6: 1253-1257, 2000), the effect of sMTS peptides derived from the signal peptide of fibroblast growth factor In vivo infiltration and anti-inflammatory and anti-apoptotic effects (DaeWoong Jo et al., Nature Medicine, 11: 892-898, 2005), by Hph-1 peptides derived from the signal peptide of human transcription factor (JM Choi et al., Nature Medicine, 12: 574-579, 2006) have been reported to inhibit the allergic inflammatory response of the nasal mucosa of the CTL-4 region.

Particularly, in a previous study of the present inventors, a method of effectively introducing a therapeutic agent such as diabetes into a cell by using an antioxidant fusion protein in which a protein transduction domain is peptide-bound to the amino terminus of metallothionein However, the fusion protein using the protein transduction domain (PTD) still has a problem that the efficacy of the fusion protein is deteriorated after being introduced into the cell after the introduction into the cell.

Therefore, the inventors of the present invention used sMTS, a short peptide of a specific amino acid sequence derived from a mitochondrial target peptide, and used a vector expressing PTD-sMTS-MT fusion protein in using a protein transduction domain (PTD) and an antioxidant fusion protein , The cell inflow and maintenance efficiency of the protein is remarkably improved, and the present invention has been completed.

A main object of the present invention is to provide a use of a peptide sMTS which significantly increases the intracellular introduction rate and retention rate of an antioxidant fusion protein composed of a metallothionein (MT) and a protein transduction domain (PTD).

Another object of the present invention is to provide a composition for treating diabetes mellitus containing a carrier of PTD-sMTS-MT fusion protein and a method for treating diabetes using the same.

In order to solve the above problems, the present invention provides a new use of sMTS peptide composed of an amino acid of MVSAL which significantly increases the intracellular introduction rate and retention ratio of the protein.

That is, the present invention relates to a novel use of a sMTS peptide containing an amino acid sequence of MVSAL as a peptide sequence (a short mitochondrial target peptide) derived from a mitochondrial target peptide. In addition to the mitochondrial targeting ability disclosed in previous studies, the sMTS peptide And a function of remarkably improving intracellular introduction and maintenance of the protein.

In particular, the intracellularly introduced protein preferably includes a protein transduction domain (PTD), and more preferably an antioxidant fusion protein comprising a metallothionein (MT) and a protein transduction domain (PTD).

The sMTS composed of the amino acids of MVSAL may be selected from the group consisting of malate dehydrogenase, human cytochrome c subunit oxydase VIII, PI subtype of human ATP synthetase subunit c, aldehyde dehydrogenase targeting sequence, human ATP synthetase subunit FI Beta and BCS1 proteins and the like can be used.

The present invention, in one embodiment,

SMTS peptides composed of amino acids of MVSAL; Metal rochionein (MT); (PTD-sMTS-MT) composition comprising a protein transduction domain (PTD) and a protein transduction domain (PTD).

Preferably, the metallothionein is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 4, and the protein transduction domain is Tat (Trans Activator of Transcription) protein of HIV-1 virus, polyarginine Arginine), Penetratin, transcription regulatory protein of HSV-1 structural protein VP22, Pep-1 peptide, and Pep-2 peptide. Preferably Tat represented by the amino acid sequence of SEQ ID NO: 5 or polyarginine represented by the amino acid sequence of SEQ ID NO: 6.

The composition of the present invention has an effect of reducing the concentration of active oxygen species (ROS) in a cell

Therefore, the present invention provides, as another embodiment, a therapeutic gene or a therapeutic drug delivery composition using the fusion protein (PTD-sMTS-MT) for introducing and maintaining the protein high-efficiency cell and a method for using the same.

The cells to be delivered are most preferably selected from the group consisting of myocardial cells, pancreatic cells, nerve cells, vascular endothelial cells, etc. which can utilize antioxidant fusion proteins composed of metallothionein (MT) and protein transduction domain (PTD) As shown in FIG.

As one example, the fusant composition, an expression vector expressing such a fusant, or a microorganism transformed with the expression vector; Or a pharmaceutical composition for the prevention and treatment of diabetes mellitus or diabetic cardiomyopathy which contains them as an active ingredient. That is, it is useful to more effectively protect pancreatic beta cells and myocardial cells from glucolipotoxicity or hyperglycemic stress.

The diabetes mellitus in the present invention includes systemic or local diseases that cause diabetes mellitus and diabetes (preferably non-insulin dependent type 2 diabetes) as a direct or indirect factor, specifically diabetic acidosis diabetic acidosis, diabetic xanthoma, diabetic amyotrophy, diabetic ketosis, diabetic coma, diabetic gastric disorder, diabetes mellitus, Diabetic gangrene, diabetic ulcer, diabetic complications, diabetic diarrhea, diabetic microangiopathy, diabetic uterine body sclerosis, diabetic neuropathy, diabetic neuropathy, Myocardial infarction, diabetic cardiomyopathy, diabetic neuropathy, diabetic nephropathy, bullosis diabeticorum, Diabetic retinopathy, diabetic cataract, diabetic dermopathy, diabetic scleredema, diabetic retinopathy, necrobiosis lipoidica diabeticorum, diabetic blood circulation disorder diabetic blood circulation disorder, and the like.

SMTS, which is a short peptide derived from the mitochondrial target peptide of the present invention, significantly increases the intracellular introduction rate and retention rate of the antioxidant fusion protein composed of the metallothionein (MT) and the protein transduction domain (PTD) Effective delivery of the gene or therapeutic agent into the cell is possible.

1 is a schematic diagram of a vector for expression of a fusion protein of the present invention.
Figure 2 shows the result of SDS-PAGE of the fusion protein of the present invention.
Figure 3 shows the result of confirming the cytotoxicity of the fusion protein of the present invention.
FIG. 4 shows the results of comparing the transfection efficiency of Tat-GFP and Tat-sMTS-GFP according to the presence or absence of the culture medium washing step.
Figure 5 shows the results of the comparison of the transfection efficiency of Tat-GFP (TG) and Tat-sMTS-GFP (TMG) after washing of the culture medium.
Figure 6 shows the results of intracellular retention time comparison of Tat-MT (TM) and Tat-sMTS-MT (TMM).
FIG. 7 is a graph showing the effect of Tat-MT (TM) and Tat-sMTS-MT (TMM) on H9c2 cells under hypoglycemia and hypoxia conditions in (a) hyperglycemia And the cell viability was confirmed.
FIG. 8 shows the effect of ROS on Hgc2 cells under hyperglycemia (HG) (b) hypoxia (HP), (c) hyperglycemia and hypoxic conditions in H9c2 cells.
FIG. 9 is a graph showing changes in blood glucose level according to injection of 3 μM TMM in a STZ-induced diabetic mouse model. FIG.
FIG. 10 shows the results of confirming the level of reactive oxygen species upon injection of 3 μM TMM in the pancreas extracted from STZ-induced diabetic mice.

