KR100877152B1 - Cell permeable p66shc fusion protein having anti-cancer activity - Google Patents

Abstract

A cell penetrating fusion protein Tat-p66shc, a gene coding the fusion protein, an expression vector containing the gene, and the transformed E. coli are provided to obtain the fusion protein having the anticancer activity. A cell penetrating fusion protein Tat-p66shc is the fusion protein that the human p66shc protein is fused with the protein transduction domain of the Tat protein, and is represented by the sequence number 1. Preferably the gene coding the fusion protein Tat-p66shc is represented by the sequence number 2. The E. coli transformed by the expression vector containing the gent is deposited (deposition number KCTC 11138BP).

Description

Cell permeable p66shc fusion protein having anti-cancer activity

The present invention relates to a fusion protein having both a function of inhibiting the generation of cancer cells and a cell permeation function by inducing the generation of free radicals and cell death, and more particularly, a human p66shc protein and a Tat (transactivator of transcription) protein. It relates to a Tat-p66shc fusion protein fused to the transduction site of.

The p66shc protein is a member of the ShcA family of adapter proteins and is known to play an important role in controlling mammal life. Animals from which the p66shc protein has been removed are known to have a lifespan of about 30% longer and resist oxidative stimuli (Migliaccio E et al, The p66shc adapter protein controls oxidative stress response and life span in mammals. Nature. 1999 Nov 18; 402 (6759): 309-13.)

p66shc protein induces increased mitochondrial free radical production (Trinei et al, A p53-p66Shc signaling pathway controls intracellular redox status, levels of oxidation-damaged DNA and oxidative stress-induced apoptosis.Oncogene. 2002 May 30; 21 (24) This increased oxygen production is closely related to the phosphorylation of serine, the 36th amino acid of the p66shc protein (Nemoto S & Finkel T. Redox regulation of forkhead proteins through a p66shc-dependent signaling pathway). Science 2002 2002 29; 295 (5564): 2450-2).

Recently, the decrease in the expression of p66shc protein increases the activation of endothelial cell type nitric oxide protein and increases the production of nitric oxide (Yamamori et al, P66shc regulates endothelial NO production and endothelium-dependent vasorelaxation: implications for age-associated vascular dysfunction.J Mol Cell Cardiol. 2005 Dec; 39 (6): 992-5). Overexpression of p66shc protein has been reported to induce cell death in T-cells and inhibit carcinogenic signaling. (Pacini S et al, p66shc promotes apoptosis and antagonizes mitogenic signaling in T cells.Molecular and Cellular Biology, 24 (4), 1747-1757, 2004)

Overexpression of genetic factors using adenoviruses and fats is used as a method of overexpressing proteins in signal transduction research, but there is a disadvantage in that expression time is required for a long time. Recently, as protein transduction sites for introducing proteins into cells are known, biological changes have been studied by fusing proteins into these introduction sites and introducing them into cells.

Although the exact cellular transduction mechanism is not known yet, Tat (transactivator of transcription) protein is known to efficiently move through the cell membrane and into the cell due to the presence of the protein transduciton domain (Murriel). CL, Dowdy SF.Influence of protein transduction domains on intracellular delivery of macromolecules.Expert Opin Drug Deliv. 2006 Nov; 3 (6): 739-46). In the case of fusion of protein using the protein transduction site of Tat protein, it is reported that the protein is easily introduced into the cell and shows the function and activity of the protein, and the effects have been reported in recent animal experiments (Cai SR, Xu G). , Becker-Hapak M, Ma M, Dowdy SF, McLeod HL.The kinetics and tissue distribution of protein transduction in mice.Eur J Pharm Sci. 2006 Mar; 27 (4): 311-9).

The p66shc protein is known to inhibit the development of cancer cells by inducing the production of free radicals and the death of cells. Therefore, it is thought that the cell-permeable p66shc protein can be utilized to suppress tumorigenesis. In addition, by using the characteristics of the p66shc protein having a function of inhibiting the growth of cells can be usefully applied to the medical industry as an anticancer drug development and utilization material.

It is an object of the present invention to provide a fusion protein in which tat, a protein transduction site, and human p66shc protein are fused to introduce p66shc protein into cells.

Another object of the present invention to provide a composition for inhibiting cancer cell growth containing the Tat-p66shc fusion protein as an active ingredient.

