KR20160000969A - Pharmaceutical composition for Parkinson's Disease containing Catalase fusion protein - Google Patents

Abstract

Parkinson's disease is a neurodegenerative disease in which dopaminergic neurons gradually disappear in the substantia nigra of the brain. The present inventors have found that PEP-1-catalase fusion proteins can protect dopamine neurons from oxidative stress in SH-SY5Y neuroblastoma cells and Parkinson's disease animal models. The PEP-1-catalyzed fusion protein was confirmed by Western blot analysis to transfer into SH-SY5Y cells and into the substantia nigra of the brain. The PEP-1-catalyzed fusion protein significantly inhibited MPP ⁺ -induced reactive oxygen species production and DNA fragmentation, resulting in the survival of SH-SY5Y cells. Further, immunohistochemistry data using TH antibody and cresyl violet staining indicate that the PEP-1-catalase fusion protein significantly protects dopamine cells in the substantia nigra against oxidative stress such as MPTP. Thus, PEP-1-catalase fusion proteins are available for the prophylactic and therapeutic use of Parkinson's disease.

Description

[0001] The present invention relates to a pharmaceutical composition for preventing and treating Parkinson's disease comprising a catalase fusion protein,

The present invention relates to the protective action of dopamine neurons of a catalase fusion protein, wherein the catalase fusion protein covalently bound to the protein transport domain smoothly migrates into the substantia nigra of the brain, significantly inhibits reactive oxygen species generation and DNA fragmentation, Lt; RTI ID = 0.0 > dopamine < / RTI > neurons in the substantia nigra, and the catalase fusion proteins are available for the prophylactic and therapeutic use of Parkinson's disease.

Parkinson's disease is a neurodegenerative disease characterized by loss of dopaminergic neurons in the substantia nigra par compacta [1]. Typical symptoms include muscle stiffness, bradykinesia, and movement disorders such as tremors and posture instability. Patients with Parkinson's disease have a characteristic back burning posture and are generally seen in elderly people over 65 years of age [2]. Parkinson's disease is characterized by a marked loss of dopaminergic neurons in the substantia nigra. Various stimuli such as excessive reactive oxygen species production, oxidative stress, misfolded protein and impaired mitochondrial function are known to be the cause of Parkinson's disease [3]. Although the exact mechanism of neuronal apoptosis is not fully understood, Parkinson's disease is associated with many risk factors including aging, inflammation, chemicals and genetic factors [1, 4-6].

Catalase is known to be an enzyme that inhibits the detrimental effects of reactive oxygen species (ROS) on cells. Catalase protects cells through the process of preventing the generation of hydroxyl radicals and decomposing hydrogen peroxide to produce water and oxygen. Catalase is crucial for maintaining oxygen-reducing conditions by removing oxygen free radicals [7].

Protein transport domains (PTDs) are small peptides that can thermally convert polymers to cells and tissues without specific receptors [8]. Recent studies have shown that the transgenic Tat-PRDX6 fusion protein can be an effective method for retarding eye disease by delaying the onset of lens epithelial cell death and transferring a protein that protects the cataracts [ 9]. In previous studies, the present inventors have shown that a fusion protein bound to PEP-1 protein transduction domain is efficiently transfected into various cells to efficiently protect it from various diseases such as skin inflammation and neurological diseases [10, 11, 12, 13 ].

[1] Choi, H.S .; Ahn, J. J .; Kim, S. Y .; Lee, S. H .; Kim, D. W .; Yoo, K. Y .; Won, M. H .; Kang, T. C .; Kwon, H.J .; Kang, J. H .; Choe, S. W .; Kwon, O.S .; Park, J. S .; Eum, W.S .; Choi, S.Y. : PEP-1-SOD fusion protein protects against paraquat-induced dopaminergic neuron damage in a Parkinson disease mouse model. Free Radical Biology & Medicine 41: 1058-1068; 2006. [2] Dawson, T. M.; Dawson, V.L. : Molecular pathways of neurodegeneration in Parkinson ' s disease. Science 302: 819-822; 2003. [3] Chinta, S.J., Andersen, J.K. : Redox imbalance in Parkinson's disease. Biochim. Biophys. Acta 1780: 1362-1367; 2008. [4] Hunot, S .; Hirsch, E. C.: Neuroinflammatory processes in Parkinson's disease. Ann. Neurol. 53: S49-60; 2003 [5] Dauer, W .; Przedborski, S.: Parkinson's disease: mechanisms and models. Neuron 39: 889-909; 2003. [6] C. Henchcliffe, et al. M. Flint Beal. : Mitochondrial biology and oxidative stress in Parkinson disease pathogenesis, Nat. Clin. Practice 4: 600-609; 2008 [7] Kim, D. W .; Jeong, H. J .; Kang, H. W .; Shin, M.J .; Sohn, E.J .; Kim, M.J .; Ahn, E. H .; Ahn, J. J .; Jang, S. H., Yoo, K. Y .; Won, M. H.; Kang, T. C .; Hwang, I. K.; Kwon, O.S .; Cho, S. W .; Park, J., Eum, W.S .; Choi, S.Y. : Transduced human PEP-1-catalase fusion protein attenuates ischemic neuronal damage. Free Radical Biology & Medicine 47: 941-952; 2009 [8] Im, S.J .; Lee, S.K. : protein transdecution domain (PTD) and its application. biowave vol. 8 No. 142006 [9] Eri Kubo .; NigarFatma .; Yoshio Akagi .; David R ,; Beier .; Sanjay; P. Singh .; Dhirendra; P. Singh. : TAT-mediated PRDX6 protein transduction protects against eye lensepithelial cell death and delays lens opacity. Am J Physiol Cell Physiol 294: C842-855: 2008. [10] Choi, S. Y.; Ahn, E.H .; Kim, D. W .; Shin, M.J .; Jo, H. S .; Eom, S.A .; Kim, D.S .; Park, E. Y .; Park, J. H .; Cho, S. W .; Park, J .; Eum, W.S .; Hwang, H.S. : Fenobam promotes the neuroprotective effect of PEP-1-FK506BP following oxidative stress through its transduction efficiency. BMB Reports. BMB Reports 46 (11): 561-566; 2013 [11] Kim, M. J .; Kim, D. W .; Park, J. H .; Kim, S. J .; Lee, C. H.; Yong, J. Y .; Ryu, E.J .; Cho, S.B .; Yeo, H.J .; Hyeon, J. Y .; Cho, S. W .; Kim, D.S .; Son, O. R .; Park, J. S .; Han, K.H; Cho, Y.S .; Eum, W.S., Choi, S.Y. : PEP-1-SIRT2 inhibits inflammatory response and oxidative stress induced cell death via expression of antioxidant enzymes inmurinemacrophages. ElsevierInc.63: 432-445; 2013 [12] Choi, J. H .; Kim, D. W .; Yoo, D.Y ;; Jeong, H.J .; Kim, W.S .; Jung, H. Y .; Nam, S. M .; Kim, J. H .; Yoon, Y.S .; Choi, S. Y .; Hwang, I.K. : Repeated Administration of PEP-1-Cu, Zn-Superoxide Dismutase and PEP-1-Peroxiredoxin-2 to Senescent Mice Induced by D-galactose Improves the Hippocampal Functions. Neurochem Res 38: 2046-2055; 2013 [13] Lee, Y. P .; Kim, D. W .; Kang, H. W .; Hwang, J. H .; Jeong, H.J .; Sohn, E.J., Kim, M.J., Ahn, E.H., Shin, M.J .; Kim, D.S .; Kang, T. C .; Kwon, O.S .; Cho, S. W .; Park, J .; Eum, W.S .; Choi, S.Y. : PEP-1-heat shock protein 27 protects from neuronaldamage in cells and in Parkinson's disease mouse models. FEBS J.279 (11): 1929-42; 2012 [14] FLORIAN M .; FREIMOSER .; CLAUDEE .; JAKOB .; MARKUS AEBI .; AND URS TUOR. : The MTT [3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide] Assay Is a Fast and Reliable Method for Colorimetric Determination of Fungal Cell Densities.Appl. Environ. Microbiol. 65 (8): 3727; 1999 [15] Kim, M.J., Kim, D.W., Jeong, H.J., Sohn, E.J., Shin, M.J., Ahn, E.H., Kwon, S.W .; Kim, Y. N .; Kim, D.S .; Park, J., Eum, W.S., Hwang, H. S., Choi, S.Y. : Tat-Frataxin protects dopaminergic neuronal cells against MPTP-induced toxicity in a mouse model of Parkinson's disease. Biochimie. 94 (11): 2448-56; 2012 [16] Ahn, E, H, Kim, D. W., Kim, Y. N., Kim, H. R., Woo, S. J .; Kim, S. M.; Park, J., Eum, W.S., Hwang, H. S., Choi, S.Y. : PEP-1-ribosomal protein S3 protects dopaminergic neurons in an MPTP-induced Parkinson's disease mouse model. Free RadicBiol Med. 55: 36-45; 2013 [17] Teresa C, Moloney .; Rhona Hyland .; Daniel O'Toole .; Alexia Paucard .; SeaKirik .; Aideen O'Doherty .; Adrienne M, Gorman .; Eil_isDowd. : Heat Shock Protein 70 Reduces a-Synuclein-Induced Predegenerative Neuronal Dystrophy in the a-Synuclein Viral Gene Transfer Rat Model of Parkinson's Disease. CNS Neuroscience & Therapeutics: 1-9; 2013 [18] R. K. B .; Pearce 1,2, A .; Owen 1, S .; Daniel 2,; P. Jenner 1 ,; and C. D. Marsden 2: Alterations in the distribution of glutathione in thesubstantianigra in Parkinson's disease. J Neural Transm 104: 661-677; 1997 [19] Siddharth Kaul .; Arthi Kanthasamy .; Masashi Kitazawa .; VellareddyAnantharam .; Anumantha G. Kanthasamy. : Caspase-3 dependent proteolytic activation of protein kinase C mediates and regulates 1-methyl-4-phenylpyridinium (MPP +) -induced apoptotic cell death in dopaminergic cells: relevance to oxidative stress. European Journal of Neuroscience, Vol. 18: 1387-1401; 2003 [20] Fuenzalida, K .; Quintanilla, R .; Ramos, P; Piderite, D; Fuentealba, R.A .; Martinez, G .; Inestrosa, N. C .; Bronfman, M. : Peroxisome proliferator-activated receptor gamma up-regulates the Bcl-2 anti-apoptotic protein in neurons and induces apoptosis. J. Biol. chem. 282: 37006-37015; 2007. [21] Ryo Hashimoto., Jing Yu .; Hideki Koizumi .; Yasuyoshi Ouchi .; Tetsuro Okabe. : Ginsenoside Rb1 Prevents MPP + -induced Apoptosis in PC12 Cellsby Stimulating Estrogen Receptors with Conjugation of ERK1 / 2, Akt and Inhibition of SAPK / JNK, p38 MAPK. Evidence-Based Complementary and Alternative Medicine Volume Article ID 693717, 8 pages2012 [22] Mates, M.: Effects of antioxidant enzymes in the molecular control of reactive oxygen species toxicology. Toxicology 153: 83-104; 2000. [23] Morris, M. C .; Depollier, J .; Mery, J .; Heitz, F .; Divita, G. A. Peptide carrier for the delivery of biologically active proteins into mammalian cells. Nat. Biotechnol. 19: 1173-1176; 2001 [24] Jehangir S Wadia .; Steven F Dowdy. : Protein transduction technology. Current Opinion in Biotechnology 13: 52-56; 2002 [25] Jian-hui Zhu .; Charge T. Chu: Regulation of Autophagy by Extracellular Signal-Regulated Protein Kinases During 1-Methyl-4-Phenylpyridinium-Induced Cell Death. AJP January 170 (1): 75-86; 2007 [26] Choi, W.S .; L. M. T. Canzoniero .; S. L. Sensi .; Karen L. O'Malley .; Gwag, B.J .; Sohn, S. H .; Kim, J. E .; Oh, T. H., B.E.H .; Oh, Y.J. : Characterization of MPP + -induced Cell Death in a Dopaminergic Neuronal Cell Line: Role of Macromolecule Synthesis, Cytosolic Calcium, Caspase, and Bcl-2-Related Proteins. Experimental Neurology 159: 274-282; 1999 [27] Shengquan Hu .; SH-SY5Y cells (Panax ginseng C.A. Meyer) were cultured in RPMI-1640 medium supplemented with 1 mM Tris-HCl (pH 7.4). Journal of Ethnopharmacology 135: 34-42; 2011 [28] Won-Seok Choi, So-Young Yoon; Tae H. Oh; Eui-Ju Choi; Karen L. O'Malley,; and Young J. Oh. : Two Distinct Mechanisms Are Involvedin 6-Hydroxydopamine- and MPP + -InducedDopaminergic Neuronal Cell Death: Role of Caspases, ROS, and JNK.Journal of Neuroscience Research 57: 86-94; 1999 [29] Heather L .; Martin, Ross B .; Mounsey ,; Sarah Mustafa ,; KinnariSathe,; Peter Teismann: Pharmacological manipulation of peroxisome proliferator-activated receptor (PPARR) in Parkinson's disease. Experimental Neurology 235: 528-538; 2012 [30] Hausenloy, D. J.; Yellon, D. M.: New directions for protecting the heart against ischaemia-reperfusion injury: targeting the reperfusion Injury Salvage Kinase (RISK) -pathway. Cardiovasc Res, 61 (3): 448-460: 2004 [31] Burke, R.E. : Inhibition of mitogen-activated protein kinase and stimulation of Akt kinase signaling pathways: Two approaches to treatment of neurodegenerative disease. Pharmacol. Ther., 114, 261-277; 2007 [32] BALACHANDAR VENKATESAN .; LENIN MAHIMAINATHAN,; FALGUNI DAS .; NANDINI GHOSH-CHOUDHURY .; AND GOUTAM GHOSH CHOUDHURY.J. : Downregulation of Catalase by Reactive Oxygen Species Via PI 3Kinase / Akt Signaling in Mesangial Cells. Cell. Physiol. 211: 457-467; 2007 [33] Lim KS .; WonYW .; Park YS .; Kim YH. : Preparationand functional analysis of recombinant protein transduction domain-metallothionein fusion proteins. Biochimie 92, 964-970. 2010 [34] St Martin JL .; Klucken J., Outeiro TF .; Nguyen P .; Keller-McGandy, Cantuti-Castelvetri I .; Grammatopoulos TN .; Standaert DG .; Hyman BT; .McLeanPJ. : Dopaminergic neuron loss and up-regulation of chaperone protein mRNA induced by over-expression of alpha-synuclein in mouse substantia nigra. J Neurochem 100: 1449-1457; 2007 [35] Wang H., Zhong CY .; Wu JF .; Huang YB .; Liu CB. : Enhancement of TAT cell membrane penetration efficiency by dimethyl sulphoxide. J Control Release143: 64-70; 2010