The terms used in the present invention are defined as follows.

"Subject" or "patient" means any single entity that requires treatment, including human, cow, dog, guinea pig, rabbit, chicken, In addition, any subject who participates in a clinical study test that does not show any disease clinical findings, or who participates in epidemiological studies or used as a control group is included.

"Tissue or cell sample" refers to a collection of similar cells obtained from a subject or tissue of a patient. The source of the tissue or cell sample may be a solid tissue from fresh, frozen and / or preserved organ or tissue sample or biopsy or aspirate; Blood or any blood components; It may be a cell at any point in the pregnancy or development of the subject. Tissue samples can also be primary or cultured cells or cell lines.

"Gene" means any nucleic acid sequence or portion thereof that has a functional role at the time of protein coding or transcription, or in the control of other gene expression. The gene may consist of only a portion of the nucleic acid encoding or expressing any nucleic acid or protein that encodes the functional protein. The nucleic acid sequence may comprise an exon, an intron, an initiation or termination region, a promoter sequence, another regulatory sequence, or a gene abnormality within a particular sequence adjacent to the gene.

"Fusion or linkage of sequences" refers to a single polypeptide chain comprising fused components. The fused components may be connected directly or indirectly. For example, another sequence (i. E., A linker or functional domain) may be located between the fused elements. In the present invention, sMTS peptides composed of amino acids of MVSAL; Metal rochionein (MT); And a protein transduction domain (PTD) can be fused.

"Expression" refers to the biological production of a product encoded by a coding sequence. In most cases, DNA sequences, including coding sequences, are transcribed to form messenger RNA (mRNA). The messenger RNA is translated to form a polypeptide having biological activity. In some cases, however, the RNA product may be considered as a gene product since it may have an associated activity. Expression may include additional processing steps of the transcriptional RNA product, such as splicing for intron removal and / or post-translational processing of the polypeptide product.

"Coding region" or "coding sequence" refers to a nucleic acid sequence, a complement thereof, or a portion thereof, which encodes a particular gene product or fragment thereof that is required to be expressed, according to a common base pairing and codon usage relationship. Coding sequences include exons in genomic DNA or immature primary RNA transcripts that are joined together by a biochemical machinery of cells to provide mature mRNA. The antisense strand is a complement of the nucleic acid, and the coding sequence can be deduced therefrom. The coding sequence is placed in the relationship of transcriptional regulatory elements and translation initiation and termination codons such that transcripts of appropriate length are generated and translated in the appropriate reading frame to produce the desired functional product

The term "polynucleotide" or "nucleic acid" refers to nucleotide polymers of any length, including ribonucleotides as well as deoxyribonucleotides. This term refers only to the primary structure of the molecule, and thus to DNA or RNA of a double or single chain. This also applies to known types of modifications, for example, labeling, methylation, "caps", nucleotide substitutions of one or more naturally occurring nucleotides, debonding (eg, methyl phosphonate, phosphotriester, phosphoamidate, carbarnate (Eg, phosphorothioate, phosphorodithioate, etc.), proteins (eg, nucleases, toxins, antibodies, signal peptides, poly-Llysine, etc.) Having an alkyl compound, a modified bond (e.g., alpha (alpha), beta (alpha) anomeric nucleic acid, etc.), as well as inter-nucleotide modification including non-modification of the polynucleotide. Generally, the nucleic acid moieties provided by the present invention may comprise fragments of a genome and a short oligonucleotide linker or series of oligonucleotides, microorganisms or viral operons, or regulatory elements derived from eukaryotic genes, Lt; RTI ID = 0.0 > nucleotides < / RTI > that can be expressed as recombinant transcription units

The term "functional equivalent" refers to, for example, one or more substitutions, deletions or additions from a reference sequence, an actual effect that does not result in various functional dissimilarities between the reference and subject sequences < / RTI > net effect) and the nucleotide sequence of the mutated mutant sequence. A substantial equivalent, e. G. Mutant, amino acid sequence according to the invention has preferably at least 80% sequence identity, more preferably at least 90% sequence identity with the listed amino acid sequence. The nucleotide sequence of the present invention, which is a substantial equivalent, may have a lower percentage of sequence identity, for example, when considering the redundancy or degeneracy of the genetic code. Preferably, the nucleotide sequence should have at least about 65% identity, more preferably at least about 75% identity, and most preferably about 95% identity. For the purposes of the present invention, sequences with substantially equivalent biological activities and substantially equivalent synthetic features are treated as substantial equivalents.

The term "vector" means a nucleic acid molecule capable of transporting another nucleic acid to where it is associated. The term " expression vector "includes a plasmid, a cosmid, or a phage capable of synthesizing a fusion protein encoded by a respective recombinant gene carried by the vector.

The term " transduction "means that the gene of the host cell is inserted into the genome of the virus during the infection process and then transferred to another host cell at the time of another infection of the virus. In a typical transduction, any gene in a host cell can be delivered by this process, but only a few specific genes are transduced in a particular transduction. Transduction is used as a microbiological technique in many other genetic experiments to create new bacterial strains or to know the gene location.

"Amino acid" and "amino acid residue" refer to naturally occurring amino acids, unnatural amino acids, and modified amino acids. Unless otherwise stated, all references to amino acids specifically include references to both D and L stereoisomers (where the structure permits such stereoisomeric forms), specifically or by name. Natural amino acids include but are not limited to alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gln), glutamic acid (Glu), histidine (Ile), Leu, lys, methionine, phenylalanine (Phe), proline, serine, threonine (Thr) (Val). Non-natural amino acids include modified amino acid residues chemically modified on the N-terminal amino group or side chain group, or reversibly or irreversibly chemically blocked, such as N-methylated D and L amino acids or side chain functional groups, Chemically modified residues.

The term " disorder "is any condition that can benefit from treatment with a molecule identified using the transgenic animal model of the invention. This includes chronic and acute diseases or diseases, including pathological conditions that make mammals susceptible to suspicious diseases. Examples of diseases to be treated herein include, but are not limited to, diabetes, obesity, metabolic syndrome, and the like.