The fusion protein of the present invention for achieving the above object is characterized in that the cell permeable fusion protein Tat-p66shc of SEQ ID NO: 1, wherein the protein transduction site of the p66shc protein and Tat protein is fused.

In order to make tat-p66shc fusion protein, human p66shc cDNA was isolated by constructing primer that connects EcoRI restriction enzyme site to start codon of p66shc and HindIII restriction enzyme site to stop codon. PTat-p66shc expression vector (FIG. 1) was prepared by inserting the isolated p66shc cNDA (1752bp) into pTAT2.1, transformed into E. coli BL21 (DE3), and the transformant BL21 (DE3) / pTat-p66shc 2007 It was deposited on June 19, 19th, at the Korea Research Institute of Bioscience and Biotechnology (BRC) (Accession No. KCTC 11138BP). Using the transformant, the Tat-p66shc fusion protein was overexpressed, isolated and denatured in a denatured condition. The Tat-p66shc fusion protein is characterized by having the amino acid sequence of SEQ ID NO: 1.

The gene encoding the Tat-p66shc fusion protein includes six histidines for protein purification, a gene encoding 9 amino acids RKKRRQRRR of the TAT transduction site, and a human p66shc gene. do. It is particularly preferred that the gene has a sequence of SEQ ID NO: 2.

In the Tat-p66shc fusion protein according to the present invention, it was observed that intracellular permeation occurred at a concentration of 1 nM. The intracellular permeation showed the maximum expression in the group treated with Tat-p66shc fusion protein in 1 hour culture in vascular endothelial cells and then gradually decreased. These results suggest that Tat-p66shc introduced into cells is slowly removed by intracellular proteolytic enzymes.

Tat-p66shc fusion protein permeated into vascular endothelial cells was found to effectively inhibit phosphorylation of eNOS mediated by TNF-α. This phenomenon seems to be due to the increased phosphorylation of p66shc (especially the phosphorylation of the 36 th serine group) as an important mediator. This effect of Tat-p66shc may be useful in the development of treatment technology for vascular diseases such as angiogenesis and angiogenesis caused by vascular signaling materials. In addition, the expression of p66shc can suppress tumorigenesis and can be used for anticancer drug development.

Pharmaceutical compositions containing the Tat-p66shc fusion protein as an active ingredient can be formulated in the form of skin smears, oral administrations, intravenous injections and intramuscular injections, in combination with carriers commonly accepted in the pharmaceutical art. The pharmaceutical composition using Tat-p66shc fusion protein as an active ingredient can be applied to the treatment of vascular diseases and the treatment of tumor diseases, and the technique can be used for the treatment of intracellular genetic diseases and new diseases.

In addition, the Tat-p66shc fusion protein according to the present invention can be used not only as a pharmaceutical composition, but also as a cosmetic composition containing the fusion protein as an active ingredient. The cosmetic composition is not particularly limited in its formulation, and may specifically have a formulation such as supple cosmetics, nourishing cosmetics, nourishing cream or massage cream. In addition, the components of the specific composition may be appropriately selected and blended by those skilled in the art according to the dosage form and the desired effect.

As described above, since the Tat-p66shc fusion protein has the properties of Tat and human p66shc at the same time, it is possible to obtain the effect of directly introducing human p66shc into the cell at the protein level, which can be used for protein treatment.

Tat-p66shc fusion protein according to the present invention can effectively inhibit the production of free radicals and the activity of eNOS, it can be effectively utilized and applied in the development of anti-cancer drugs and the treatment of vascular diseases related to free radicals and eNOS .

In addition, Tat-p66shc fusion protein of the present invention can be used in the development of products such as cosmetics and health food in addition to the treatment of diseases.

The present invention will be described in detail through the following examples. However, these examples are for illustrative purposes only and the present invention is not limited thereto.

Example

Example 1 Preparation and Transformation of Tat-p66shc Expression Vector

To overexpress the Tat-p66shc fusion protein, a Tat-p66shc expression vector was prepared using the pTat2.1 vector containing six histidine genes, the Tat transduction site (RKKRRQRRR), and the human p66shc cDNA. . 1 shows a cleavage map of the Tat-p66shc expression vector.