The present inventors have found that a catalase fusion protein can effectively inhibit neuronal cell death in a Parkinson's disease model induced by MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) And to provide a pharmaceutical composition for preventing and treating Parkinson's disease comprising a catalase fusion protein.

The present inventors examined the protective effect of PEP-1-catalase fusion protein in animal models of SH-SY5Y cells and Parkinson's disease. Parkinson's disease is a neurodegenerative disease in which dopaminergic neurons gradually disappear in the substantia nigra of the brain. The inventors have found that catalase fusion proteins can protect dopamine neurons from oxidative stress in SH-SY5Y neuroblastoma cells and Parkinson's disease animal models. The catalase fusion protein was confirmed by Western blot analysis to transfer into the SH-SY5Y cells and into the substantia nigra of the brain. The catalase fusion protein markedly inhibited the production of MPP ⁺ -induced reactive oxygen species and DNA fragmentation, resulting in the survival of SH-SY5Y cells. The neuroprotective effect is obtained by the catalase fusion protein affecting the levels of pro-apoptotic and anti-apoptotic mediators. Furthermore, immunohistochemistry data using TH antibody and cresyl violet staining indicate that the catalase fusion protein significantly protects dopamine cells in the substantia nigra against oxidative stress such as MPTP. Thus, catalase fusion proteins are available for the prevention and treatment of Parkinson's disease.

The pharmaceutical composition containing the catalase fusion protein as an active ingredient can be formulated together with a carrier that is conventionally accepted in the pharmaceutical field and can be formulated into the oral or injectable form by a conventional method. Oral compositions include, for example, tablets and gelatin capsules, which may contain, in addition to the active ingredient, a diluent such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine, , Magnesium stearate, stearic acid and its magnesium or calcium salt and / or polyethylene glycol) and the tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone ), And may optionally contain a disintegrant (e.g., starch, agar, alginic acid or a sodium salt thereof) or a boiling mixture and / or an absorbent, a colorant, a flavoring agent and a sweetening agent. The injectable composition is preferably an isotonic aqueous solution or suspension, and the composition mentioned is sterilized and / or contains adjuvants such as preservatives, stabilizers, wetting or emulsifying solution accelerators, salts for controlling osmotic pressure and / or buffering agents. They may also contain other therapeutically valuable substances.

The pharmaceutical preparations thus prepared may be administered orally or parenterally, that is, intravenously, subcutaneously, intraperitoneally, or topically, as desired. The dose may be administered in a single dose of 0.01 to 100 mg / kg per day. The dosage level for a particular patient may vary depending on the patient's body weight, age, sex, health condition, time of administration, method of administration, excretion rate, severity of disease, and the like.

The definitions of the main terms used in the description of the present invention and the like are as follows.

"Catalase fusion protein" means a covalent complex formed by genetic fusion or chemical bonding between a protein transport domain and a cargo molecule (i.e., catalase in the present invention), including a protein transport domain and a catalase. As one specific example in the present specification and drawings, "PEP-1-catalase" refers to the PEP-1 protein transport domain bound to the N-terminus of the catalase fusion protein.

In addition, the term "genetic fusion" means a link consisting of a linear, covalent bond formed through genetic expression of a DNA sequence encoding a protein.

The term "target cell" refers to a cell to which a cargo molecule is delivered by a transport domain. That is, the target cell is a cell, that is, a living organism or a cell or organ It is meant to include microorganisms that are found. In addition, the target cell means an extracellular cell, that is, a cultured animal cell, a human cell or a microorganism. Specifically, in the present specification, the target cell means a brain cell or a nerve cell.

As used herein, the term "protein transport domain" refers to a peptide or protein that is covalently bonded to a cargo molecule (target protein) peptide or protein and can introduce the peptide or protein into cells without requiring additional receptor, carrier, energy, For example, PEP-1 peptide (SEQ ID NO: 3).

As used herein, the term "target protein" refers to a molecule that is covalently bound to a PEP-1 protein transduction domain and introduced into cells to exhibit activity. It is synonymous with "cargo molecule".

Also, in the present specification, the term "introduction", "transport", "penetration", "transport", "transfer", "permeation", "pass" Respectively.

The present invention relates to a pharmaceutical composition for preventing and treating Parkinson's disease comprising a catalase fusion protein in which a protein transport domain is covalently bonded to one or more of N-terminal and C-terminal of human catalase protein.

In addition, the present invention relates to a pharmaceutical composition for preventing and treating Parkinson's disease containing a catalase fusion protein, wherein the protein transport domain is a PEP-1 peptide.

In addition, the present invention relates to a pharmaceutical composition for preventing and treating Parkinson's disease containing a fused protein of catalase, wherein the fusion protein is SEQ ID NO: 16.

The PEP-1-catalase fusion protein of the present invention was permeabilized intracellularly in a time-dependent, dose-dependent manner into SH-SY5Y cells, furthermore, the level of intracellular PEP-1-catalase fusion protein was maintained significantly for 48 hours.

In addition, the PEP-1-catalase fusion protein of the present invention significantly inhibited the production of reactive oxygen species, which is a response to MPP ⁺ .

The PEP-1-catalase fusion protein of the present invention can protect cells from oxidative stress induced by MPP ⁺ .

The PEP-1-catalase fusion protein of the present invention can effectively cross the cerebral blood vessel barrier and protects dopamine neurons against MPTP toxicity in the MPTP-induced Parkinson's disease mouse model. In MPTP-induced blackness Of dopaminergic neurons and to decrease motor dysfunction.

Figure 1 shows the purification of PEP-1-catalase fusion protein and the permeation of PEP-1-catalase fusion protein into SH-SY5Y cells. (A) Purification of catalase protein and PEP-1-catalase fusion protein. (B) Purified protein after purification of catalase protein and PEP-1-catalase fusion protein was analyzed by 12% SDS-PAGE and Western blotting using anti-histidine antibody. Lane 1: Marker, lane 2 to 5: non-specificity, lane 3 to 6: induction, lane 4 to 7: purified (C) Shows the permeation of PEP-1-catalase fusion protein into SH-SY5Y cells. SH-SY5Y cells were cultured with PEP-1-catalase fusion protein and catalase protein (0.5 to 3 μM) for 3 hours and cultured with PEP-1-catalase fusion protein and catalase protein (3 μM) for 1 to 3 hours . The PEP-1-catalase fusion protein or catalase protein in each sample was analyzed by Western blot. (D) were observed with a confocal fluorescence microscope. Scale bar = 50 탆. (E) shows the SH-SY5Y intracellular stability of the PEP-1-catalyzed fusion protein introduced into the cell. The PEP-1-catalyzed fusion protein (3 [mu] M) was treated for 3 to 60 hours, the intracellular level was measured by Western blot analysis, and then the band intensity was measured with a density meter.
Figure 2 shows the inhibitory effect of PEP-1-catalase fusion protein on apoptosis and production of reactive oxygen species by MPP ⁺ . (A) cells were treated with PEP-1-catalase fusion protein, catalase protein (3.0 μM) for 3 hours and treated with MPP ⁺ (4.0 mM) for 17 hours. Cell viability was assessed using MTT assay. ^* P < 0.01, compared to MPP ⁺ treated cells. (B) After pretreatment, SH-SY5Y cells were treated with 3 μM PEP-1-catalase and catalase protein for 3 hours and MPP ⁺ 4 mM for 40 minutes. The level of active oxygen in the cells was stained with DCF-DA and detected and measured with an ELISA plate reader. Scale bar = 50 탆. ^** P < 0.01, compared to MPP ⁺ treated cells.
FIG. 3 shows the effect of PEP-1-catalase fusion protein inhibiting DNA fragmentation and cell death by MPP ⁺ in SH-SY5Y cells. After pretreatment, 3 μM PEP-1-catalase fusion protein, catalase protein, was treated with SH-SY5Y cells for 3 hours and cells were treated with MPP ⁺ (4 mM) for 14 hours 30 minutes. DNA fragmentation was detected by TUNEL staining and fluorescence intensity was measured by ELISA plate reader. Scale bar == 50 占 퐉. ^** P < 0.01, compared to MPP ⁺ treated cells. (B) PEP-1-catalase fusion protein and catalase protein (3 μM) were treated with SH-SY5Y cells, treated with MPP ⁺ 4 mM for 10 minutes (pAkt), 3 hours (p-p38). The level of protein displayed was measured by Western blot analysis and the band intensity was measured by a densitometer. ^** P < 0.01, compared to MPP ⁺ treated cells.
Figure 4 shows the effect of PEP-1-catalase fusion protein protecting dopaminergic neurons in an animal model of Parkinson's disease. (A) PEP-1-catalase fusion protein crossed the cerebral vascular barrier. Mice were intraperitoneally injected with PEP-1-catalase fusion protein (2 mg / kg). After 6 hours, mice were sacrificed and the brain was sectioned and stained with anti-histidine antibody. Scale bar = 100 탆. (B) The protective function was confirmed by staining immunoreactive neurons with tyrosine hydroxylase (TH) and cresyl violet (CV). Brain tissues were obtained 7 days after intraperitoneal injection in each group (N = 5) of MPTP (20 mg / kg) injection, catalase protein, PEP-1 peptide and PEP-1-catalase fusion protein (2 mg / kg). Scale bar = 100 탆 and 50 탆. (C) Protection and penetration were confirmed by their antibody double staining and TH immunity. MPTP (20 mg / kg) injection, catalase protein, PEP-1 peptide and PEP-1-catalase fusion protein (2 mg / kg), respectively, and sacrificed after 12 hours. The PEP-1-catalase fusion protein and catalase protein were then detected using its antibody and secondary antibody FITC-label and DA neurons were detected using TH and tetramethylrhodamine isothiocyanate secondary antibody. DA neurons (TH antibody), red; Control catalase protein or PEP-1-catalase fusion protein (antibody thereof), green. Scale bar = 100 탆 and 18.8 탆.