"Treatment" is an approach to obtaining beneficial or desired results, including clinical results. "Treatment" or "alleviation" of a disease, disorder or condition refers to a decrease or decrease in the severity of a condition, disorder, or disease state and / or undesirable clinical symptoms, Is slow or long.

A "therapeutically effective amount" refers to the amount of active compound in a composition, system, subject, or composition that will elicit a biological or medical response in a human, system, subject, or human being sought by a researcher, veterinarian, .

A "prophylactically effective amount" refers to an amount of a biological or medicinal product in a tissue, system, subject or human being sought by a researcher, veterinarian, physician or other clinician to prevent the onset of the disease in a subject at risk for the relevant disorder, condition, Quot; means the amount of active compound in the composition from which the response will be derived.

"Gene therapy" refers to treating a disease by correcting a mutated gene, treating a genetic disease, or regulating protein expression using a gene or RNAi. In other words, it is a method of treating a disease by transplanting a normal gene from the outside into a patient's cell and changing the phenotype of the cell. In gene therapy, research on gene vectors and systems that transduce genes into the body is essential.

"About" means that the reference quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight or length is 30, 25, 20, 25, 10, 9, 8, 7, , Level, value, number, frequency, percent, dimension, size, quantity, weight, or length that varies from one to three, two, or one percent.

Throughout this specification, the words " comprising "and" comprising ", unless the context requires otherwise, include the stated step or element, or group of steps or elements, but not to any other step or element, And that they are not excluded.

Hereinafter, the present invention will be described in detail.

The present invention relates to an intracellular biomass delivery system, and more particularly to a novel use of a short mitochondrial targeting peptide (sMTS) containing an amino acid sequence of MVSAL, as used herein.

 Preferably, the function of sMTS containing the amino acid sequence of MVSAL, which significantly improves the intracellular introduction rate and maintenance rate of the antioxidant fusion protein capable of protecting the destruction of pancreatic beta cells and cardiomyocytes from sugar oxidation and glycolipid stress .

In a specific embodiment, a sMTS peptide consisting of an amino acid of MVSAL; Metal rochionein (MT); (PTD-sMTS-MT) composition composed of a protein transduction domain (PTD) and a protein transduction domain (PTD), a recombinant polynucleotide construct such as a recombinant expression vector containing the same (collectively referred to as "expression vector" , A microorganism transformed with the above expression vector, and a composition for the treatment of diabetic diseases comprising one of the above, as well as to an intracellular introduction and maintenance system of the PTD-sMTS-MT fusion.

The present invention relates to a method for screening a sMTS peptide consisting of an amino acid of MVSAL with a metallothionein (MT) -PTD sequence and using a standard cloning technique known to those of ordinary skill in the art and a conventional method, And expressing the fusion protein in vivo.

MVSAL Consisting of sMTS Peptides

In one aspect, the present invention relates to a novel function of enhancing intracellular protein introduction and retention ability of sMTS peptide composed of amino acid of MVSAL.

That is, the present invention aims at enhancing the ability to introduce and maintain the cell into the cell, not merely using the target function into mitochondria of known sMTS.

(M) -valine (V) -serine (S) -alanine (A) - alanine (A) - alanine (A) - alanine Leucine (L) ". In the specification of the present invention, " MVSAL ", which is an abbreviation of the amino acids, is used.

In this case, the sMTS sequence MVSAL is not particularly limited in the present invention as long as it is derived from a mitochondrial protein.

Typically, the mitochondrial targeting sequence is selected from the group consisting of malate dehydrogenase, human cytochrome c subunit oxidase VIII, PI subtype of human ATP synthetase subunit c, aldehyde dehydrogenase targeting sequence, human ATP synthase subunit FI beta And a BCS1 protein. Preferably from malate < RTI ID = 0.0 > dehydrogenase. ≪ / RTI >

PTD - sMTS - MT  Composition

As described above, the sMTS peptide composed of the MVSAL amino acid of the present invention significantly increases the ability to introduce and maintain the protein into a desired cell, for example, a myocardial cell, a pancreatic cell, a neuron, a vascular endothelial cell, .

As a preferable example, the antioxidant fusion protein composed of the deurocholine (MT) and the protein transduction domain (PTD) binds to sMTS of MVSAL, thereby significantly increasing the ability to introduce and maintain the desired cell.

The antioxidant fusion protein used in one embodiment of the present invention is peptide-coupled with a protein transduction domain at the amino terminus of metallothionein. The peptide is bound to the pancreatic beta cells and the myocardium from saccharide- And the function of protecting cell destruction.

The metallothionein may be any of the types I, II, III and IV, and its isoforms, if derived from human. Preferably those represented by the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

SEQ ID NO: 1: Met Asp Pro Asn Cys Ser Cys Ser Thr Gly Gly Ser Cys Thr Cys Thr Ser Cys Ala Cys Lys Asn Cys Lys Cys Thr Ser Cys Lys Lys Ser Cys Cys Ser Cys Cys Pro Val Gly Cys Ser Lys Cys Ala Gln Gly Cys Val Cys Lys Gly Ala Ala Asp Lys Cys Thr Cys Cys Ala

SEQ ID NO: 2: Met Asp Pro Asn Cys Ser Cys Ala Ala Gly Asp Ser Cys Thr Cys Ala Gly Ser Cys Lys Cys Lys Glu Cys Lys Cys Thr Ser Cys Lys Lys Ser Cys Cys Ser Cys Cys Pro Val Gly Cys Ala Lys Cys Ala Gln Gly Cys Ile Cys Lys Gly Ala Ser Asp Lys Cys Ser Cys Cys Ala

SEQ ID NO: 3: Met Asp Pro Glu Thr Cys Pro Cys Pro Ser Gly Gly Ser Cys Thr Cys Ala Asp Ser Cys Lys Cys Glu Gys Cys Lys Cys Thr Ser Cys Lys Lys Ser Cys Cys Ser Cys Cys Pro Ala Glu Cys Glu Lys Cys Ala Lys Asp Cys Val Cys Lys Gly Gly Glu Ala Ala Glu Ala Glu Ala Glu Lys Cys Ser Cys Cys Gln

SEQ ID NO: 4: Met Asp Pro Arg Glu Cys Val Cys Met Ser Gly Gly Ile Cys Met Cys Gly Asp Asn Cys Lys Cys Thr Thyr Cys Asn Cys Lys Thr Cys Arg Lys Ser Cys Cys Pro Cys Cys Pro Pro Gly Cys Ala Lys Cys Ala Arg Gly Cys Ile Cys Lys Gly Gly Ser Asp Lys Cys Ser Cys Cys Pro

In addition, the protein transduction domain (PTD) can be fused with the metallothionein and penetrate into the cell. Any known substance in the art can be used without limitation.