First, primers of SEQ ID NO: 3 linking the ecoRI restriction enzyme site to the start codon of p66shc and primers of SEQ ID NO: 4 linking the HindIII restriction enzyme site to the stop codon include human p66shc genes. Separation of p66shc-pcDNA (Provided by Professor Kaikobad Irani, Pittsburgh University) using polymerase chain reaction (polymerase chain reaction conditions; 95 ° C, 30 seconds, 62 ° C 1 minute, 72 ° C 2 minutes, 30 cycles) It was. The isolated p66shc cDNA was 1752bp and the sequence was set forth in SEQ ID NO: 5.

SEQ ID NO: 5'-CGG AAT TCG AAG GAT CTC CTG CCC CCC-3 '(sense primer)

SEQ ID NO: 5'-CGA AGC TTT CAC AGT TTC CGC TCC ACA GG-3 '(anti-sense primer)

The pTAT-2.1 vector (5343 bp, donated by Professor Steven Dowdy, University of California, USA) was digested using EcoRI, HindIII restriction enzymes, and p66shc cDNA was inserted into the pTat2.1 vector to complete the Tat-p66shc vector. pTat-p66shc is characterized by including six histidine genes for protein purification, 9 amino acids RKKRRQRRR in the TAT transduction region, and human p66shc genes.

The vector was transformed into Escherichia coli BL21 (DE3) to prepare Escherichia coli transformant BL21 (DE3) / pTat-p66shc, and deposited on June 19, 2007 at the Korea Institute of Bioscience and Biotechnology Center (Accession No. KCTC 11138BP). ).

Example 2 Isolation and Purification of Recombinant Tat-p66shc Fusion Proteins

E. coli transformant BL21 (DE3) / pTat-p66shc prepared in Example 1 was purified by 50mg / ml of kanamycin. Plates were added to the added LB medium and incubated for 18 hours with stirring at 200-250 rpm at 37 ° C. Transformed colonies were selected and added to 1 L of medium and incubated at 37-200 rpm at 37-200 rpm with an OD value of 0.4-0.6 while stirring at 200-250 rpm at 37 ° C. When the OD value was 0.4-0.6, 1 mM of IPTG (isopropylthio-β-D-galactoside) was administered and further cultured at 37 ° C. for 4 hours to induce the expression of Tat-p66shc.

When the expression of the Tat-p66shc protein is induced, centrifugation at 5,000 rpm at 4 ° C. for 5 minutes, followed by washing with phosphate buffered saline and centrifugation again yields a precipitate fraction. 10 ml of buffer Z (8M Urea, 100 mM NaCl, 20 mM HEPES pH8.0) was added to the precipitate fraction, followed by sonication. The mixture was centrifuged again and the supernatant was taken and loaded into a protein column (Bio-rad, Cat No: 731-1550) to which 2-3 ml of Ni-NTA (Qiagen, Cat No: 30210) was added. After washing with buffer Z containing 10 mM imidazole, the fusion protein was eluted with each elution buffer (buffer Z containing 100 mM, 250 mM, 500 mM or 1M of Imidazole). Figure 2 is an electrophoresis picture showing the results of purification of the expressed Tat-p66shc fusion protein using each elution buffer, the most Tat-p66shc fusion protein eluted in the buffer Z containing imidazole 250mM imidazole It was confirmed that the buffer solution Z having a concentration of 250 mM was the most suitable elution buffer for elution of the fusion protein. It was also found that the molecular weight of the Tat-p66shc fusion protein was about 71 kDa.

The eluted fusion protein was desalted using a PD10 column kit (GE Healthcare Bio Sciences AB, Cat No: 17-0851-01). The purified fusion protein without salt was stored at -70 ° C by adding 10% glycerol, and the concentration of protein was compared with bovine serum albumin by coomassie blue staining using SDS-PAGE. After dilution was used according to the concentration used in the following examples.

Example 3 Intracellular Permeation of Tat-p66shc Fusion Proteins in Vascular Endothelial Cells

Western blot analysis was performed in the following examples of the present invention as follows. Cell homogenates were separated by 10% SDS polyacrylamide gel electrophoresis and electrophoresed to the gel with a microcellulose membrane. The protein-transferred membrane was treated with 5% skim milk and then treated with human Shc antibody (1: 1000 dilution), phospho-eNOS (1: 1000 dilution), or phospho-p66shc (1: 1000 dilution). After the reaction with horseradish peroxidase-conjugated secondary antibody (1: 2000 dilution) for 1 hour, the expression level of the protein was confirmed using the enhanced chemiluminescence-scence kit.