Hereinafter, the configuration of the present invention will be described in more detail with reference to specific embodiments. However, it is apparent to those skilled in the art that the scope of the present invention is not limited to the description of the embodiments. Particularly, as a result of each experimental example, data on the PEP-1-catalase fusion protein among the various fusion proteins prepared in the specific examples were described, but other protein transport domains such as Tat, oligo lysine and oligoarginine were used Fusion proteins also show results similar to those obtained with PEP-1-catalase fusion proteins as samples.

Materials and cell culture

p38, p-p38, Akt, p-Akt, Bax and Bcl-2 rabbit antibodies were purchased from Cell signaling (Denvers, MA, USA). Other primary antibodies and secondary anti-rabbit antibodies were purchased from Santa Cruz Biotechnology (USA). Other chemicals and reagents not specified were purchased at the highest level in Sigma-Aldrich (St. Louis, Mo., USA). PEP-1 peptides were synthesized in Peptron (Daejeon, Korea).

SH-SY5Y human neuroblastoma cells were cultured in EMEM medium containing heat-activated activated rabbit serum (10% FBS) and antibiotics (100 μg / ml streptomycin, 100 U / ml penicillin) ₂ of And cultured under a humid environment.

PEP -One- Catalase Fusion protein  refine

Construction and over-expression of PEP-1-catalase fusion proteins were purified as previously studied [7]. Briefly, transformed cell growth and IPTG were induced. The harvested cells were sonicated in binding buffer (5 mM imidazole, 500 mM NaCl, 20 mM Tris-HCl, PH 7.9) at 4 ° C with an ultrasonic disruptor. The supernatant was immediately centrifuged Ni ^{2 +} - acrylonitrile triazol setik acid Sepharose Super flow ^{(Ni 2 + -nitrilotriacetic acid sepharose super} flow) load the column and binding buffer of 10 times by volume with 6-fold volume wash buffer (60mM of (500 mM imidazole, 500 mM NaCl, 20 mM Tris-HCl, pH 7.9) and eluted with elution buffer (500 mM imidazole, 500 mM NaCl, pH 7.9). Fractions containing the fusion protein were then pooled and subjected to PD-10 column chromatography for 24 hours to remove the salts contained in the fraction. The purified protein concentration was measured by the Bradford method using bovine serum albumin as a standard [7].

SH - SY5Y  Neuroblastoma cell PEP -One- Catalase Of the fusion protein  Cell introduction

SH-SY5Y cells were cultured in a 60 mm dish to permeabilize the PEP-1-catalase fusion protein. Cells were treated with various concentrations of PEP-1-catalase fusion protein (0.5-3 μM) for 3 hours and treated at different concentrations of PEP-1-catalase fusion protein at 3 μM (1 to 3 hours). Cells were treated with trypsin-EDTA and washed with PBS. Cells were harvested to produce cell extracts and used for Western blot analysis.

Western Blat  analysis

For Western blot analysis, the proteins in the cell lysate were separated by 12% SDS polyacrylamide gel, and the proteins in the gels were electrochromatically transferred to a nitrocellulose membrane (Amersham, UK). The membranes were blocked with 5% skim milk powder in TBS-T buffer (25 mM Tris-HCl, 140 mM NaCl, 0.1% Tween 20, pH 7.5) for 1 hour and reacted with primary and secondary antibodies. (1: 10,000 dilution) coupled with rabbit anti-histidine antibody, p38, p-p38, Akt, p-Akt, Bax and Bcl-2 antibodies (1: 10,000 dilution) and horseradish peroxidase Lt; / RTI &gt; The conjugated antibody complexes were detected using a chemiluminescence detection kit according to the manufacturer's instructions (Amersham, Piscataway, NJ, USA) [7].

Cell survival analysis

Cell viability was evaluated by MTT {3- (4,5-dimethylthiazol-2-yl) -2,5-dipheyltetrazolium bromide} [14]. Cells were seeded in 96-well plates and treated with PEP-1-catalase fusion protein (1-3 μM) for 3 h, then washed with PBS and incubated with 4 mM MPP ⁺ (1-methyl-4-phenylpyridinium) Was added to the culture medium. Cell viability was evaluated by color development using MTT {3- (4,5-dimethylthiazol-2-yl) -2,5-dipheyltetrazolium bromide}. Absorbance was measured at 570 nm using an ELISA microplate reader (Labsystems Multiskan MCC / 340) and the cell viability was expressed as a percentage of control cells that did not receive the fusion protein.

Confocal  Microscopic observation

To detect PEP-1-catalase fusion proteins and catalase proteins in SH-SY5Y cells, cells were seeded onto glass cover slips and treated with PEP-1-catalase fusion protein and 3 μM catalase protein for 3 hours. Cells were washed twice with PBS, fixed with 4% paraformaldehyde for 5 minutes at room temperature, and incubated with anti-histidine primary antibody and Alexa Fluor 488 conjugated secondary antibody (Invitrogen, Carlsbad, Calif., USA). The nuclei were stained with 1 [mu] g / ml of 4'6-diamidino-2-phenylindole (Roche Applied Science, Basel, Switzerland) for 5 minutes. Fluorescence was analyzed with an Olympus FV-300 confocal fluorescence microscope (Olympus, Tokyo, Japan) [15].

Active oxygen species  Measure

(2 ', 7'-dichlorofluorescein diacetate) sensitive to reactive oxygen species, which is converted to DCF (2', 7'-dichlorofluorescein) by reactive oxygen species, . In order to test the effect of PEP-1-catalase fusion protein on the production of reactive oxygen species, SH-SY5Y cells were treated with MPP ⁺ ((3 μM) 4 mM) was added for 40 minutes. SH-SY5Y cells were washed twice with PBS, treated with 30 μM DCF-DA for 30 minutes, and washed twice with DPBS. Fluorescence images of the cells were taken with an Eclopse 80i fluorescence microscope (Nikon, Tokyo, Japan).

TUNEL  ( Terminal deoxynucleotidyl transferase - mediated dUTP nick - end -labeling) analysis

SH-SY5Y cells on glass cover slips were incubated at 37 ° C for 3 hours with PEP-1-catalase fusion protein and catalase protein (2 mM), respectively, and exposed to MPP ⁺ (4.0 mM) for 14 hours and 30 minutes. TUNEL staining was performed using a cell death kits (Roche Applied Science, Basel, Switzerland). Fluorescence microscopy photographs were taken on an Eclipse 80i fluorescence microscope (Nikon, Tokyo, Japan) [16].

Experimental animal

Male, 6 weeks old, C57BL / 6 mice were purchased from Hallym University Laboratory Animals Center. The mice were bred at a constant temperature (23 ° C) and at a constant relative humidity (60%) for 12 hours. Water and feed were freely accessible. All laboratory procedures and controls related to animals have been followed by the National Veterinary Research and Quarantine Service of Korea, which is guided by the Committee on Animal Protection and Utilization. Parkinson's disease models were constructed as previously described [17]. Mice were injected with MPTP at a dose of 20 mg / kg every two hours. To confirm the protective effect of PEP-1-catalase fusion protein on Parkinson's disease, 2.0 mg / kg protein was subcutaneously injected 12 hours before MPTP treatment. MPTP ⁺ PEP-1-catalyzed fusion protein treated group, and MPTP ⁺ PEP (n = 5 for each group) were divided into five groups as follows: 1) untreated control group 2) MPTP treated group 3) MPTP ⁺ -1 peptide treated group, and 5) MPTP ⁺ control group treated with catalase. Mice were sacrificed one week after the last injection.

Immunohistochemistry

Immunostaining was described in previous studies [1, 18]. Tissue sections were blocked by treatment with PBS containing 3% bovine serum albumin for 30 minutes at room temperature and labeled with tyrosine hydroxylase to detect histidine antibodies or dopamine neurons to detect PEP-1-catalase fusion proteins and catalase proteins hydroxylase (TH) antibody. Biotin - conjugated goat anti - rabbit antibody was used as a secondary antibody. Cresyl violet staining was performed to stain Nissl bodies, which are granules found in living neurons and considered as rough endoplasmic reticulum. The sections were visualized with DAB (3,3'-diaminobenzidine) in 0.1 M Tris buffer and placed on gelatin-coated slides. The images were taken and analyzed with an Olympus DP72 digital camera and DP2-BSW microscope digital camera software. The drawing was made using Adobe Photoshop 7.0 (San Jose, CA, USA).

Statistical analysis

Data were expressed as mean ± standard deviation from each independent experiment. The differences between the mean values were analyzed using one - way ANOVA. Newman-Keuls post hoc analyzes were employed when differences were observed in the ANOVA test ( p <0.01).

Result 1: PEP -One- Catalase Fusion protein  Build and refine

A catalase protein and a PEP-1-catalase fusion protein expression vector were constructed, and a schematic diagram thereof is shown in FIG. 1A. PEP-1- catalase and catalase protein was overexpressed in IPGT, Ni ^{2 +} - a nitrile triazol setik acid Sepharose was purified by Super flow ^{(Ni 2 + -nitrilotriacetic acid sepharose super} flow) column. The purified protein was washed with PBS buffer for 24 hours to remove salt. As shown in FIG. 1B, overexpression and purification of PEP-1-catalase fusion protein were confirmed by SDS-PAGE and Western blot analysis.

