WO2006122512A1 - Proteine de fusion comprenant un domaine de transduction de proteine, une proteine auxiliaire et une proteine bioactive, production et utilisations - Google Patents
Proteine de fusion comprenant un domaine de transduction de proteine, une proteine auxiliaire et une proteine bioactive, production et utilisations Download PDFInfo
- Publication number
- WO2006122512A1 WO2006122512A1 PCT/CN2006/001048 CN2006001048W WO2006122512A1 WO 2006122512 A1 WO2006122512 A1 WO 2006122512A1 CN 2006001048 W CN2006001048 W CN 2006001048W WO 2006122512 A1 WO2006122512 A1 WO 2006122512A1
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- WO
- WIPO (PCT)
- Prior art keywords
- protein
- transduction domain
- tat
- gst
- connexin
- Prior art date
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/18—Antioxidants, e.g. antiradicals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/06—Fusion polypeptide containing a localisation/targetting motif containing a lysosomal/endosomal localisation signal
Definitions
- Fusion protein comprising protein transduction domain, accessory protein and biologically active protein, and preparation method and application thereof
- the invention relates to a kind of fusion protein (Protein Fusion) and a preparation method and application thereof.
- Cells are the basic unit of life. Proteins are involved in almost all life processes. The absence or deficiency of normal proteins in cells often leads to abnormalities in the pathology and life activities of cells. Although gene therapy is expected to complement these proteins through transcription and translation, the conditions for efficient localization of target genes and long-term expression of target proteins remain to be further explored. Therefore, protein therapy that directly introduces biologically active proteins from cells outside the cell to improve the physiological state of the cells is a very interesting alternative.
- ANTP RQIKIWFQNRRM WKK compares the amino acid sequences of the core regions of these three protein transduction domains and finds that they are all rich in basic amino acids (arginine and lysine), which can be positively charged. The action of electrostatic attraction to each other binds to the cell membrane, thereby promoting transmembrane delivery of the foreign protein fused thereto.
- accessory proteins in fusion proteins - Fusion proteins are composed of two or more linked and distinct proteins that are produced by fusion genes. When a fusion protein is expressed in a host cell, problems such as low expression level and difficulty in isolation and purification tend to occur. Adding a helper protein to the fusion protein will help to increase the expression level of the fusion protein and the efficiency of separation and purification.
- accessory proteins can also be used to increase the time of action of the fusion protein in the cell, as well as to regulate the reaction products of biologically active proteins, particularly enzyme molecules, thereby increasing the use of biologically active proteins.
- One of the objects of the present invention is to provide a class of biologically active proteins of a connexin transduction domain and a helper protein having efficient transmembrane delivery capability.
- a second object of the present invention is to provide a biologically active protein of a connexin transduction domain and an accessory protein which can increase the expression level and isolation and purification efficiency of the fusion protein.
- the third object of the present invention is to provide a connexin transduction domain capable of increasing the action time of a fusion protein in a cell and regulating the reaction product of a biologically active protein, particularly an enzyme molecule, thereby improving the use effect of the biologically active protein. And biologically active proteins of accessory proteins.
- the technical scheme of the present invention is as follows: a biologically active protein of a connexin transduction domain and an accessory protein, which comprises a protein transduction domain, a biologically active protein, characterized in that it further comprises an accessory protein.
- Figure 1 Effect of cell types on transmembrane delivery of GST-TAT-GFP and GST-GFP.
- Four different cell cultures were cultured in fresh RMPI 1640 medium containing 500 ug/mL GST-TAT-GFP or GST-GFP.
- the fluorescence value was quantitatively determined by a fluorescence spectrophotometer after incubation at % C0 2 and 37 ° C for 6 hours.
- Figure 2, Figure 3, and Figure 4 are the graphs of serum uric acid, lactate degassing enzyme, and lactic acid content of the mice in Experiment 7.
- the labels on the abscissas 1, 2, and 3 represent the administrations at 30 minutes, 120 minutes, and 240 minutes before exercise.
- Figure 5 is a graph of the repair effect of the fusion protein GST-TAT-SOD on the damage of alloxan-induced free radical damage to MDCK cells.
- Figure 6 is a graph showing the protective effect of the fusion protein GST-TAT-SOD on UV-induced radiation damage of MDCK cells.
- the connexin transduction domain of the invention and the biologically active protein of the accessory protein which comprises a protein transduction domain, an accessory protein, a biologically active protein, covalently linked, and the linkage occurs in any structure along the biologically active protein structure.