1) Tat-p66shc fusion protein permeation of vascular endothelial cells

The degree of Tat-p66shc protein influx into vascular endothelial cells was investigated. As a vascular endothelial cell, the rat endothelial cell line MS (MILE SVEN) -1 dispensed from American Type Culture Collection (Rockvile, MD) was used. The mouse pancreatic endothelial cell line, MS-1 cell line, was maintained and cultured in a 37 ° C. water supply incubator with 5% carbon dioxide and 95% air. Dulbecco's Modified Eagle's Medium (Gibco, Grand Island, NY) supplemented with 10% Fetal Bovine Serum and Penicillin 100 U / ml was used as a culture.

The cultured cells were used diluted in the culture solution at a concentration of 10 ⁵ cells / ml.

The diluted culture cells were dispensed into 6 wells of 2 ml, and Tat-p66shc was added at 0, 1, 5, 10 and 20 nM concentrations, respectively, and incubated in a 37 ° C. water incubator for 2 hours. After washing three times with phosphorylated buffered saline and pulverized with a cell crusher and centrifuged for 15 minutes at 12000rpm to obtain a supernatant. Protein amount was quantified using a Bio-rad assay. 30 μg of the quantified protein was sampled, heated at 100 ° C. for 5 minutes, and quenched for 10 minutes. The amount of Tat-p66shc fusion protein permeated into cells was measured by Western blot using ShcA monoclonal antibody, and the results are shown in FIG. 3. As can be seen in FIG. 3, the Tat-p66shc fusion protein was found to be intracellularly permeated from 1 nM concentration, and as the concentration increased, it was introduced into the cell in a concentration-dependent manner.

2) Duration after vascular endothelial cell penetration of Tat-p66shc fusion protein

In order to know how long the Tat-p66shc introduced from vascular endothelial cells existed in the cells, the duration of the cells was investigated by treating 20 nM Tat-p66shc fusion protein to the culture medium by time. The persistence of Tat-p66shc introduced into the cells was measured by Western blotting, and the results are shown in FIG. 4. However, the sampling process is the same as in Example 1).

As can be seen in Figure 4, in the vascular endothelial cells treated with Tat-p66shc for 1 hour culture showed the maximum expression and then showed a tendency to decrease gradually. These results suggest that Tat-p66shc introduced into cells is slowly removed by intracellular proteolytic enzymes.

Through this example, it was confirmed that the Tat-p66shc fusion protein according to the present invention was successfully introduced into vascular endothelial cells.

Example 4 Determination of eNOS Protein Phosphorylation Inhibitory Effect of Tat-p66shc Fusion Protein

In order to investigate whether the Tat-p66shc protein introduced into cells has normal physiological function, the effect on the phosphorylation of eNOS protein induced by TNF-α in vascular endothelial cells was measured.

Example 1 1) In the endothelial cell culture dilution, 20 nM Tat-p66shc for 2 hours and in the untreated control group, 15 ng / ml of TNF-α for 5 to 60 minutes after phosphorylation of eNOS And the degree of phosphorylation of p66shc was measured by Western blot analysis, and the results are shown in FIG. 5. However, the sampling process is the same as in Example 1).

From FIG. 5, it was confirmed that Tat-p66shc fusion protein was stably overexpressed in cells when 20 nM of Tat-p66shc fusion protein was treated in the cell culture medium for 2 hours. The introduced Tat-p66shc fusion protein effectively inhibited eNOS phosphorylation increased by TNF-α in cells.

1 is a cleavage map of a Tat-p66shc expression vector.

Figure 2 is an electrophoresis picture showing the results of the purification of Tat-p66shc fusion protein by elution buffer containing various concentrations of imidazole.

Figure 3 is a photograph showing the results confirmed by Western blotting the degree of vascular endothelial cell permeation by concentration of Tat-p66shc fusion protein.

Figure 4 is a photograph showing the results confirmed by Western blot after the intravascular endothelial cell permeation of Tat-p66shc fusion protein.

Figure 5 is a photograph showing the results of confirming the degree of phosphorylation of eNOS in vascular endothelial cells after treatment with vascular endothelial cells Tat-p66shc by concentration using Western blot.