For example, Tat protein of HIV-1 virus, poly arginine (more than 6 arginine), Penetratin, transcription regulatory protein of HSV-1 structural protein VP22, Pep-1 peptide, or Pep- It is possible.

Preferably, Tat (Trans Activator of Transcription) represented by the amino acid sequence of SEQ ID NO: 5 or polyarginine represented by the amino acid sequence of SEQ ID NO: 6 can be used.

SEQ ID NO: 5: Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg

SEQ ID NO: 6: Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg

Methods for chemically or biologically fusing the components are well known in the art, and therefore, a detailed description thereof will be omitted herein.

Therefore, in a specific embodiment of the present invention, a sMTS peptide consisting of an amino acid of MVSAL; Metal rochionein (MT); (PTD-sMTS-MT) composition comprising a protein transduction domain (PTD) and a protein transduction domain (PTD).

The fusant composition may bind a poly-His region to the amino terminus of the protein transduction domain.

The poly-His region is one of the labeled peptides, and can be used to separate and purify the fusion protein of the present invention by binding with a histidine binding resin. Preferably, hexahistidine is used as the polyhistidine region.

At this time, cDNA encoding the protein transduction domain is bound to the 5 'end of the cDNA encoding the metallothionein protein, and the recombinant polynucleotide encoding the antioxidant fusion protein of the present invention is included.

Such polynucleotides can be prepared from the known nucleic acid sequences encoding metallothionein, a protein transduction domain, a mitochondrial targeting sequence, according to conventional methods.

The present invention also provides, as another specific embodiment, an expression vector expressing a fusion substance for high-efficiency cell introduction and maintenance (PTD-sMTS-MT).

The vector is not particularly limited in the present invention as long as it can be used as a cloning vector. For example, pRSET, pET3, pET11, pBAD, pThioHis, pTrcHis and the like are possible. A vector having a DNA encoding a poly-His region inserted therein may be used.

Such a vector can be prepared by inserting the gene encoding the fusion protein into a cloning vector truncated with an appropriate restriction enzyme by a conventional method known in the art.

The present invention can also provide a microorganism transformed with said vector.

Any of the microorganisms can be used as long as the fusion protein can be effectively expressed. Typically, E. coli, Pichia genus, and the like can be exemplified.

The fusant composition of the present invention can be prepared, for example, by the following method:

(a) a metallothionein represented by one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 4, a sMTS composed of the amino acid of MVSAL, and a protein represented by the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6 Transforming a microorganism with a recombinant expression vector comprising a recombinant polynucleotide encoding a fusion substance of the present invention to which a transduction domain (PTD) is bound;

(b) culturing the transformed microorganism to express an antioxidant fusion; And

(c) purifying the expressed antioxidant fusions.

In the above method, the transformation step may be carried out by carrying the expression vector into a host cell. When the host cell is a prokaryotic cell, the transforming method may be carried out by a CaCl ₂ method, a Hanahan method, or an electroporation method. When the host cell is a eukaryotic cell, the expression vector is injected into the host microorganism by microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, and gene spring buddement can do. The expression vector injected into the microorganism is expressed in the microorganism to obtain the desired fusion protein.

The expressed fusion protein can be purified in a denatured state or in a natural state through a general purification method known in the art. Purification methods include, for example, fractionation by ammonium sulfate, size-specific filtration (ultrafiltration) and metal chelating affinity chromatography.

Pharmaceutical composition

The present invention provides the use of fused compositions for the introduction and maintenance of highly efficient cells of proteins containing sMTS consisting of the amino acids of MVSAL.

As a preferred example, sMTS of MVSAL; Metal rochionein (MT); And a protein transduction domain (PTD) for the prevention or treatment of diabetes or diabetic cardiomyopathy. That is, the present invention provides a pharmaceutical composition for preventing or treating diabetes mellitus or diabetic cardiomyopathy, or a therapeutic method using the same.

The diabetes mellitus in the present invention includes systemic or local diseases that cause diabetes mellitus and diabetes (preferably non-insulin dependent type 2 diabetes) as a direct or indirect factor, specifically diabetic acidosis diabetic acidosis, diabetic xanthoma, diabetic amyotrophy, diabetic ketosis, diabetic coma, diabetic gastric disorder, diabetes mellitus, Diabetic gangrene, diabetic ulcer, diabetic complications, diabetic diarrhea, diabetic microangiopathy, diabetic uterine body sclerosis, diabetic neuropathy, diabetic neuropathy, Myocardial infarction, diabetic cardiomyopathy, diabetic neuropathy, diabetic nephropathy, bullosis diabeticorum, Diabetic retinopathy, diabetic cataract, diabetic dermopathy, diabetic scleredema, diabetic retinopathy, necrobiosis lipoidica diabeticorum, diabetic blood circulation disorder diabetic blood circulation disorder, and the like.

The metallocenone (MT) of the present invention; And protein transduction domain (PTD) are highly efficiently introduced into and maintained intracellularly by sMTS of MVSAL, exhibiting the antioxidative and anti-apoptotic effects of metallothionein, It protects beta cells and myocardial cells. In particular, this effect is achieved by reducing the concentration of intracellular reactive oxygen species (ROS).

In addition, the pharmaceutical composition of the present invention may further comprise at least one pharmaceutically acceptable carrier in addition to the above-described effective ingredient for administration.

The pharmaceutically acceptable carrier may be a mixture of saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components. If necessary, an antioxidant, , And other conventional additives such as a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Further, it can be suitably formulated according to each disease or ingredient, using appropriate methods in the art or by the method disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA.

The composition according to the present invention can be administered orally or parenterally at the time of clinical administration and can be used in the form of a general pharmaceutical preparation. In the case of pharmaceutical preparation, the composition can be used as fillers, extenders, binders, wetting agents, disintegrants, Of diluent or excipient may be used.

The solid preparation for oral administration can be prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose, lactose or gelatin into the fusion protein according to the present invention. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions or syrups. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances and preservatives can be used have.