To investigate PEP-1-catalase fusion protein delivery to SH-SY5Y cells, PEP-1-catalase fusion protein was treated in culture medium at different concentrations (0.5-3 μM) and time (1-3 h) . Then, western blot analysis was performed to confirm the transfection efficiency. The PEP-1-catalase fusion protein was transduced intracellularly in a dose and time dependent manner of the protein. However, the catalase protein was not transduced into the cells (Fig. 1C). To further confirm the transduction of the PEP-1-catalase fusion protein into cells, we performed a confocal fluorescence microscopy analysis using its antibody DAPI, Alexa fluor-488 complex secondary antibody. As shown in FIG. 1D, no fluorescent substance was detected in the cells treated with the catalase protein, whereas a strong fluorescent substance was detected in the cells treated with the PEP-1-catalase fusion protein. These results indicate that the PEP-1-catalase fusion protein efficiently penetrated SH-SY5Y cells. To evaluate the stability of the PEP-1-catalyzed fusion protein transmitted into SH-SY5Y cells, PEP-1-catalase fusion protein (3 [mu] M) was transfected for 3 hours. The intracellular PEP-1-catalase fusion protein was incubated for 60 hours. Intracellular levels of PEP-1-catalase fusion protein increased up to 48 hours (Fig. 1E). Thus, this data suggests that the PEP-1-catalyzed fusion protein penetrates into SH-SY5Y cells and remains in the cells for 48 hours.

Result 2: PEP -One- Catalase The fusion protein MPP ⁺ On by Active oxygen species  It inhibits production and cell death.

MPP ⁺ induces the production of intracellular reactive oxygen species, resulting in DNA damage and apoptosis [19]. Therefore, the effect of MPP ⁺ on cell survival was examined to see if permeable PEP-1-catalase fusion protein protects cells against neurotoxicity-induced neuronal cell death. The cell viability of the PEP-1-catalyzed fusion protein was increased in a dose dependent manner on the fusion protein. Cells were treated with MPP ⁺ (4 mM) for 17 h. As in Figure 2A, only 46% of the cells survived. However, the survival rate of cells pretreated with PEP-1-catalase fusion protein increased by a dose-dependent manner and was 76% or more when the highest concentration was used. On the other hand, the control catalase protein did not show a protective effect under the same conditions.

In addition, the present inventors have examined whether the PEP-1-catalase fusion protein inhibits MPP ⁺ -induced intracellular production of reactive oxygen species. When cells were exposed to MPP ⁺ for 40 min, MPP ⁺ significantly increased the DCF signal. Cells treated with MMPP ⁺ showed higher intensity of fluorescence than control cells showing the production of MPP ⁺ induced intracellular reactive oxygen species. Activated oxygen species production was similar to MPP ⁺ in cells treated with the control catalase protein. However, the PEP-1-catalase fusion protein inhibited the production of intracellular reactive oxygen species by MPP ⁺ (Fig. 2B). These results suggest that the PEP-1-catalase fusion protein inhibits the production of reactive oxygen species induced by MPP ⁺ .

Result 3: PEP -One- Catalase The fusion protein MPP ⁺ On by DNA  Protects against damage and regulation of cell signaling.

The present inventors performed TUNEL analysis to examine the protective effect of PEP-1-catalase fusion protein on DNA fragmentation by MPP ⁺ . After treatment with PEP-1-catalase fusion protein, cells were exposed to MPP ⁺ for 14 hours 30 minutes. DNA fragmentation was then observed using a fluorescence microscope. As shown in Fig. 3A, the PEP-1-catalase fusion protein significantly inhibited DNA fragmentation. These data suggest that the PEP-1-catalase fusion protein protects nerve cells by inhibiting self-apoptotic cell death. Next, the present inventors confirmed the presence of the cell signal of the PEP-1-catalase fusion protein induced by MPP ⁺ . In some studies, cell damage from MPP ^{+ has} been shown to increase levels of Akt, Bax, and p38 phosphorylation and increase levels of apoptotic proteins and BCL-2 expression in cells [20, 21]. As shown in FIG. 3B, the levels of phosphorylation of Akt, Bax and P38 were significantly higher in MPP ⁺ treated cells Respectively. However, the expression levels of Akt, Bax and p38 were decreased in a concentration dependent on PEP-1-catalase fusion protein concentration. On the other hand, the expression level of BCL-2 decreased in MPP ⁺ treated cells. However, the PEP-1-catalase fusion protein increased BCL-2 expression. These results indicate that PEP-1-catalase fusion protein inhibits MPP ⁺ -induced DNA fragmentation in SH-SY5Y cells by regulating Akt, Bax, BCL-2 and P38 cell signaling.

Result 4: PEP -One- Catalase The fusion protein  Protects dopamine neurons in Parkinson's disease animal models.

PEP-1-catalase fusion protein and catalase protein (2 mg / kg) were subcutaneously injected to evaluate the ability of PEP-1-catalyzed fusion protein to penetrate the cerebral blood vessel barrier, and after 6 hours, The positions of PEP-1-catalase fusion proteins and catalase proteins in the cells were confirmed using histidine antibodies. Immunohistochemical data showed that only the PEP-1-catalyzed fusion protein-treated samples showed strong immunoreactivity scattered in the parenchyma containing the substantia nigra, so that only the PEP-1-catalase fusion protein crossed the cerebrovascular barrier (Fig. 4A). These results indicate that the PEP-1-catalase fusion protein crosses the brain and cerebrovascular barrier.

In addition, we examined whether MPEP-induced dopamine neuronal cell death of PEP-1-catalase fusion protein was prevented. Cell viability was assessed by immunohistochemical staining using a tyrosine hydroxylase (TH) antibody and cresyl violet (CV) staining (Fig. 4B). In MPTP treated mice, dopamine neuronal cell death was significantly increased compared to the Siamese control mice. In addition, control group proteinase treated group and PEP-1 peptide treated group mice were similar to MPTP treated mice. However, the survival rate of dopamine neurons was significantly increased in mice treated with PEP-1-catalase fusion protein.

In addition, the present inventors have examined whether PEP-1-catalase fusion protein can protect neural cells from dopamine neuronal cell death induced by MPTP around the substantia nigra beyond the blood-brain barrier. PEP-1-catalase protein (2 mg / kg) was intraperitoneally injected and 12 hours after MPTP injection, the brain was harvested and stained twice with a TH antibody (red) and a histidine antibody (green). As shown in FIG. 4C, the Siam surgical control group and the other groups had no difference in TH staining. However, green fluorescence was only detected in the PEP-1-catalase fusion protein treated group. These results show that the PEP-1-catalase fusion protein efficiently crosses the blood-brain barrier and has a protective effect against MPTP-induced dopamine neuronal cell death.