<110> The Industry & Academic Cooperation in Chungnam National University (IAC) <120> Cell permeable p66shc fusion protein having anti-cancer activity <160> 5 <170> KopatentIn 1.71 <210> 1 <211> 619 <212> PRT <213> Artificial Sequence <220> <223> Tat-p66shc <400> 1 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro   1 5 10 15 Arg Gly Ser His Met Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Ser              20 25 30 Asp Pro Asn Ser Met Asp Leu Leu Pro Pro Lys Pro Lys Tyr Asn Pro          35 40 45 Leu Arg Asn Glu Ser Leu Ser Ser Met Glu Glu Gly Ala Ser Gly Ser      50 55 60 Thr Pro Pro Glu Glu Leu Pro Ser Pro Pro Ala Ser Ser Leu Gly Pro  65 70 75 80 Ile Leu Pro Pro Leu Pro Gly Asp Asp Ser Pro Thr Thr Leu Cys Ser                  85 90 95 Phe Phe Pro Arg Met Ser Asn Leu Arg Leu Ala Asn Pro Ala Gly Gly             100 105 110 Arg Pro Gly Ser Lys Gly Glu Pro Gly Arg Ala Ala Asp Asp Gly Glu         115 120 125 Gly Ile Val Gly Ala Ala Met Pro Asp Ser Gly Pro Leu Pro Leu Leu     130 135 140 Gln Asp Met Asn Lys Leu Ser Gly Gly Gly Gly Arg Arg Thr Arg Val 145 150 155 160 Glu Gly Gly Gln Leu Gly Gly Glu Glu Trp Thr Arg His Gly Ser Phe                 165 170 175 Val Asn Lys Pro Thr Arg Gly Trp Leu His Pro Asn Asp Lys Val Met             180 185 190 Gly Pro Gly Val Ser Tyr Leu Val Arg Tyr Met Gly Cys Val Glu Val         195 200 205 Leu Gln Ser Met Arg Ala Leu Asp Phe Asn Thr Arg Thr Gln Val Thr     210 215 220 Arg Glu Ala Ile Ser Leu Val Cys Glu Ala Val Pro Gly Ala Lys Gly 225 230 235 240 Ala Thr Arg Arg Arg Lys Pro Cys Ser Arg Pro Leu Ser Ser Ile Leu                 245 250 255 Gly Arg Ser Asn Leu Lys Phe Ala Gly Met Pro Ile Thr Leu Thr Val             260 265 270 Ser Thr Ser Ser Leu Asn Leu Met Ala Ala Asp Cys Lys Gln Ile Ile         275 280 285 Ala Asn His His Met Gln Ser Ile Ser Phe Ala Ser Gly Gly Asp Pro     290 295 300 Asp Thr Ala Glu Tyr Val Ala Tyr Val Ala Lys Asp Pro Val Asn Gln 305 310 315 320 Arg Ala Cys His Ile Leu Glu Cys Pro Glu Gly Leu Ala Gln Asp Val                 325 330 335 Ile Ser Thr Ile Gly Gln Ala Phe Glu Leu Arg Phe Lys Gln Tyr Leu             340 345 350 Arg Asn Pro Pro Lys Leu Val Thr Pro His Asp Arg Met Ala Gly Phe         355 360 365 Asp Gly Ser Ala Trp Asp Glu Glu Glu Glu Glu Pro Pro Asp His Gln     370 375 380 Tyr Tyr Asn Asp Phe Pro Gly Lys Glu Pro Pro Leu Gly Gly Val Val 385 390 395 400 Asp Met Arg Leu Arg Glu Gly Ala Ala Pro Gly Ala Ala Arg Pro Thr                 405 410 415 Ala Pro Asn Ala Gln Thr Pro Ser His Leu Gly Ala Thr Leu Pro Val             420 425 430 Gly Gln Pro Val Gly Gly Asp Pro Glu Val Arg Lys Gln Met Pro Pro         435 440 445 Pro Pro Pro Cys Pro Gly Arg Glu Leu Phe Asp Asp Pro Ser Tyr Val     450 455 460 Asn Val Gln Asn Leu Asp Lys Ala Arg Gln Ala Val Gly Gly Ala Gly 465 470 475 480 Pro Pro Asn Pro Ala Ile Asn Gly Ser Ala Pro Arg Asp Leu Phe Asp                 485 490 495 Met Lys Pro Phe Glu Asp Ala Leu Arg Val Pro Pro Pro