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the non-aqueous solvent or suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, etc. Examples of the suppository include withexol, macrogol, tween 61, Cacao paper, laurin paper, glycerol paper, gelatin paper, and the like.

The composition of the present invention may be administered parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) or orally, depending on the intended method. The dose may be 0.01 to 1000 mg per kg of body weight It can be administered in divided doses. The dosage may be adjusted depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of disease.

[Example]

Example

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

material

The pRSET vector was purchased from Invitrogen (La Jolla, Calif.). All restriction enzymes and PCR cloning kits were obtained from Takara (Tokyo, Japan). IMAC (immobilized Ni-affinity chromatography column) was purchased from Bio-Rad (Hercules, Calif.) And NaAsO2 (sodium arsenite) and glucose were purchased from Sigma-Aldrich (St. Louis, Mo.). H2DCFDA (2 ', 7'-Dichlorodihydrofluorescein diacetate) was purchased from Invitrogen (Carlsbad, Calif.).

Statistical analysis

All data were expressed as mean ± standard deviation (n = 4). Statistical comparisons were performed using Student's t-test. P-values <0.05 and <0.01 were considered statistically significant.

Example  One: TM  And TMM  Production of Recombinant Fusion Protein

MT, TM and TMM fusion proteins were cloned, expressed and purified.

1-1 Cloning

Plasmid vectors expressing Tat-MT fusion proteins were constructed using pRSET vectors designed for high-level protein expression and purification in E. coli. The Tat-MT fusion construct was constructed with reference to the prior art [K. Lim, Y. Won, Y. Park, Y. Kim, Preparation and functional analysis of recombinant protein transduction domain-metallothionein fusion proteins, Biochimie, 92 (2010) 964-970]

Tat oligonucleotides and MT cDNA were inserted into a pRSET bacterial expression vector (Invitrogen, La Jolla, Calif.) To produce a Tat-MT fusion protein expression vector. Tat (TAG GGC AGG AAG AAG CGG AGA CAG CGA CGA CGA) was annealed and cloned into pRSET vector to generate pRSET-Tat vector. The MT coding sequence was amplified by PCR and cloned into pRSET-Tat vector, and then sMTS was serially annealed to obtain short malate dehydrogenase (ATG GTG TCC GCT CTC, Met-Val-Ser-Ala-Leu amino acid sequence ) Was prepared. The purified sMTS fragments were cloned into the Tat-MT expression vector at the Xho I site. The Tat-sMTS-MT structure was confirmed by DNA sequencing (Cosmogene Tech, Seoul, Korea).

In a similar manner, the Tat-GFP and Tat-sMTS-GFP DNA sequences were cloned into the pRSET-A vector and the retention time and transduction efficiency of the Tat and Tat-sMTS fusion proteins were determined. Nucleotide sequences of Tat-GFP (TG) and Tat-sMTS-GFP (TMG) were confirmed by DNA sequencing (Consmogenetech, Seoul, Korea). Fig. 1 shows a structural schematic diagram of the expression vector prepared.

1-2 Of the fusion protein  Expression and purification

MT, Tat-MT (TM), and Tat-sMTS-MT (TMM) were produced with reference to the prior art.

Briefly, all fusion protein expression vectors were transfected into Escherichia coli strain BL21 (DE) pLysS (Novagen, Madison, WI) and cultured in LB medium (Becton Dickinson and Company, MD) for 4 hours at 37 ° C.

(1 mM) and ZnSO ₄ (1 mM) were added to induce the expression and stabilization of each protein. Cell pellets were collected by centrifugation and suspended in lysis buffer (300 mM KCl, 49 mM KH2PO4, 4.9 mM Imidazole). The suspended pellets were sonicated and filtered through a 0.45-um filter. The proteins were then purified by IMAC (Immobilized Metal Affinity Chromatography) on FPLC (Bio-Rad, Hercules, Calif.) Using Ni-NTA resin column. Finally, the protein solution was dialyzed against pH 7.4 PBS (phosphate buffered saline) using a 3,500 MWCO (Molecular weight cut off) membrane (Spectrum Laboratories, CA).

Tat-GFP (TG) and Tat-sMTS-GFP (TMG) were prepared in a similar manner. The expressed Tat-GFP and Tat-sMTS-GFP fusion proteins were purified using IMAC and dialyzed against pH 7.4 PBS using 12,000 - 14,000 MWCO membranes. All fusion proteins were electrophoresed on a 12% SDS-PAGE gel and stained with coumarin brilliant blue (Bio-Rad, Hercules, CA) for MT detection for 60 min.

1-3 TM  And TMM  Preparation of recombinant fusion proteins and cytotoxicity

The results are shown in Fig. 2 and Fig.

All recombinant proteins were prepared in the presence of zinc, requiring stabilized folding and MT activity. And as a result, a single band was obtained on SDS-PAGE. The fusion protein bands corresponding to TM and TMM were visualized near predicted sizes of 12.6 kDa and 13.2 kDa, respectively (Fig. 2).

Treatment of H9c2 cells with MT, TM, and TMM at concentrations ranging from 1 to 10 [mu] M did not result in significant cytotoxicity to 10 [mu] M (Fig. 3). Subsequent experiments were performed at 3 μM.

Example  2: Transduction

2-1. Cell culture

Cardiac-derived mouse H9c2 cardiomyocytes (Korean Cell Line Bank, Seoul, Korea) were inoculated into DMEM containing 10% FBS (fetal bovine serum) supplemented with 100 U / ml penicillin and 100 mg / ml streptomycin (Dulbecco's modified Eagle's medium (5% CO2 atmosphere) and 37 &lt; 0 &gt; C

2-2. TG  And TMG Of transfection efficiency and cell lag time ( cell retention time )

H9c2 cells were seeded onto 6-well plates at a density of 2 x 10 &lt; 5 &gt; cells per well. Cells were treated with TG and TMG at 1 and 3 μM for 10 min to 60 min, then the cells were trypsinized and washed twice with pH 7.4 PBS. The cells were fixed with FBS in fixed buffer containing FACS buffer

Transfection efficiency was measured using FACSCalibur (BD Biosciences, San Diego, Calif.) And data analysis was performed with CellQuest software (BD PharMingen, San Jose, CA). To measure the enhanced delay time of GFP by sMTS in the cells, TG and TMG were incubated with H9c2 for 1 h, replaced with fresh medium and the cells were fixed after 2 hours. The delay time of TG and TMG was measured using FACS.