<110> Industry Academic Cooperation Foundation, Hallym University <120> Pharmaceutical composition for Parkinson's Disease containing          Catalase fusion protein <130> hallym-sychoi-CAT-PD-140624 <160> 18 <170> Kopatentin 1.71 <210> 1 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 ctcgagatgg ctgacagccg ggatcccgcc 30 <210> 2 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 ggatcctcac agatttgccr rctcccttgc c 31 <210> 3 <211> 1671 <212> DNA <213> Artificial Sequence <220> <223> coding sequence for Tat-catalase fusion protein <400> 3 catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagccatag gaagaagcgg 60 agacagcgac gaagactcga gatggctgac agccgggatc ccgccagcga ccagatgcag 120 cactggaagg agcagcgggc cgcgcagaaa gctgatgtcc tgaccactgg agctggtaac 180 ccagtaggag acaaacttaa tgttattaca gtagggcccc gtgggcccct tcttgttcag 240 gatgtggttt tcactgatga aatggctcat tttgaccgag agagaattcc tgagagagtt 300 gtgcatgcta aaggagcagg ggcctttggc tactttgagg tcacacatga cattaccaaa 360 tactccaagg caaaggtatt tgagcatatt ggaaagaaga ctcccatcgc agttcggttc 420 tccactgttg ctggagaatc gggttcagct gacacagttc gggaccctcg tgggtttgca 480 gtgaaatttt acacagaaga tggtaactgg gatctcgttg gaaataacac ccccattttc 540 ttcatcaggg atcccatatt gtttccatct tttatccaca gccaaaagag aaatcctcag 600 acacatctga aggatccgga catggtctgg gacttctgga gcctacgtcc tgagtctctg 660 catcaggttt ctttcttgtt cagtgatcgg gggattccag atggacatcg ccacatgaat 720 ggatatggat cacatacttt caagctggtt aatgcaaatg gggaggcagt ttattgcaaa 780 ttccattata agactgacca gggcatcaaa aacctttctg ttgaagatgc ggcgagactt 840 tcccaggaag atcctgacta tggcatccgg gatcttttta acgccattgc cacaggaaag 900 tacccctcct ggacttttta catccaggtc atgacattta atcaggcaga aacttttcca 960 tttaatccat tcgatctcac caaggtttgg cctcacaagg actaccctct catcccagtt 1020 ggtaaactgg tcttaaaccg gaatccagtt aattactttg ctgaggttga acagatagcc 1080 ttcgacccaa gcaacatgcc acctggcatt gaggccagtc ctgacaaaat gcttcagggc 1140 cgcctttttg cctatcctga cactcaccgc catcgcctgg gacccaatta tcttcatata 1200 cctgtgaact gtccctaccg tgctcgagtg gccaactacc agcgtgacgg cccgatgtgc 1260 atgaggaca atcagggtgg tgctccaaat tactacccca acagctttgg tgctccggaa 1320 caacagcctt ctgccctgga gcacagcatc caatattctg gagaagtgcg gagattcaac 1380 actgccaatg atgataacgt tactcaggtg cgggcattct atgtgaacgt gctgaatgag 1440 gaacagagga aacgtctgtg tgagaacatt gccggccacc tgaaggatgc acaaattttc 1500 atccagaaga aagcggtcaa gaacttcact gaggtccacc ctgactacgg gagccacatc 1560 caggctcttc tggacaagta caatgctgag aagcctaaga atgcgattca cacctttgtg 1620 cagtccggat ctcacttggc ggcaagggag aaggcaaatc tgtgaggatc c 1671 <210> 4 <211> 554 <212> PRT <213> Artificial Sequence <220> <223> Tat-catalase fusion protein <400> 4 His His His His His Ser Ser Gly Leu Val Pro Arg Gly Ser His   1 5 10 15 Arg Lys Lys Arg Arg Gln Arg Arg Arg Leu Glu Met Ala Asp Ser Arg              20 25 30 Asp Pro Ala Ser Asp Gln Met Gln His Trp Lys Glu Gln Arg Ala Ala          35 40 45 Gln Lys Ala Asp Val Leu Thr Thr Gly Ala Gly Asn Pro Val Gly Asp      50 55 60 Lys Leu Asn Val Ile Thr Val Gly Pro Arg Gly Pro Leu Leu Val Gln  65 70 75 80 Asp Val Val Phe Thr Asp Glu Met Ala His Phe Asp Arg Glu Arg Ile                  85 90 95 Pro Glu Arg Val Val His Ala Lys Gly Ala Gly Ala Phe Gly Tyr Phe             100 105 110 Glu Val Thr His Asp Ile Thr Lys Tyr Ser Lys Ala Lys Val Phe Glu         115 120 125 His Ile Gly Lys Lys Thr Pro Ile Ala Val Arg Phe Ser Thr Val Ala     130 135 140 Gly Glu Ser Gly Ser Ala Asp Thr Val Arg Asp Pro Arg Gly Phe Ala 145 150 155 160 Val Lys Phe Tyr Thr Glu Asp Gly Asn Trp Asp Leu Val Gly Asn Asn                 165 170 175 Thr Pro Ile Phe Phe Ile Arg Asp Pro Ile Leu Phe Pro Ser Phe Ile             180 185 190 His Ser Gln Lys Arg Asn Pro Gln Thr His Leu Lys Asp Pro Asp Met         195 200 205 Val Trp Asp Phe Trp Ser Leu Arg Pro Glu Ser Leu His Gln Val Ser     210 215 220 Phe Leu Phe Ser Asp Arg Gly Ile Pro Asp Gly His Arg His Met Asn 225 230 235 240 Gly Tyr Gly Ser His Thr Phe Lys Leu Val Asn Ala Asn Gly Glu Ala                 245 250 255 Val Tyr Cys Lys Phe His Tyr Lys Thr Asp Gln Gly Ile Lys Asn Leu             260 265 270 Ser Val Glu Asp Ala Ala Arg Leu Ser Gln Glu Asp Pro Asp Tyr Gly         275 280 285 Ile Arg Asp Leu Phe Asn Ale Ile Ala Thr Gly Lys Tyr Pro Ser Trp     290 295 300 Thr Phe Tyr Ile Gln Val Met Thr Phe Asn Gln Ala Glu Thr Phe Pro 305 310 315 320 Phe Asn Pro Phe Asp Leu Thr Lys Val Trp Pro His Lys Asp Tyr Pro                 325 330 335 Leu Ile Pro Val Gly Lys Leu Val Leu Asn Arg Asn Pro Val Asn Tyr             340 345 350 Phe Ala Glu Val Glu Gln Ile Ala Phe Asp Pro Ser Asn Met Pro Pro         355 360 365 Gly Ile Glu Ala Ser Pro Asp Lys Met Leu Gln Gly Arg Leu Phe Ala     370 375 380 Tyr Pro Asp Thr His Arg His Arg Leu Gly Pro Asn Tyr Leu His Ile 385 390 395 400 Pro Val Asn Cys Pro Tyr Arg Ala Arg Val Ala Asn Tyr Gln Arg Asp                 405 410 415 Gly Pro Met Cys Met Gln Asp Asn Gln Gly Gly Ala Pro Asn Tyr Tyr             420 425 430 Pro Asn Ser Phe Gly Ala Pro Glu Gln Gln Pro Ser Ala Leu Glu His         435 440 445 Ser Ile Gln Tyr Ser Gly Glu Val Arg Arg Phe Asn Thr Ala Asn Asp     450 455 460 Asp Asn Val Thr Gln Val Arg Ala Phe Tyr Val Asn Val Leu Asn Glu 465 470 475 480 Glu Gln Arg Lys Arg Leu Cys Glu Asn Ile Ala Gly His Leu Lys Asp                 485 490 495 Ala Gln Ile Phe Ile Gln Lys Lys Ala Val Lys Asn Phe Thr Glu Val             500 505 510 His Pro Asp Tyr Gly Ser His Ile Gln Ala Leu Leu Asp Lys Tyr Asn         515 520 525 Ala Glu Lys Pro Lys Asn Ale Ile His Thr Phe Val Gln Ser Gly Ser     530 535 540 His Leu Ala Ala Arg Glu Lys Ala Asn Leu 545 550 <210> 5 <211> 1670 <212> DNA <213> Artificial Sequence <220> <223> coding sequence for catalase-Tat fusion protein <400> 5 catcatcatc atcatcacag cagcggcctg gtgccggcgg cagccactcg agatggctga 60 cagccgggat cccgccagcg accagatgca gcactggaag gagcagcggg ccgcgcagaa 120 agctgatgtc ctgaccactg gagctggtaa cccagtagga gacaaactta atgttattac 180 agtagggccc cgtgggcccc ttcttgttca ggatgtggtt ttcactgatg aaatggctca 240 ttttgaccga gagagaattc ctgagagagt tgtgcatgct aaaggagcag gggcctttgg 300 ctactttgag gtcacacatg acattaccaa atactccaag gcaaaggtat ttgagcatat 360 cgggttcagc 420 tgacacagtt cgggaccctc gtgggtttgc agtgaaattt tacacagaag atggtaactg 480 ggatctcgtt ggaaataaca cccccatttt cttcatcagg gatcccatat tgtttccatc 540 ttttatccac agccaaaaga gaaatcctca gacacatctg aaggatccgg acatggtctg 600 ggacttctgg agcctacgtc ctgagtctct gcatcaggtt tctttcttgt tcagtgatcg 660 ggggattcca gatggacatc gccacatgaa tggatatgga tcacatactt tcaagctggt 720 taatgcaaat ggggaggcag tttattgcaa attccattat aagactgacc agggcatcaa 780 aaacctttct gttgaagatg cggcgagact ttcccaggaa gatcctgact atggcatccg 840 ggatcttttt aacgccattg ccacaggaaa gtacccctcc tggacttttt acatccaggt 900 catgacattt aatcaggcag aaacttttcc atttaatcca ttcgatctca ccaaggtttg 960 gcctcacaag gactaccctc tcatcccagt tggtaaactg gtcttaaacc ggaatccagt 1020 taattacttt gctgaggttg aacagatagc cttcgaccca agcaacatgc cacctggcat 1080 tgaggccagt cctgacaaaa tgcttcaggg ccgccttttt gcctatcctg acactcaccg 1140 ccatcgcctg ggacccaatt atcttcatat acctgtgaac tgtccctacc gtgctcgagt 1200 ggccaactac cagcgtgacg gcccgatgtg catgcaggac aatcagggtg gtgctccaaa 1260 ttactacccc aacagctttg gtgctccgga acaacagcct tctgccctgg agcacagcat 1320 ccaatattct ggagaagtgc ggagattcaa cactgccaat gatgataacg ttactcaggt 1380 gggggcattc tatgtgaacg tgctgaatga ggaacagagg aaacgtctgt gtgagaacat 1440 tgccggccac ctgaaggatg cacaaatttt catccagaag aaagcggtca agaacttcac 1500 tgaggtccac cctgactacg ggagccacat ccaggctctt ctggacaagt acaatgctga 1560 gaagcctaag aatgcgattc acacctttgt gcagtccgga tctcacttgg cggcaaggga 1620 gaaggcaaat ctgggatcct aggaagaagc ggagacagcg acgaagatag 1670 <210> 6 <211> 536 <212> PRT <213> Artificial Sequence <220> <223> catalase-Tat fusion protein <400> 6 Met Ala Asp Ser Arg Asp Pro Ala Ser Asp Gln Met Gln His Trp Lys   1 5 10 15 Glu Gln Arg Ala Gln Lys Ala Asp Val Leu Thr Thr Gly Ala Gly              20 25 30 Asn Pro Val Gly Asp Lys Leu Asn Val Ile Thr Val Gly Pro Arg Gly          35 40 45 Pro Leu Leu Val Gln Asp Val Val Phe Thr Asp Glu Met Ala His Phe      50 55 60 Asp Arg Glu Arg Ile Pro Glu Arg Val Val His Ala Lys Gly Ala Gly  65 70 75 80 Ala Phe Gly Tyr Phe Glu Val Thr His Asp Ile Thr Lys Tyr Ser Lys                  85 90 95 Ala Lys Val Phe Glu His Ile Gly Lys Lys Thr Pro Ile Ala Val Arg             100 105 110 Phe Ser Thr Val Ala Gly