- the connexin transduction domain of the present invention and the bioactive protein of the accessory protein can be applied to cosmetics, or can be applied to the treatment of inflammatory diseases, or can be applied to functional foods, or can be applied to the treatment of radiation damage. .
- Example 1 Preparation of fusion protein GST-TAT-GFP by DNA recombination technique (GST: glutathione-S-transferase as accessory protein; TAT: protein transduction domain of TAT protein; GFP: green fluorescent protein) .
- the 5' primer 1 and the 3' primer 2 of the GFP-encoding gene in the bacterial plasmid were used for PCR amplification, and the coding sequence of TAT was also contained at the 5' end of the primer 1, thereby amplifying the fusion gene of TAT and GFP. .
- the amplified PCR product was digested by enzyme digestion and electrophoresis, inserted into the multiple cloning site of plasmid pGEX, and the expression plasmid pTAT-GFP was constructed and transformed into E. coli BL21 (DE3). The positive clone was screened and identified as an expression strain.
- TAT can enhance biological activity in the fusion protein GST-TAT-GFP (GST: accessory protein glutathione-S-transferase; TAT: protein transduction domain of TAT protein; GFP: green fluorescent protein) Transmembrane delivery of protein GFP.
- GST-TAT-GFP GST: accessory protein glutathione-S-transferase
- TAT protein transduction domain of TAT protein
- GFP green fluorescent protein
- This experiment investigated the transmembrane delivery of the fusion protein GST-TAT-GFP to L-02, SMMC-7721, Hela and BEL-7402 cells. After the 500 ⁇ g/mL fusion protein was incubated with these different kinds of cells for 6 h, the four cells showed bright green under the fluorescence microscope, although their fluorescence intensity was slightly different. The four cells treated with the control TAT-free fusion protein GST-GFP showed only the background color of the cells under fluorescence microscopy, and no fluorescence appeared, even if the incubation time was extended to 24 hours.
- the fluorescence intensity of these cells was quantitatively determined by a fluorescence spectrophotometer (Fig. 1), and it was found that the fluorescence intensity of the cells of the GST-TAT-GFP experimental group was more than 25 times that of the untreated cells, whereas the control TAT-free fusion protein GST- The fluorescence intensity of the cells of the experimental group of GFP was not much different from that of the untreated cells. The above results indicate that TAT can enhance the ability of GFP to cross-membrane into different cells.
- Mouse skin application test - Skin is the first barrier between the body and nature. Its main function is to protect against environmental harmful substances and the loss of body fluids.
- the frozen protein of mouse abdominal skin was directly placed under a fluorescence microscope to investigate the transmembrane delivery of the fusion protein GST-Tat-GFP into the skin.
- GST-Tat-GFP the fusion protein
- TAT helps GST-Tat-GFP to enter the skin of mice through skin application.
- the fusion protein GST-Tat-GFP was used to investigate its tissue delivery in mice. According to the amount of 10 mg / kg (protein / mouse), intraperitoneal injection was performed. After 17 hours, the tissue was dissected and frozen, and frozen sections were also observed. The heart, liver and kidney tissue sections were also observed under the fluorescence microscope. Green fluorescence was observed and distributed in the tissues, while no fluorescence was observed in the negative control protein GST-GFP. In addition, green fluorescence was also observed in brain tissue, indicating that the fusion protein successfully crossed the blood-brain barrier (BBB).
- BBB blood-brain barrier
- TAT enhances biologically active proteins in the fusion protein GST-TAT-SOD (GST: accessory protein glutathione-S-transferase; TAT: protein transduction domain of TAT protein, SOD: superoxide dismutase) Transmembrane delivery of SOD.
- GST-TAT-SOD accessory protein glutathione-S-transferase
- TAT protein transduction domain of TAT protein
- SOD superoxide dismutase
- GST-TAT-SOD and GST-SOD control protein were added to different types of cells (L-02, BEL-7402 and Hela) for transmembrane transduction experiments.
- the final concentration of the fusion protein was 500 ug/mL. .
- the SOD enzyme activity in the cell lysate was measured. It was found that compared with the cell blank control and the cells treated with GST-SOD,
- Example 1 The large amount of induced bacterial liquid described in Example 1 was collected by centrifugation, and the cells were sonicated by high speed centrifugation under ice bath conditions, and the supernatant was bright green; SDS-PAGE electrophoresis revealed that about 95% of the fusion protein GST-TAT- GFP was soluble; the same preparation method produced GST-free fusion protein TAT-GFP, SDS-PAGE electrophoresis showed that less than 5% of the fusion protein TAT-GFP was soluble, and the bacterial solution was ultrasonically disrupted. The supernatant basically does not see green.