Gln Ser             500 505 510 Val Ser Met Ala Glu Gln Leu Arg Gly Glu Pro Trp Phe His Gly Lys         515 520 525 Leu Ser Arg Arg Glu Ala Glu Ala Leu Leu Gln Leu Asn Gly Asp Phe     530 535 540 Leu Val Arg Glu Ser Thr Thr Thr Pro Gly Gln Tyr Val Leu Thr Gly 545 550 555 560 Leu Gln Ser Gly Gln Pro Lys His Leu Leu Leu Val Asp Pro Glu Gly                 565 570 575 Val Val Arg Thr Lys Asp His Arg Phe Glu Ser Val Ser His Leu Ile             580 585 590 Ser Tyr His Met Asp Asn His Leu Pro Ile Ile Ser Ala Gly Ser Glu         595 600 605 Leu Cys Leu Gln Gln Pro Val Glu Arg Lys Leu     610 615 <210> 2 <211> 1800 <212> DNA <213> Artificial Sequence <220> <223> gene coding Tat-p66shc <400> 2 atgaggaaga agcggagaca gcgacgaaga ggctcggatc cgaattcgat ggatctcctg 60 ccccccaagc ccaagtacaa tccactccgg aatgagtctc tgtcatcgat ggaggaaggg 120 gcttctgggt ccaccccccc ggaggagctg ccttccccac cagcttcatc cctggggccc 180 atcctgcctc ctctgcctgg ggacgatagt cccactaccc tgtgctcctt cttcccccgg 240 atgagcaacc tgaggctggc caacccggct ggggggcgcc cagggtctaa gggggagcca 300 ggaagggcag ctgatgatgg ggaggggatc gtaggggcag ccatgccaga ctcaggcccc 360 ctacccctcc tccaggacat gaacaagctg agtggaggcg gcgggcgcag gactcgggtg 420 gaagggggcc agcttggggg cgaggagtgg acccgccacg ggagctttgt caataagccc 480 acgcggggct ggctgcatcc caacgacaaa gtcatgggac ccggggtttc ctacttggtt 540 cggtacatgg gttgtgtgga ggtcctccag tcaatgcgtg ccctggactt caacacccgg 600 actcaggtca ccagggaggc catcagtctg gtgtgtgagg ctgtgccggg tgctaagggg 660 gcgacaagga ggagaaagcc ctgtagccgc ccgctcagct ctatcctggg gaggagtaac 720 ctgaaatttg ctggaatgcc aatcactctc accgtctcca ccagcagcct caacctcatg 780 gccgcagact gcaaacagat catcgccaac caccacatgc aatctatctc atttgcatcc 840 ggcggggatc cggacacagc cgagtatgtc gcctatgttg ccaaagaccc tgtgaatcag 900 agagcctgcc acattctgga gtgtcccgaa gggcttgccc aggatgtcat cagcaccatt 960 ggccaggcct tcgagttgcg cttcaaacaa tacctcagga acccacccaa actggtcacc 1020 cctcatgaca ggatggctgg ctttgatggc tcagcatggg atgaggagga ggaagagcca 1080 cctgaccatc agtactataa tgacttcccg gggaaggaac cccccttggg gggggtggta 1140 gacatgaggc ttcgggaagg agccgctcca ggggctgctc gacccactgc acccaatgcc 1200 cagaccccca gccacttggg agctacattg cctgtaggac agcctgttgg gggagatcca 1260 gaagtccgca aacagatgcc acctccacca ccctgtccag gcagagagct ttttgatgat 1320 ccctcctatg tcaacgtcca gaacctagac aaggcccggc aagcagtggg tggtgctggg 1380 ccccccaatc ctgctatcaa tggcagtgca ccccgggacc tgtttgacat gaagcccttc 1440 gaagatgctc ttcgggtgcc tccacctccc cagtcggtgt ccatggctga gcagctccga 1500 ggggagccct ggttccatgg gaagctgagc cggcgggagg ctgaggcact gctgcagctc 1560 aatggggact tcttggtacg ggagagcacg accacacctg gccagtatgt gctcactggc 1620 ttgcagagtg ggcagcctaa gcatttgcta ctggtggacc ctgagggtgt ggttcggact 1680 aaggatcacc gctttgaaag tgtcagtcac cttatcagct accacatgga caatcacttg 1740 cccatcatct ctgcgggcag cgaactgtgt ctacagcaac ctgtggagcg gaaactgtga 1800                                                                         1800 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> sense