Glu Ser Gly Ser Ala Asp Thr Val Arg Asp         115 120 125 Pro Arg Gly Phe Ala Val Lys Phe Tyr Thr Glu Asp Gly Asn Trp Asp     130 135 140 Leu Val Gly Asn Asn Thr Pro Ile Phe Phe Ile Arg Asp Pro Ile Leu 145 150 155 160 Phe Pro Ser Phe Ile His Ser Gln Lys Arg Asn Pro Gln Thr His Leu                 165 170 175 Lys Asp Pro Asp Met Val Trp Asp Phe Trp Ser Leu Arg Pro Glu Ser             180 185 190 Leu His Gln Val Ser Phe Leu Phe Ser Asp Arg Gly Ile Pro Asp Gly         195 200 205 His Arg His Met Asn Gly Tyr Gly Ser His Thr Phe Lys Leu Val Asn     210 215 220 Ala Asn Gly Glu Ala Val Tyr Cys Lys Phe His Tyr Lys Thr Asp Gln 225 230 235 240 Gly Ile Lys Asn Leu Ser Val Glu Asp Ala Ala Arg Leu Ser Gln Glu                 245 250 255 Asp Pro Asp Tyr Gly Ile Arg Asp Leu Phe Asn Ala Ile Ala Thr Gly             260 265 270 Lys Tyr Pro Ser Trp Thr Phe Tyr Ile Gln Val Met Thr Phe Asn Gln         275 280 285 Ala Glu Thr Phe Pro Phe Asn Pro Phe Asp Leu Thr Lys Val Trp Pro     290 295 300 His Lys Asp Tyr Pro Leu Ile Pro Val Gly Lys Leu Val Leu Asn Arg 305 310 315 320 Asn Pro Val Asn Tyr Phe Ala Glu Val Glu Gln Ile Ala Phe Asp Pro                 325 330 335 Ser Asn Met Pro Pro Gly Ile Glu Ala Ser Pro Asp Lys Met Leu Gln             340 345 350 Gly Arg Leu Phe Ala Tyr Pro Asp Thr His Arg Arg His Arg Leu Gly Pro         355 360 365 Asn Tyr Leu His Ile Pro Val Asn Cys Pro Tyr Arg Ala Arg Val Ala     370 375 380 Asn Tyr Gln Arg Asp Gly Pro Met Cys Met Gln Asp Asn Gln Gly Gly 385 390 395 400 Ala Pro Asn Tyr Tyr Pro Asn Ser Phe Gly Ala Pro Glu Gln Gln Pro                 405 410 415 Ser Ala Leu Glu His Ser Ile Gln Tyr Ser Gly Glu Val Arg Arg Phe             420 425 430 Asn Thr Ala Asn Asp Asp Asn Val Thr Gln Val Arg Ala Phe Tyr Val         435 440 445 Asn Val Leu Asn Glu Glu Gln Arg Lys Arg Leu Cys Glu Asn Ile Ala     450 455 460 Gly His Leu Lys Asp Ala Gln Ile Phe Ile Gln Lys Lys Ala Val Lys 465 470 475 480 Asn Phe Thr Glu Val His Pro Asp Tyr Gly Ser His Ile Gln Ala Leu                 485 490 495 Leu Asp Lys Tyr Asn Ala Glu Lys Pro Lys Asn Ala Ile His Thr Phe             500 505 510 Val Gln Ser Gly Ser Le Leu Ala Ala Arg Glu Lys Ala Asn Leu Arg         515 520 525 Lys Lys Arg Arg Gln Arg Arg Arg     530 535 <210> 7 <211> 1699 <212> DNA <213> Artificial Sequence <220> <223> coding sequence for Tat-catalase-Tat fusion protein <400> 7 catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagccatag gaagaagcgg 60 agacagcgac gaagactcga gatggctgac agccgggatc ccgccagcga ccagatgcag 120 cactggaagg agcagcgggc cgcgcagaaa gctgatgtcc tgaccactgg agctggtaac 180 ccagtaggag acaaacttaa tgttattaca gtagggcccc gtgggcccct tcttgttcag 240 gatgtggttt tcactgatga aatggctcat tttgaccgag agagaattcc tgagagagtt 300 gtgcatgcta aaggagcagg ggcctttggc tactttgagg tcacacatga cattaccaaa 360 tactccaagg caaaggtatt tgagcatatt ggaaagaaga ctcccatcgc agttcggttc 420 tccactgttg ctggagaatc gggttcagct gacacagttc gggaccctcg tgggtttgca 480 gtgaaatttt acacagaaga tggtaactgg gatctcgttg gaaataacac ccccattttc 540 ttcatcaggg atcccatatt gtttccatct tttatccaca gccaaaagag aaatcctcag 600 acacatctga aggatccgga catggtctgg gacttctgga gcctacgtcc tgagtctctg 660 catcaggttt ctttcttgtt cagtgatcgg gggattccag atggacatcg ccacatgaat 720 ggatatggat cacatacttt caagctggtt aatgcaaatg gggaggcagt ttattgcaaa 780 ttccattata agactgacca gggcatcaaa aacctttctg ttgaagatgc ggcgagactt 840 tcccaggaag atcctgacta tggcatccgg gatcttttta acgccattgc cacaggaaag 900 tacccctcct ggacttttta catccaggtc atgacattta atcaggcaga aacttttcca 960 tttaatccat tcgatctcac caaggtttgg cctcacaagg actaccctct catcccagtt 1020 ggtaaactgg tcttaaaccg gaatccagtt aattactttg ctgaggttga acagatagcc 1080 ttcgacccaa gcaacatgcc acctggcatt gaggccagtc ctgacaaaat gcttcagggc 1140 cgcctttttg cctatcctga cactcaccgc catcgcctgg gacccaatta tcttcatata 1200 cctgtgaact gtccctaccg tgctcgagtg gccaactacc agcgtgacgg cccgatgtgc 1260 atgaggaca atcagggtgg tgctccaaat tactacccca acagctttgg tgctccggaa 1320 caacagcctt ctgccctgga gcacagcatc caatattctg gagaagtgcg gagattcaac 1380 actgccaatg atgataacgt tactcaggtg cgggcattct atgtgaacgt gctgaatgag 1440 gaacagagga aacgtctgtg tgagaacatt gccggccacc tgaaggatgc acaaattttc 1500 atccagaaga aagcggtcaa gaacttcact gaggtccacc ctgactacgg gagccacatc 1560 caggctcttc tggacaagta caatgctgag aagcctaaga atgcgattca cacctttgtg 1620 cagtccggat ctcacttggc ggcaagggag aaggcaaatc tgggatccta ggaagaagcg 1680 gagacagcga cgaagatag 1699 <210> 8 <211> 549 <212> PRT <213> Artificial Sequence <220> <223> Tat-catalase-Tat fusion protein <400> 8 Arg Lys Lys Arg Arg Gln Arg Arg Arg Leu Glu Met Ala Asp Ser Arg   1 5 10 15 Asp Pro Ala Ser Asp Gln Met Gln His Trp Lys Glu Gln Arg Ala Ala              20 25 30 Gln Lys Ala Asp Val Leu Thr Thr Gly Ala Gly Asn Pro Val Gly Asp          35 40 45 Lys Leu Asn Val Ile Thr Val Gly Pro Arg Gly Pro Leu Leu Val Gln      50 55 60 Asp Val Val Phe Thr Asp Glu Met Ala His Phe Asp Arg Glu Arg Ile  65 70 75 80 Pro Glu Arg Val Val His Ala Lys Gly Ala Gly Ala Phe Gly Tyr Phe                  85 90 95 Glu Val Thr His Asp Ile Thr Lys Tyr Ser Lys Ala Lys Val Phe Glu             100 105 110 His Ile Gly Lys Lys Thr Pro Ile Ala Val Arg Phe Ser Thr Val Ala         115 120 125 Gly Glu Ser Gly Ser Ala Asp Thr Val Arg Asp Pro Arg Gly Phe Ala     130 135 140 Val Lys Phe Tyr Thr Glu Asp Gly Asn Trp Asp Leu Val Gly Asn Asn 145 150 155 160 Thr Pro Ile Phe Phe Ile Arg Asp Pro Ile Leu Phe Pro Ser Phe Ile                 165 170 175 His Ser Gln Lys Arg Asn Pro Gln Thr His Leu Lys Asp Pro Asp Met             180 185 190 Val Trp Asp Phe Trp Ser Leu Arg Pro Glu Ser Leu His Gln Val Ser         195 200 205 Phe Leu Phe Ser Asp Arg Gly Ile Pro Asp Gly His Arg His Met Asn     210 215 220 Gly Tyr Gly Ser His Thr Phe Lys Leu Val Asn Ala Asn Gly Glu Ala 225 230 235 240 Val Tyr Cys Lys Phe His Tyr Lys Thr Asp Gln Gly Ile Lys Asn Leu                 245 250 255 Ser Val Glu Asp Ala Ala Arg Leu Ser Gln Glu Asp Pro Asp Tyr Gly             260 265 270 Ile Arg Asp Leu Phe Asn Ale Ile Ala Thr Gly Lys Tyr Pro Ser Trp         275 280 285 Thr Phe Tyr Ile Gln Val Met Thr Phe Asn Gln Ala Glu Thr Phe Pro     290 295 300 Phe Asn Pro Phe Asp Leu Thr Lys Val Trp Pro His Lys Asp Tyr Pro 305 310 315 320 Leu Ile Pro Val Gly Lys Leu Val Leu Asn Arg Asn Pro Val Asn Tyr                 325 330 335 Phe Ala Glu Val Glu Gln Ile Ala Phe Asp Pro Ser Asn Met Pro Pro             340 345 350 Gly Ile Glu Ala Ser Pro Asp Lys Met Leu Gln Gly Arg Leu Phe Ala         355 360 365 Tyr Pro Asp Thr His Arg His Arg Leu Gly Pro Asn Tyr Leu His Ile     370 375 380 Pro Val Asn Cys Pro Tyr Arg Ala Arg Val Ala Asn Tyr Gln Arg Asp 385 390 395 400 Gly Pro Met Cys Met Gln Asp Asn Gln Gly Gly Ala Pro Asn Tyr Tyr                 405 410 415 Pro Asn Ser Phe Gly Ala Pro Glu Gln Gln Pro Ser Ala Leu Glu His             420 425 430 Ser Ile Gln Tyr Ser Gly Glu Val Arg Arg Phe Asn Thr Ala Asn Asp         435 440 445 Asp Asn Val Thr Gln Val Arg Ala Phe Tyr Val Asn Val Leu Asn Glu     450 455 460 Glu Gln Arg Lys Arg Leu Cys Glu Asn Ile Ala Gly His Leu Lys Asp 465 470 475 480 Ala Gln Ile Phe Ile Gln Lys Lys Ala Val Lys Asn Phe Thr Glu Val                 485 490 495 His Pro Asp Tyr Gly Ser His Ile Gln Ala Leu Leu Asp Lys Tyr Asn             500 505 510 Ala Glu Lys Pro Lys Asn Ale Ile His Thr Phe Val Gln Ser Gly Ser         515 520 525 His Leu Ala Ala Arg Glu Lys Ala Asn Leu Gly Ser Arg Lys Lys Arg     530 535 540 Arg Gln Arg Arg Arg 545 <210> 9 <211> 1708 <212> DNA <213> Artificial Sequence <220> <223> coding sequence for PEP-1-catalase fusion protein <400> 9 catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagccataa aagaaacctg 60 gtgggaaacc tggtggaccg aatggtctca gccgaaaaaa aaacgtaaag tgctcgagat 120 ggctgacagc cgggatcccg ccagcgacca gatgcagcac tggaaggagc agcgggccgc 180 gcagaaagct gatgtcctga ccactggagc tggtaaccca gtaggagaca aacttaatgt 240 tattacagta gggccccgtg ggccccttct tgttcaggat gtggttttca ctgatgaaat 300 ggctcatttt gaccgagaga gaattcctga gagagttgtg catgctaaag gagcaggggc 360 ctttggctac tttgaggtca cacatgacat taccaaatac tccaaggcaa aggtatttga 420 gcatattgga aagaagactc ccatcgcagt tcggttctcc actgttgctg gagaatcggg 480 ttcagctgac acagttcggg accctcgtgg gtttgcagtg aaattttaca cagaagatgg 540 taactgggat ctcgttggaa ataacacccc cattttcttc atcagggatc ccatattgtt 600 tccatctttt atccacagcc aaaagagaaa tcctcagaca catctgaagg atccggacat 660 