2-3. TG  And TMG of Intracellular  Congestion image ( retention image )

H9c2 cells were seeded onto a 60-mm dish at a density of 1 x 104 cells / dish. Cells were cultured for one day and fusion protein transduction was performed.

The cells were treated with TG and TMG at 3 μM for 1 h, and then the medium was fresh. The cells were washed three times with PBS after 2 hours and fixed with 4% formaldehyde solution for 49 minutes. After fixation, the cells were washed twice with PBS and mounted on a slide glass with DAPI-fluoromount G (Southern Biotech, Birmingham, AL) for nuclear staining. TG and TMG were measured by using a multi-photon confocal laser scanning microscope (LSM780 META NLO; Carl Zeiss Jena, Germany; Korea Basic Science Institute, Chuncheon Center, Chuncheon-city, Korea) Respectively. To detect TG and TMG in H9c2 cells without immobilization, H9c2 cells were treated with 3 μM TG and TMG for 1 h and the media was washed. After replacing the medium, the cells were washed twice with PBS and stained with pentahydrate (bis-Benzimise) (Molecular Probes, Carlsbad, Calif.) For 15 min.

Intracellular TG and TMG were detected using a multi-proton confocal laser scanning microscope (LSM510 META NLO; Carl Zeiss Jena, Germnay, Korea Basic Science Institute, Chuncheon Center, Chuncheon, Korea).

2-4. From transduction efficiency Taste  And Taste - sMTS  effect

To compare the activity of Tat and Tat-sMTS in transfection efficiency, MT was replaced by GFP in the same plasmid vector and the transfection efficiency was measured by FACS.

As a result, as shown in Table 1, GFP alone showed negative transduction results without Tat or Tat-sMTS, while Tat-GFP (TG) and Tat-sMTS-GFP (TMG) Efficiency increased significantly in dose - and culture time - dependent manner. The transfection efficiency of TG ranged from 8.78 ± 12.28% to 73.4 ± 9.54% at 1 μM over 10 min to 1 h incubation time and reached 92.90 ± 7.18% at 1 μM at 3 μM

The addition of sMTS increased the transfection efficiency and shortened the transfection time to 91.92 ± 8.17% after 30 min incubation at 1 μM and reached 92.91 ± 4.51% within 3 min (Table 1) .

Thus, sMTS peptides derived from the mitochondrial malate dehydrogenase (mMDH) signal sequence enhanced protein transduction and mitochondrial targeting capabilities. Since the addition of sMTS to the fusion protein significantly improves GFP transfection ability and shortens the transfection time, the present inventors have anticipated and confirmed that the sMTS peptide can increase the stagnation time of the fusion protein in the cell.

H9c2 cells were treated with GFP, TG, and TMG for 1 h, and the medium was replaced with fresh medium. After 2 h, the cells were fixed with fixed buffer and the transfection efficiency was compared with the cells not subjected to washing step 3 The results are shown in Fig. 4 and Fig.

1 and 3 [mu] M, GFP showed no ability to transduce cells. There was no apparent decrease in TMG at TMG at 3 [mu] M, while the transduction capacity of TG was significantly reduced from 92.9 + 7.18% to 40.08 + 3.94%. However, when the cells were treated with TG and TMG for 3 h without the washing step, the transduction efficiencies of TG and TMG were both 94.89 ± 5.87% and 98.08 ± 0.63%, respectively (FIG. 4).

And, as shown in FIG. 5 showing CLSM, the fluorescence intensity of TMG was maintained in the cells after the culture medium replacement, and the case of TG was remarkably decreased. Suggesting that sMTS-mediated cell stagnation was enhanced.

Live cell images without immobilization showed the same results, showing that the difference between TG and TMG was not due to differences in the immobilization process. The ability of mitochondrial localization by sMTS was confirmed by CLSM, but sMTS was also not sufficient to localize to mitochondria (data not shown)

These results show that the addition of sMTS peptides to Tat-protein increases transduction efficiency by shortening the transduction time and increasing the intracellular stasis time.

Example  3: MT  Cell trafficking of fusion proteins Cellular trafficking )

3-1. Taste - MT  And Taste - sMTS - MT Cell Trafficking

H9c2 cells were seeded in two groups on a glass cover in 6-well plates and 1 + 2 h and 3 h incubation protocols were performed. At 3 μM MT, Tat-MT (TM) and Tat-sMTS-MT (TMM) were treated with H9c2 cells for 1 + 2 h and 3 h, respectively. Samples were then prepared for immunohistochemical analysis and the following protocol was performed.

Cells were washed with PBS and stained for 20 min with 300 nM MitoTracker Orange (Molecular Probes Eugene, OR) in complete medium containing 10% FBS. All cells were fixed with 4% paraformaldehyde for 10 min in ice, permeabilized with 0.1% Triton X-100, covered with 5% BSA at room temperature for 1 h in TBS (Tris-buffered saline) The cells were treated with 6x-his antibody (Cell Signaling Technology, Beverly, Mass., USA; 1: 500).

Cells were washed and probed with a suitable secondary antibody conjugated to Alexa Fluor 488 (1: 1000, Molecular Probes, Eugene, Oreg.) At room temperature for 1 hour. Nuclei were stained with Hoechst (2 μg / ml, Molecular Probes, Eugene, OR) at room temperature for 5 min in PBS. The slides were washed twice with PBS and mounted using a DAKO fluorescent mounting medium (DAKO corporation, Carpinteria, Calif.). Specimens were measured at 405 nm, 488 nm and 555 nm for Hoechst, 6x-His, and Mitotracker, respectively, using a confocal laser scanning microscope (Carl Zeiss, Germany).

3-2. result

Although we show that sMTS improves cellular uptake and stasis time of GFP, we anticipate that bleaching of GFP will affect this outcome and Tat-sMTS-MT (TMM ) And Tat-MT (TM) intracellular trafficking with Alexa Fluor 488-conjugated antibody using CLSM.

As shown in FIG. 6, after 1 + 2 h washing step, the TMM was retained in the cells and showed strong fluorescence intensity in the cytosol after the culture medium replacement. However, Tat-MT (TM) showed a significantly reduced strength, indicating less efficient stiction activity compared to TMM.