primer <400> 3 cggaattcga aggatctcct gcccccc 27 <210> 4 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> anti-sense primer <400> 4 cgaagctttc acagtttccg ctccacagg 29 <210> 5 <211> 1752 <212> DNA <213> human <400> 5 atggatctcc tgccccccaa gcccaagtac aatccactcc ggaatgagtc tctgtcatcg 60 atggaggaag gggcttctgg gtccaccccc ccggaggagc tgccttcccc accagcttca 120 tccctggggc ccatcctgcc tcctctgcct ggggacgata gtcccactac cctgtgctcc 180 ttcttccccc ggatgagcaa cctgaggctg gccaacccgg ctggggggcg cccagggtct 240 aagggggagc caggaagggc agctgatgat ggggagggga tcgtaggggc agccatgcca 300 gactcaggcc ccctacccct cctccaggac atgaacaagc tgagtggagg cggcgggcgc 360 aggactcggg tggaaggggg ccagcttggg ggcgaggagt ggacccgcca cgggagcttt 420 gtcaataagc ccacgcgggg ctggctgcat cccaacgaca aagtcatggg acccggggtt 480 tcctacttgg ttcggtacat gggttgtgtg gaggtcctcc agtcaatgcg tgccctggac 540 ttcaacaccc ggactcaggt caccagggag gccatcagtc tggtgtgtga ggctgtgccg 600 ggtgctaagg gggcgacaag gaggagaaag ccctgtagcc gcccgctcag ctctatcctg 660 gggaggagta acctgaaatt tgctggaatg ccaatcactc tcaccgtctc caccagcagc 720 ctcaacctca tggccgcaga ctgcaaacag atcatcgcca accaccacat gcaatctatc 780 tcatttgcat ccggcgggga tccggacaca gccgagtatg tcgcctatgt tgccaaagac 840 cctgtgaatc agagagcctg ccacattctg gagtgtcccg aagggcttgc ccaggatgtc 900 atcagcacca ttggccaggc cttcgagttg cgcttcaaac aatacctcag gaacccaccc 960 aaactggtca cccctcatga caggatggct ggctttgatg gctcagcatg ggatgaggag 1020 gaggaagagc cacctgacca tcagtactat aatgacttcc cggggaagga accccccttg 1080 gggggggtgg tagacatgag gcttcgggaa ggagccgctc caggggctgc tcgacccact 1140 gcacccaatg cccagacccc cagccacttg ggagctacat tgcctgtagg acagcctgtt 1200 gggggagatc cagaagtccg caaacagatg ccacctccac caccctgtcc aggcagagag 1260 ctttttgatg atccctccta tgtcaacgtc cagaacctag acaaggcccg gcaagcagtg 1320 ggtggtgctg ggccccccaa tcctgctatc aatggcagtg caccccggga cctgtttgac 1380 atgaagccct tcgaagatgc tcttcgggtg cctccacctc cccagtcggt gtccatggct 1440 gagcagctcc gaggggagcc ctggttccat gggaagctga gccggcggga ggctgaggca 1500 ctgctgcagc tcaatgggga cttcttggta cgggagagca cgaccacacc tggccagtat 1560 gtgctcactg gcttgcagag tgggcagcct aagcatttgc tactggtgga ccctgagggt 1620 gtggttcgga ctaaggatca ccgctttgaa agtgtcagtc accttatcag ctaccacatg 1680 gacaatcact tgcccatcat ctctgcgggc agcgaactgt gtctacagca acctgtggag 1740 cggaaactgt ga 1752  

Claims (8)

The cell permeable fusion protein Tat-p66shc of SEQ ID NO: 1 in which the protein transduction site of human p66shc protein and Tat protein was fused. The gene encoding the fusion protein Tat-p66shc of claim 1. The method of claim 2, Gene encoding the fusion protein Tat-p66shc, characterized in that the sequence of the gene is SEQ ID NO: 2. An expression vector comprising the gene of claim 2 or claim 3 expressing the fusion protein of claim 1. E. coli transformants transformed with the expression vector of claim 4 (Accession No. KCTC 11138BP). delete delete delete

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