ggtctgggac ttctggagcc tacgtcctga gtctctgcat caggtttctt tcttgttcag 720 tgatcggggg attccagatg gacatcgcca catgaatgga tatggatcac atactttcaa 780 gctggttaat gcaaatgggg aggcagttta ttgcaaattc cattataaga ctgaccaggg 840 catcaaaaac ctttctgttg aagatgcggc gagactttcc caggaagatc ctgactatgg 900 catccgggat ctttttaacg ccattgccac aggaaagtac ccctcctgga ctttttacat 960 ccaggtcatg acatttaatc aggcagaaac ttttccattt aatccattcg atctcaccaa 1020 ggtttggcct cacaaggact accctctcat cccagttggt aaactggtct taaaccggaa 1080 tccagttaat tactttgctg aggttgaaca gatagccttc gacccaagca acatgccacc 1140 tggcattgag gccagtcctg acaaaatgct tcagggccgc ctttttgcct atcctgacac 1200 tcaccgccat cgcctgggac ccaattatct tcatatacct gtgaactgtc cctaccgtgc 1260 tcgagtggcc aactaccagc gtgacggccc gatgtgcatg caggacaatc agggtggtgc 1320 tccaaattac taccccaaca gctttggtgc tccggaacaa cagccttctg ccctggagca 1380 cagcatccaa tattctggag aagtgcggag attcaacact gccaatgatg ataacgttac 1440 tcaggtgcgg gcattctatg tgaacgtgct gaatgaggaa cagaggaaac gtctgtgtga 1500 gaacattgcc ggccacctga aggatgcaca aattttcatc cagaagaaag cggtcaagaa 1560 cttcactgag gtccaccctg actacgggag ccacatccag gctcttctgg acaagtacaa 1620 tgctgagaag cctaagaatg cgattcacac ctttgtgcag tccggatctc acttggcggc 1680 aagggagaag gcaaatctgt gaggatcc 1708 <210> 10 <211> 550 <212> PRT <213> Artificial Sequence <220> <223> PEP-1-catalase fusion protein <400> 10 Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys   1 5 10 15 Lys Lys Arg Lys Val Leu Glu Met Ala Asp Ser Arg Asp Pro Ala Ser              20 25 30 Asp Gln Met Gln His Trp Lys Glu Gln Arg Ala Gln Lys Ala Asp          35 40 45 Val Leu Thr Thr Gly Ala Gly Asn Pro Val Gly Asp Lys Leu Asn Val      50 55 60 Ile Thr Val Gly Pro Arg Gly Pro Leu Leu Val Gln Asp Val Val Phe  65 70 75 80 Thr Asp Glu Met Ala His Phe Asp Arg Glu Arg Ile Pro Glu Arg Val                  85 90 95 Val His Ala Lys Gly Ala Gly Ala Phe Gly Tyr Phe Glu Val Thr His             100 105 110 Asp Ile Thr Lys Tyr Ser Lys Ala Lys Val Phe Glu His Ile Gly Lys         115 120 125 Lys Thr Pro Ile Ala Val Arg Phe Ser Thr Val Ala Gly Glu Ser Gly     130 135 140 Ser Ala Asp Thr Val Arg Asp Pro Arg Gly Phe Ala Val Lys Phe Tyr 145 150 155 160 Thr Glu Asp Gly Asn Trp Asp Leu Val Gly Asn Asn Thr Pro Ile Phe                 165 170 175 Phe Ile Arg Asp Pro Ile Leu Phe Pro Ser Phe Ile His Ser Gln Lys             180 185 190 Arg Asn Pro Gln Thr His Leu Lys Asp Pro Asp Met Val Trp Asp Phe         195 200 205 Trp Ser Leu Arg Pro Glu Ser Leu His Gln Val Ser Phe Leu Phe Ser     210 215 220 Asp Arg Gly Ile Pro Asp Gly His Arg His Met Asn Gly Tyr Gly Ser 225 230 235 240 His Thr Phe Lys Leu Val Asn Ala Asn Gly Glu Ala Val Tyr Cys Lys                 245 250 255 Phe His Tyr Lys Thr Asp Gln Gly Ile Lys Asn Leu Ser Val Glu Asp             260 265 270 Ala Ala Arg Leu Ser Gln Glu Asp Pro Asp Tyr Gly Ile Arg Asp Leu         275 280 285 Phe Asn Ala Ile Ala Thr Gly Lys Tyr Pro Ser Trp Thr Phe Tyr Ile     290 295 300 Gln Val Met Thr Phe Asn Gln Ala Glu Thr Phe Pro Phe Asn Pro Phe 305 310 315 320 Asp Leu Thr Lys Val Trp Pro His Lys Asp Tyr Pro Leu Ile Pro Val                 325 330 335 Gly Lys Leu Val Leu Asn Arg Asn Pro Val Asn Tyr Phe Ala Glu Val             340 345 350 Glu Gln Ile Ala Phe Asp Pro Ser Asn Met Pro Pro Gly Ile Glu Ala         355 360 365 Ser Pro Asp Lys Met Leu Gln Gly Arg Leu Phe Ala Tyr Pro Asp Thr     370 375 380 His Arg His Arg Leu Gly Pro Asn Tyr Leu His Ile Pro Val Asn Cys 385 390 395 400 Pro Tyr Arg Ala Arg Val Ala Asn Tyr Gln Arg Asp Gly Pro Met Cys                 405 410 415 Met Gln Asp Asn Gln Gly Gly Ala Pro Asn Tyr Tyr Pro Asn Ser Phe             420 425 430 Gly Ala Pro Glu Gln Gln Pro Ser Ala Leu Glu His Ser Ile Gln Tyr         435 440 445 Ser Gly Glu Val Arg Arg Phe Asn Thr Ala Asn Asp Asp Asn Val Thr     450 455 460 Gln Val Arg Ala Phe Tyr Val Asn Val Leu Asn Glu Glu Gln Arg Lys 465 470 475 480 Arg Leu Cys Glu Asn Ile Ala Gly His Leu Lys Asp Ala Gln Ile Phe                 485 490 495 Ile Gln Lys Lys Ala Val Lys Asn Phe Thr Glu Val His Pro Asp Tyr             500 505 510 Gly Ser His Ile Gln Ala Leu Leu Asp Lys Tyr Asn Ala Glu Lys Pro         515 520 525 Lys Asn Ala Ile His Thr Phe Val Gln Ser Gly Ser His Leu Ala Ala     530 535 540 Arg Glu Lys Ala Asn Leu 545 550 <210> 11 <211> 1707 <212> DNA <213> Artificial Sequence <220> <223> coding sequence for catalase-PEP-1 fusion protein <400> 11 catcatcatc atcatcacag cagcggcctg gtgccggcgg cagccactcg agatggctga 60 cagccgggat cccgccagcg accagatgca gcactggaag gagcagcggg ccgcgcagaa 120 agctgatgtc ctgaccactg gagctggtaa cccagtagga gacaaactta atgttattac 180 agtagggccc cgtgggcccc ttcttgttca ggatgtggtt ttcactgatg aaatggctca 240 ttttgaccga gagagaattc ctgagagagt tgtgcatgct aaaggagcag gggcctttgg 300 ctactttgag gtcacacatg acattaccaa atactccaag gcaaaggtat ttgagcatat 360 cgggttcagc 420 tgacacagtt cgggaccctc gtgggtttgc agtgaaattt tacacagaag atggtaactg 480 ggatctcgtt ggaaataaca cccccatttt cttcatcagg gatcccatat tgtttccatc 540 ttttatccac agccaaaaga gaaatcctca gacacatctg aaggatccgg acatggtctg 600 ggacttctgg agcctacgtc ctgagtctct gcatcaggtt tctttcttgt tcagtgatcg 660 ggggattcca gatggacatc gccacatgaa tggatatgga tcacatactt tcaagctggt 720 taatgcaaat ggggaggcag tttattgcaa attccattat aagactgacc agggcatcaa 780 aaacctttct gttgaagatg cggcgagact ttcccaggaa gatcctgact atggcatccg 840 ggatcttttt aacgccattg ccacaggaaa gtacccctcc tggacttttt acatccaggt 900 catgacattt aatcaggcag aaacttttcc atttaatcca ttcgatctca ccaaggtttg 960 gcctcacaag gactaccctc tcatcccagt tggtaaactg gtcttaaacc ggaatccagt 1020 taattacttt gctgaggttg aacagatagc cttcgaccca agcaacatgc cacctggcat 1080 tgaggccagt cctgacaaaa tgcttcaggg ccgccttttt gcctatcctg acactcaccg 1140 ccatcgcctg ggacccaatt atcttcatat acctgtgaac tgtccctacc gtgctcgagt 1200 ggccaactac cagcgtgacg gcccgatgtg catgcaggac aatcagggtg gtgctccaaa 1260 ttactacccc aacagctttg gtgctccgga acaacagcct tctgccctgg agcacagcat 1320 ccaatattct ggagaagtgc ggagattcaa cactgccaat gatgataacg ttactcaggt 1380 gggggcattc tatgtgaacg tgctgaatga ggaacagagg aaacgtctgt gtgagaacat 1440 tgccggccac ctgaaggatg cacaaatttt catccagaag aaagcggtca agaacttcac 1500 tgaggtccac cctgactacg ggagccacat ccaggctctt ctggacaagt acaatgctga 1560 gaagcctaag aatgcgattc acacctttgt gcagtccgga tctcacttgg cggcaaggga 1620 gaaggcaaat ctgggatcct aaaagaaacc tggtgggaaa cctggtggac cgaatggtct 1680 cagccgaaaa aaaaacgtaa agtgtag 1707 <210> 12 <211> 566 <212> PRT <213> Artificial Sequence <220> <223> catalase-PEP-1 fusion protein <400> 12 His His His His His Ser Ser Gly Leu Val Pro Arg Gly Ser His   1 5 10 15 Leu Glu Met Ala Asp Ser Arg Asp Pro Ala Ser Asp Gln Met Gln His              20 25 30 Trp Lys Glu Gln Arg Ala Gln Lys Ala Asp Val Leu Thr Thr Gly          35 40 45 Ala Gly Asn Pro Val Gly Asp Lys Leu Asn Val Ile Thr Val Gly Pro      50 55 60 Arg Gly Pro Leu Leu Val Gln Asp Val Val Phe Thr Asp Glu Met Ala  65 70 75 80 His Phe Asp Arg Glu Arg Ile Pro Glu Arg Val Val His Ala Lys Gly                  85 90 95 Ala Gly Ala Phe Gly Tyr Phe Glu Val Thr His Asp Ile Thr Lys Tyr             100 105 110 Ser Lys Ala Lys Val Phe Glu His Ile Gly Lys Lys Thr Pro Ile Ala         115 120 125 Val Arg Phe Ser Thr Val Ala Gly Glu Ser Ser Ser Ser Ala Asp Thr Val     130 135 140 Arg Asp Pro Arg Gly Phe Ala Val Lys Phe Tyr Thr Glu Asp Gly Asn 145 150 155 160 Trp Asp Leu Val Gly Asn Asn Thr Pro Ile Phe Phe Ile Arg Asp Pro                 165 170 175 Ile Leu Phe Pro Ser Phe Ile His Ser Gln Lys Arg Asn Pro Gln Thr             180 185 190 His Leu Lys Asp Pro Asp Met Val Trp Asp Phe Trp Ser Leu Arg Pro         195 200 205 Glu Ser Leu His Gln Val Ser Phe Leu Phe Ser Asp Arg Gly Ile Pro     210 215 220 Asp Gly His Arg His Met Asn Gly Tyr Gly Ser His Thr Phe Lys Leu 225 230 235 240 Val Asn Ala Asn Gly Glu Ala Val Tyr Cys Lys Phe His Tyr Lys Thr                 245 250 255 Asp Gln Gly Ile Lys Asn Leu Ser Val Glu Asp Ala Ala Arg Leu Ser             260 265 270 Gln Glu Asp Pro Asp Tyr Gly Ile Arg Asp Leu Phe Asn Ala Ile Ala         275 280 285 Thr Gly Lys Tyr Pro Ser Trp Thr Phe Tyr Ile Gln Val Met Thr Phe     290 295 300 Asn Gln Ala Glu Thr Phe Pro Phe Asn Pro Phe Asp Leu Thr Lys Val 305 310 315 320 Trp Pro His Lys Asp Tyr Pro Leu Ile Pro Val Gly Lys Leu Val Leu                 325 330 335 Asn Arg Asn Pro Val Asn Tyr Phe Ala Glu Val Glu Gln Ile Ala Phe             340 345 350 Asp Pro