Although the primary antibody to histidine recognized both histidine in the nucleus and its tagged MT fusion protein, MT and PBS treated groups showed fluorescence intensities in the nucleus. It is also interesting that the sMTS sequence is derived from the mitochondrial targeting sequence but is not capable of localizing the fusion protein into the mitochondria. These results suggest that the fusion of sMTS peptides accelerates the intracellular stagnation time of the fusion protein.

Example  4: Hyperglycemia - and Hypoxia -Induced cell death Protective  effect

4-1. Induce hyperglycemia and hypoxia

To examine the ability of fusion proteins to reduce hyperglycemia-induced apoptosis in H9c2 cells, cells were exposed to hyperglycemic conditions by incubation in 350 mM glucose (Sigma, St. Louis, MO) solution for 24 h. Hyperglycemic-exposed cells were also incubated for 24 h under hypoxic conditions (1% O 2 and 5% CO 2). Cell viability in H9c2 cells exposed to these conditions was measured by Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan).

4-2. H9c2  In a cell Hyperglycemia - and Hypoxia -Induced cell death MT  Of the recombinant fusion protein Protective  effect

It is known that hyperglycemia and hypoxic conditions are closely related to causing type 1 and 2 diabetes by increasing the production of ROS and oxidative stress. Therefore, in order to examine the protective effect of MT protein on hyperglycemia, cells were exposed to hyperglycemia by incubation in a 350 mM glucose solution for 24 hours.

The results are shown in Fig.

H9c2 cell viability was reduced to 43.9 +/- 1.9% under hyperglycemic conditions (Figure 7a). When cells were exposed to hyperglycemia with protein, the cell viability increased to 56.4 ± 2.5% for TM and to 65.0 ± 2.9% for TMM. TM and TMM fusion proteins also showed protective effects against hypoxic stress (Fig. 7B). And, as shown in FIG. 7C, they showed cell viability from 48.7 ± 10.4% to 63.3 ± 6.9% for TM and 73.1 ± 3.2% for TMM under hypoxic and hyperglycemic conditions. For hyperglycemic- and hypoxia-induced apoptosis, MT was less efficient in increasing cell viability due to its low transduction efficiency

Example  5: MT  Antioxidant effect of recombinant fusion protein

5-1. H2DCFDA  Used ROS ( Reactive oxygen species ) Fluorescence analysis

To confirm the antioxidant effect of MT fusion protein, NaAsO2 (sodium arsenite) was used to increase intracellular ROS.

H9c2 cells were exposed to 10 μM NaAsO2 for 24 h. After removing the medium from the cells, H9c2 cells were washed twice with PBS. Cell-permeable H2DCFDA probes (10 [mu] M) were added to each well of the plate and incubated at 37 [deg.] C for 30 minutes. Fluorescence intensity was measured at 499 nm excitation and 522 nm emission wavelength with a UV / Vis fluorescence spectrophotometer (SpectraMax M2e; Molecular Devices, Sunnyvale, Calif.). ROS levels in H9c2 cells exposed to hyperglycemia and hyperglycemia / hypoxia were also measured by the same method.

5-2. H9c2  In a cell MT  Antioxidant effect of recombinant fusion protein

The antioxidative effect of MT fusion protein was observed by measuring the level of reactive oxygen species (ROS) in H9c2 cells exposed to chemical ROS inductors (NaAsO2), hyperglycemia, hypoxia, hyperglycemia + hypoxia.

The results are shown in Fig.

ROS levels in cells exposed to oxidative stress-induced conditions without protein increased to 9.3 +/- 7.3% (Figure 8-a). The ROS intensity decreased to 5.1 ± 4.8% for TM and to 0.6 ± 1.5% for TMM. After H9c2 cells were exposed to hypoxic conditions for 24 hours, the ROS intensity was increased by 10 +/- 1% compared to the control (Figure 8-b). TMM-treated cells showed a significant decrease in ROS levels after 24 hours. TM and TMM also decreased ROS intensity from 10.3 ± 1.5% of the untreated group to 7.9 ± 1.4% and 4.5 ± 1.9%, respectively, under hyperglycemic and hypoxic conditions (Figure 8-c). The combination of hyperglycemia and hypoxia did not show a synergistic effect on ROS increase. The difference was not great, but TMM apparently reduced ROS levels in the cells.

NaAsO ₂ is known to be a chemical ROS inducer and is widely used in oxidative stress-related studies. After coadministering H9c2 cells with 10 μM NaAsO ₂ for 24 h, co-administered TMM reduced the ROS intensity to 9.7 ± 2.3%. On the other hand, MT did not induce a statistically significant decrease in ROS levels (Fig. 8-d).

Example  6: In diabetic mouse models Protective  effect

6-1. Streptozotocin ( STZ ) -Induced diabetic mouse model

STZ was dissolved in 50 mM citrate buffer (pH 4.5) and injected intraperitoneally (IP). STZ was injected into 18 - 21 g of 5 - week - old balb / c mice at a dose of 80 mg / kg to induce diabetes mellitus. Diabetes was induced by STZ within 3 days by beta cell destruction. TM and TMM 3 μM (48 μl) were administered to four mice in each group before and after injection of STZ daily for 5 days. Blood glucose was monitored using a retro-orbital blood collection and measured using an automated glucose analyzer (Roche, Basel, Switzerland). Blood glucose levels in diabetic mice were over 300 mg / dl.

6-2. From the diabetic pancreas Ex vivo ROS  Measure

For detection of ROS levels in the pancreas, TMM was injected into STZ-induced diabetic mice and H2DCFDA was also injected into the STZ-induced diabetic mouse model abdominal cavity.

After one hour, the pancreas was extracted from the mice. ROS intensity was measured immediately using a Kodak Image Station (Kodak Image Station 4000MM, Eastman Kodak Company, Scientific Imaging Systems, New Haven, Conn.).

6-3. Streptozotocin ( STZ ) -Induced diabetic mouse model MT  Of the recombinant fusion protein Protective  effect

An STZ-induced diabetic animal model was prepared by injecting STZ in the abdominal cavity at a dose of 80 mg / kg as described above to induce diabetes. Three days after the injection, the blood glucose level reached 300 mg / dl or more. To determine the effective timing of MT fusion protein to lower blood glucose levels, TMM was injected intraperitoneally at 3 [mu] M before and after STZ administration

As a result, as shown in Fig. 9-a, the protein treatment before STZ administration was effective in maintaining normal glucose levels.

Blood glucose levels (BGL) of MT fusion protein-treated mice were also monitored by prolonging the time until BGL exceeded 300 mg / dl by daily protein administration.