- Experiment 4 Experiment 4
- This experiment demonstrates the reaction product of CAT by decomposing bioactive protein SOD in the fusion protein TAT-SOD-CAT (TAT: protein transduction domain of TAT protein; SOD: superoxide dismutase; CAT: Catalase, catalase)
- a hydrogen peroxide further improves the biological activity of SOD.
- TAT-SOD-CAT and TAT-SOD control protein. After maintaining at 37 ° C for 10 minutes, the superoxide radicals in the reaction system were measured by EPR (Electron paramagnetic Resonance) method. It was found that TAT-SOD-CAT can not only eliminate the superoxide anion radical 0 2 _ 100%, compared with the phosphate buffer without the fusion protein, and
- This experiment demonstrates the application of the fusion protein GST-TAT-SOD (TAT: protein transduction domain of TAT protein, SOD: superoxide dismutase) as an active ingredient of facial cream in cosmetics.
- TAT protein transduction domain of TAT protein
- SOD superoxide dismutase
- This experiment demonstrates the application of the fusion protein GST-TAT-SOD (TAT: protein transduction domain of TAT protein, SOD: superoxide dismutase) in the regulation of inflammatory diseases such as rheumatoid arthritis, atopic dermatitis and tenosynovitis.
- TAT protein transduction domain of TAT protein
- SOD superoxide dismutase
- GST-TAT-SOD medicinal cream 1000 U superoxide dismutase activity per gram of emulsion
- apply a proper amount of GST-TAT-SOD cream stored in a refrigerator or a cool place at 4 degrees), observe once a day, and evaluate the effect as one week later.
- mice were swimming in the swimming pool, water depth 40 cm, 7J temperature 25 degrees Celsius, rats The tail root is loaded with 5% body weight of lead. Load After 5 minutes of swimming, 12 mice in each group were bled and dried to remove blood.
- Test items blood lactic acid, lactate dehydrogenase, uric acid.
- MDCK dog kidney epithelial cell line was inoculated into a 96-well plate overnight, and disrupted with lOumol/L alloxan for 1 hour, the disruptant group was added to the medium, and the SOD group was added to the medium and GST-S0D (final The concentration of 0. 6mg/ml), the sample group was added to the medium and GST-TAT-SOD (final concentrations were 0.05, 0. 1, 0. 2, 0.4, and 0.6 mg/ml, respectively, and cultured for 24 hours).
- MTT method measured 0D590. 1, 2, 3, 4, 5 groups respectively indicate that the final concentration of GST-TAT-S0D is 0.05, 0. 1, 0. 2, 0. 3, 0.4, and 0.6 mg/ml, and the normal group indicates Cells that are not destroyed by alloxan.
- Test items The number of remaining cells.
- MDCK dog kidney epithelial cell line was inoculated into 96-well plate overnight, and then PBS, GST-S0D or GST-TAT-S0D was added to the medium for 3 hours, then fresh medium was added to destroy 90 with ultraviolet light. Minutes, continue to culture for 24 hours, and the MTT method measures 0D590.
- the protection ratio indicates the percentage of cells protected by GST-S0D or GST-TAT-S0D as a whole.
- Test items The number of remaining cells.
- the fusion protein GST-TAT-S0D protects against cellular radiation damage caused by UV rays, and this protective effect increases with the increase in S0D activity. Therefore, the bioactive protein of the connexin transduction domain and accessory proteins can be used in the treatment of radiation damage.
- bioactive protein of the connexin transduction domain and the accessory protein of the invention have been subjected to a large number of experiments by the applicant, have good effects, can be industrially produced, and have good industrial applicability.