As shown in Figure 9-b, the MT- and TM-treated groups reached 300 mg / dl after 5 days and 7 days, respectively, but the TMM-treated group grew at or below 200 mg / BGL was maintained

And the positive effects of TMM were also observed in the pancreas extracted from STZ-induced diabetic mice.

The level of ROS was higher in the pancreas of untreated mice after STZ administration, but the Tat-sMTS-MT treated group showed a decreased ROS intensity (Fig. 10-a). The ROI value of ROS in the pancreas was increased to 145 ± 10% (n = 3) as compared with the control group (FIG. 10-b). TMM treatment significantly reduced ROS intensity up to 94 ± 5%.

These results indicate that the introduction of sMTS of the present invention enhances the intracellular influx of the fusion protein and enhances the intracellular persistence effect. Therefore, this function of sMTS suggests that it can be applied to drug delivery systems such as fusion proteins, gene carriers, and nanoparticles, thereby enhancing therapeutic efficacy.

<110> HANYANG UNIVERSITY INDUSTRY COOPERATION FOUNDATION <120> A Peptide for Efficiently Introducing Proteins into Cells and          Remaining them <130> 2013-P-091 <160> 6 <170> Kopatentin 2.0 <210> 1 <211> 61 <212> PRT <213> Artificial Sequence <220> <223> metallothionein <400> 1 Met Asp Pro Asn Cys Ser Cys Ser Thr Gly Gly Ser Cys Thr Cys Thr   1 5 10 15 Ser Ser Cys Ala Cys Lys Asn Cys Lys Cys Thr Ser Cys Lys Lys Ser              20 25 30 Cys Cys Ser Cys Cys Pro Val Gly Cys Ser Lys Cys Ala Gln Gly Cys          35 40 45 Val Cys Lys Gly Ala Ala Asp Lys Cys Thr Cys Cys Ala      50 55 60 <210> 2 <211> 61 <212> PRT <213> Artificial Sequence <220> <223> metallothionein <400> 2 Met Asp Pro Asn Cys Ser Cys Ala Ala Gly Asp Ser Cys Thr Cys Ala   1 5 10 15 Gly Ser Cys Lys Cys Lys Glu Cys Lys Cys Thr Ser Cys Lys Lys Ser              20 25 30 Cys Cys Ser Cys Cys Pro Val Gly Cys Ala Lys Cys Ala Gln Gly Cys          35 40 45 Ile Cys Lys Gly Ala Ser Asp Lys Cys Ser Cys Cys Ala      50 55 60 <210> 3 <211> 68 <212> PRT <213> Artificial Sequence <220> <223> metallothionein <400> 3 Met Asp Pro Glu Thr Cys Pro Cys Pro Ser Gly Gly Ser Cys Thr Cys   1 5 10 15 Ala Asp Ser Cys Lys Cys Glu Gly Cys Lys Cys Thr Ser Cys Lys Lys              20 25 30 Ser Cys Cys Ser Cys Cys Pro Ala Glu Cys Glu Lys Cys Ala Lys Asp          35 40 45 Cys Val Cys Lys Gly Gly Glu Ala Glu Ala Glu Ala Glu Lys Cys      50 55 60 Ser Cys Cys Gln  65 <210> 4 <211> 62 <212> PRT <213> Artificial Sequence <220> <223> metallothionein <400> 4 Met Asp Pro Arg Glu Cys Val Cys Met Ser Gly Gly Ile Cys Met Cys   1 5 10 15 Gly Asp Asn Cys Lys Cys Thr Thr Cys Asn Cys Lys Thr Cys Arg Lys              20 25 30 Ser Cys Cys Pro Cys Cys Pro Pro Gly Cys Ala Lys Cys Ala Arg Gly          35 40 45 Cys Ile Cys Lys Gly Gly Ser Asp Lys Cys Ser Cys Cys Pro      50 55 60 <210> 5 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Trans Activator of Transcription <400> 5 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg   1 5 10 <210> 6 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> poly arginine <400> 6 Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg   1 5 10

Claims (16)

Efficient fusion of an antioxidant fusion protein consisting of a metallothionein and a Tat protein transduction domain containing a short mitochondrial target peptide (sMTS) composed of amino acids arranged in the order of MVSAL, Composition.
delete The composition according to claim 1, wherein the metallothionein is represented by one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 4.
delete The method according to claim 1, wherein the sMTS composed of the amino acids arranged in the order of MVSAL
From the group consisting of the human cytochrome c subunit oxidase VIII, the PI subtype of the human ATP synthase subunit c, the aldehyde dehydrogenase targeting sequence, the human ATP synthase subunit FI beta and the BCS1 protein Wherein the composition is derived from one selected protein.
delete delete delete The composition of claim 1, wherein the Tat protein transduction domain is represented by the amino acid sequence of SEQ ID NO: 5.
delete The composition of claim 1, wherein the cell is selected from the group consisting of myocardial cells, pancreatic cells, nerve cells, vascular endothelial cells and skin cells.
2. The composition of claim 1, wherein the composition reduces the concentration of active oxygen species in the cell.
A vector expressing the fusant composition of claim 1.
13. A microorganism transformed with the vector of claim 13.
A pharmaceutical composition for the prevention and treatment of diabetes comprising the fused composition of claim 1, the vector of claim 13 or the microorganism of claim 14 as an active ingredient.
A diabetic acidosis, a diabetic xanthoma, a diabetic amyotrophy, diabetes mellitus, diabetes mellitus, diabetes mellitus, diabetes mellitus, diabetes mellitus, Diabetic complications such as diabetic ketosis, diabetic coma, diabetic gastric disorder, diabetic gangrene, diabetic ulcer, diabetic complications, diabetic ulcer diabetic microangiopathy, diabetic uterine body sclerosis, diabetic cardiomyopathy, diabetic neuropathy, diabetic nephropathy, diabetic neuropathy, diabetic neuropathy, diabetic neuropathy, diabetic neuropathy, diabetic neuropathy, diabetic neuropathy, diabetic neuropathy, diabetic neuropathy, Diabetic cataract, diabetic dermopathy, diabetic scleredema, diabetic keratitis, diabetic keratitis, diabetic keratitis, A pharmaceutical composition for preventing and treating at least one diabetic complication selected from the group consisting of diabetic retinopathy, necrobiosis lipoidica diabeticorum, and diabetic blood circulation disorder.

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