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- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Animal Behavior & Ethology (AREA)
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- Public Health (AREA)
- Veterinary Medicine (AREA)
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- Pharmacology & Pharmacy (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
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- Dermatology (AREA)
- Polymers & Plastics (AREA)
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- Physics & Mathematics (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
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Abstract
Les protéines de fusion comprenant un domaine de transduction de protéine, une protéine auxiliaire, une protéine bioactive, leur production et leurs utilisations. Le domaine de transduction de protéine sert à améliorer la capacité de la protéine bioactive de pénétrer dans une cellule, alors que la protéine auxiliaire sert à améliorer le niveau d'expression, la séparation et la purification, la stabilité thermique et la période dans la cellule des protéines de fusion. Ces dernières peuvent être utilisées comme composants efficaces dans le secteur de la cosmétique, des médicaments de traitement d'inflammations ou de lésions causées par un rayonnement et des aliments fonctionnels.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CNB2005100187540A CN100387623C (zh) | 2005-05-20 | 2005-05-20 | 连接蛋白转导结构域及辅助蛋白的生物活性蛋白、它的制备方法和应用 |
CN200510018754.0 | 2005-05-20 |
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WO2006122512A1 true WO2006122512A1 (fr) | 2006-11-23 |
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PCT/CN2006/001048 WO2006122512A1 (fr) | 2005-05-20 | 2006-05-19 | Proteine de fusion comprenant un domaine de transduction de proteine, une proteine auxiliaire et une proteine bioactive, production et utilisations |
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CN (1) | CN100387623C (fr) |
WO (1) | WO2006122512A1 (fr) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
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CN101812439B (zh) * | 2009-02-25 | 2012-07-04 | 张舒羽 | 一种透皮结构域及其衍生的具有透皮作用的融合蛋白和其制备方法 |
CN101985478A (zh) * | 2010-10-29 | 2011-03-16 | 福州大学 | 一种融合蛋白及其在缺血性脑卒中中的应用 |
CN103789278A (zh) * | 2013-07-26 | 2014-05-14 | 薛荣亮 | 新型PTD4-Cu,Zn-SOD融合蛋白及其制备方法 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2004039846A1 (fr) * | 2002-10-31 | 2004-05-13 | Hallym University | Proteine hybride elaboree de transport par transduction cellulaire a domaine de transport proteique entre domaine et cible |
TW200411056A (en) * | 2002-12-30 | 2004-07-01 | qi-cai Lin | Method for clone, expression, purification, analysis and application of Cu/Zn superoxide dismutase |
CN1648135A (zh) * | 2004-01-20 | 2005-08-03 | 中国人民解放军军事医学科学院毒物药物研究所 | 转导肽-人源胆碱乙酰基转移酶融合蛋白及其应用 |
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2005
- 2005-05-20 CN CNB2005100187540A patent/CN100387623C/zh not_active Expired - Fee Related
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2006
- 2006-05-19 WO PCT/CN2006/001048 patent/WO2006122512A1/fr active Application Filing
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2004039846A1 (fr) * | 2002-10-31 | 2004-05-13 | Hallym University | Proteine hybride elaboree de transport par transduction cellulaire a domaine de transport proteique entre domaine et cible |
TW200411056A (en) * | 2002-12-30 | 2004-07-01 | qi-cai Lin | Method for clone, expression, purification, analysis and application of Cu/Zn superoxide dismutase |
CN1648135A (zh) * | 2004-01-20 | 2005-08-03 | 中国人民解放军军事医学科学院毒物药物研究所 | 转导肽-人源胆碱乙酰基转移酶融合蛋白及其应用 |
Non-Patent Citations (4)
Title |
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GUO S. ET AL.: "Heterogenic Bcl-Xs Fused with Tat Basic Domain can Induce Programmed Cell Death After Transient Incubation", CHIN. J. BIOCHEM. MOL. BIOL., vol. 16, no. 5, October 2000 (2000-10-01), pages 674 - 679, XP008072958 * |
KWON H.Y. ET AL.: "Transduction of Cu,Zn-superoxide dismutase mediated by an HIV-1 Tat protein basic domain into mammalian cells", FEBS LETTERS, vol. 485, 2000, pages 163 - 167, XP001002544 * |
PARK J. ET AL.: "9-Polylysine Protein Tranduction Domain: Enhanced Penetration Efficiency of Superoxide Dismutase into Mammalian Cells and Skin", MOL. CELLS, vol. 13, no. 2, 2002, pages 202 - 208, XP009016372 * |
WANG F. ET AL.: "Expression and Trans-membrane Activity of Human Cu,Zn-SOD-TAT PTD", PHARMACEUTICAL BIOTECHNOLOGY, vol. 11, no. 1, February 2004 (2004-02-01), pages 11 - 15, XP008072946 * |
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Publication number | Publication date |
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CN1712415A (zh) | 2005-12-28 |
CN100387623C (zh) | 2008-05-14 |
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