Ser Asn Met Pro Pro Gly Ile Glu Ala Ser Pro Asp Lys Met         355 360 365 Leu Gln Gly Arg Leu Phe Ala Tyr Pro Asp Thr His Arg His His Leu     370 375 380 Gly Pro Asn Tyr Leu His Ile Pro Val Asn Cys Pro Tyr Arg Ala Arg 385 390 395 400 Val Ala Asn Tyr Gln Arg Asp Gly Pro Met Cys Met Gln Asp Asn Gln                 405 410 415 Gly Gly Ala Pro Asn Tyr Tyr Pro Asn Ser Phe Gly Ala Pro Glu Gln             420 425 430 Gln Pro Ser Ala Leu Glu His Ser Ile Gln Tyr Ser Gly Glu Val Arg         435 440 445 Arg Phe Asn Thr Ala Asn Asp Asp Asn Val Thr Gln Val Arg Ala Phe     450 455 460 Tyr Val Asn Val Leu Asn Glu Glu Gln Arg Lys Arg Leu Cys Glu Asn 465 470 475 480 Ile Ala Gly His Leu Lys Asp Ala Gln Ile Phe Ile Gln Lys Lys Ala                 485 490 495 Val Lys Asn Phe Thr Glu Val His Pro Asp Tyr Gly Ser His Ile Gln             500 505 510 Ala Leu Leu Asp Lys Tyr Asn Ala Glu Lys Pro Lys Asn Ala Ile His         515 520 525 Thr Phe Val Gln Ser Gly Ser His Leu Ala Ala Arg Glu Lys Ala Asn     530 535 540 Leu Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro 545 550 555 560 Lys Lys Lys Arg Lys Val                 565 <210> 13 <211> 1773 <212> DNA <213> Artificial Sequence <220> <223> coding sequence for PEP-1-catalase-PEP-1 fusion protein <400> 13 catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagccataa aagaaacctg 60 gtgggaaacc tggtggaccg aatggtctca gccgaaaaaa aaacgtaaag tgctcgagat 120 ggctgacagc cgggatcccg ccagcgacca gatgcagcac tggaaggagc agcgggccgc 180 gcagaaagct gatgtcctga ccactggagc tggtaaccca gtaggagaca aacttaatgt 240 tattacagta gggccccgtg ggccccttct tgttcaggat gtggttttca ctgatgaaat 300 ggctcatttt gaccgagaga gaattcctga gagagttgtg catgctaaag gagcaggggc 360 ctttggctac tttgaggtca cacatgacat taccaaatac tccaaggcaa aggtatttga 420 gcatattgga aagaagactc ccatcgcagt tcggttctcc actgttgctg gagaatcggg 480 ttcagctgac acagttcggg accctcgtgg gtttgcagtg aaattttaca cagaagatgg 540 taactgggat ctcgttggaa ataacacccc cattttcttc atcagggatc ccatattgtt 600 tccatctttt atccacagcc aaaagagaaa tcctcagaca catctgaagg atccggacat 660 ggtctgggac ttctggagcc tacgtcctga gtctctgcat caggtttctt tcttgttcag 720 tgatcggggg attccagatg gacatcgcca catgaatgga tatggatcac atactttcaa 780 gctggttaat gcaaatgggg aggcagttta ttgcaaattc cattataaga ctgaccaggg 840 catcaaaaac ctttctgttg aagatgcggc gagactttcc caggaagatc ctgactatgg 900 catccgggat ctttttaacg ccattgccac aggaaagtac ccctcctgga ctttttacat 960 ccaggtcatg acatttaatc aggcagaaac ttttccattt aatccattcg atctcaccaa 1020 ggtttggcct cacaaggact accctctcat cccagttggt aaactggtct taaaccggaa 1080 tccagttaat tactttgctg aggttgaaca gatagccttc gacccaagca acatgccacc 1140 tggcattgag gccagtcctg acaaaatgct tcagggccgc ctttttgcct atcctgacac 1200 tcaccgccat cgcctgggac ccaattatct tcatatacct gtgaactgtc cctaccgtgc 1260 tcgagtggcc aactaccagc gtgacggccc gatgtgcatg caggacaatc agggtggtgc 1320 tccaaattac taccccaaca gctttggtgc tccggaacaa cagccttctg ccctggagca 1380 cagcatccaa tattctggag aagtgcggag attcaacact gccaatgatg ataacgttac 1440 tcaggtgcgg gcattctatg tgaacgtgct gaatgaggaa cagaggaaac gtctgtgtga 1500 gaacattgcc ggccacctga aggatgcaca aattttcatc cagaagaaag cggtcaagaa 1560 cttcactgag gtccaccctg actacgggag ccacatccag gctcttctgg acaagtacaa 1620 tgctgagaag cctaagaatg cgattcacac ctttgtgcag tccggatctc acttggcggc 1680 aagggagaag gcaaatctgg gatcctaaaa gaaacctggt gggaaacctg gtggaccgaa 1740 tggtctcagc cgaaaaaaaa acgtaaagtg tag 1773 <210> 14 <211> 573 <212> PRT <213> Artificial Sequence <220> <223> PEP-1-catalase-PEP-1 fusion protein <400> 14 Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys   1 5 10 15 Lys Lys Arg Lys Val Leu Glu Met Ala Asp Ser Arg Asp Pro Ala Ser              20 25 30 Asp Gln Met Gln His Trp Lys Glu Gln Arg Ala Gln Lys Ala Asp          35 40 45 Val Leu Thr Thr Gly Ala Gly Asn Pro Val Gly Asp Lys Leu Asn Val      50 55 60 Ile Thr Val Gly Pro Arg Gly Pro Leu Leu Val Gln Asp Val Val Phe  65 70 75 80 Thr Asp Glu Met Ala His Phe Asp Arg Glu Arg Ile Pro Glu Arg Val                  85 90 95 Val His Ala Lys Gly Ala Gly Ala Phe Gly Tyr Phe Glu Val Thr His             100 105 110 Asp Ile Thr Lys Tyr Ser Lys Ala Lys Val Phe Glu His Ile Gly Lys         115 120 125 Lys Thr Pro Ile Ala Val Arg Phe Ser Thr Val Ala Gly Glu Ser Gly     130 135 140 Ser Ala Asp Thr Val Arg Asp Pro Arg Gly Phe Ala Val Lys Phe Tyr 145 150 155 160 Thr Glu Asp Gly Asn Trp Asp Leu Val Gly Asn Asn Thr Pro Ile Phe                 165 170 175 Phe Ile Arg Asp Pro Ile Leu Phe Pro Ser Phe Ile His Ser Gln Lys             180 185 190 Arg Asn Pro Gln Thr His Leu Lys Asp Pro Asp Met Val Trp Asp Phe         195 200 205 Trp Ser Leu Arg Pro Glu Ser Leu His Gln Val Ser Phe Leu Phe Ser     210 215 220 Asp Arg Gly Ile Pro Asp Gly His Arg His Met Asn Gly Tyr Gly Ser 225 230 235 240 His Thr Phe Lys Leu Val Asn Ala Asn Gly Glu Ala Val Tyr Cys Lys                 245 250 255 Phe His Tyr Lys Thr Asp Gln Gly Ile Lys Asn Leu Ser Val Glu Asp             260 265 270 Ala Ala Arg Leu Ser Gln Glu Asp Pro Asp Tyr Gly Ile Arg Asp Leu         275 280 285 Phe Asn Ala Ile Ala Thr Gly Lys Tyr Pro Ser Trp Thr Phe Tyr Ile     290 295 300 Gln Val Met Thr Phe Asn Gln Ala Glu Thr Phe Pro Phe Asn Pro Phe 305 310 315 320 Asp Leu Thr Lys Val Trp Pro His Lys Asp Tyr Pro Leu Ile Pro Val                 325 330 335 Gly Lys Leu Val Leu Asn Arg Asn Pro Val Asn Tyr Phe Ala Glu Val             340 345 350 Glu Gln Ile Ala Phe Asp Pro Ser Asn Met Pro Pro Gly Ile Glu Ala         355 360 365 Ser Pro Asp Lys Met Leu Gln Gly Arg Leu Phe Ala Tyr Pro Asp Thr     370 375 380 His Arg His Arg Leu Gly Pro Asn Tyr Leu His Ile Pro Val Asn Cys 385 390 395 400 Pro Tyr Arg Ala Arg Val Ala Asn Tyr Gln Arg Asp Gly Pro Met Cys                 405 410 415 Met Gln Asp Asn Gln Gly Gly Ala Pro Asn Tyr Tyr Pro Asn Ser Phe             420 425 430 Gly Ala Pro Glu Gln Gln Pro Ser Ala Leu Glu His Ser Ile Gln Tyr         435 440 445 Ser Gly Glu Val Arg Arg Phe Asn Thr Ala Asn Asp Asp Asn Val Thr     450 455 460 Gln Val Arg Ala Phe Tyr Val Asn Val Leu Asn Glu Glu Gln Arg Lys 465 470 475 480 Arg Leu Cys Glu Asn Ile Ala Gly His Leu Lys Asp Ala Gln Ile Phe                 485 490 495 Ile Gln Lys Lys Ala Val Lys Asn Phe Thr Glu Val His Pro Asp Tyr             500 505 510 Gly Ser His Ile Gln Ala Leu Leu Asp Lys Tyr Asn Ala Glu Lys Pro         515 520 525 Lys Asn Ala Ile His Thr Phe Val Gln Ser Gly Ser His Leu Ala Ala     530 535 540 Arg Glu Lys Ala Asn Leu Gly Ser Lys Glu Thr Trp Trp Glu Thr Trp 545 550 555 560 Trp Thr Glu Trp Ser Gln Pro Lys Lys Lys Arg Lys Val                 565 570 <210> 15 <211> 1664 <212> DNA <213> Artificial Sequence <220> <223> coding sequence for olygolysine-catalase fusion protein <400> 15 catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagccaaaa aaaaaaaaaa 60 aaaaaaaaaa aaaactcgag atggctgaca gccgggatcc cgccagcgac cagatgcagc 120 actggaagga gcagcgggcc gcgcagaaag ctgatgtcct gaccactgga gctggtaacc 180 cagtaggaga caaacttaat gttattacag tagggccccg tgggcccctt cttgttcagg 240 atgtggtttt cactgatgaa atggctcatt ttgaccgaga gagaattcct gagagagttg 300 tgcatgctaa aggagcaggg gcctttggct actttgaggt cacacatgac attaccaaat 360 actccaaggc aaaggtattt gagcatattg gaaagaagac tcccatcgca gttcggttct 420 ccactgttgc tggagaatcg ggttcagctg acacagttcg ggaccctcgt gggtttgcag 480 tgaaatttta cacagaagat ggtaactggg atctcgttgg aaataacacc cccattttct 540 tcatcaggga tcccatattg tttccatctt ttatccacag ccaaaagaga aatcctcaga 600 cacatctgaa ggatccggac atggtctggg acttctggag cctacgtcct gagtctctgc 660 atcaggtttc tttcttgttc agtgatcggg ggattccaga tggacatcgc cacatgaatg 720 gatatggatc acatactttc aagctggtta atgcaaatgg ggaggcagtt tattgcaaat 780 tccattataa gactgaccag ggcatcaaaa acctttctgt tgaagatgcg gcgagacttt 840 cccaggaaga tcctgactat ggcatccggg atctttttaa cgccattgcc acaggaaagt 900 acccctcctg gactttttac atccaggtca tgacatttaa tcaggcagaa acttttccat 960 ttaatccatt cgatctcacc aaggtttggc ctcacaagga ctaccctctc atcccagttg 1020 gtaaactggt cttaaaccgg aatccagtta attactttgc tgaggttgaa cagatagcct 1080 tcgacccaag caacatgcca cctggcattg aggccagtcc tgacaaaatg cttcagggcc 1140 gcctttttgc ctatcctgac actcaccgcc atcgcctggg acccaattat cttcatatac 1200 ctgtgaactg tccctaccgt gctcgagtgg ccaactacca gcgtgacggc ccgatgtgca 1260 tgcaggacaa tcagggtggt gctccaaatt actaccccaa cagctttggt gctccggaac 1320 aacagccttc tgccctggag cacagcatcc aatattctgg agaagtgcgg agattcaaca 1380 ctgccaatga tgataacgtt actcaggtgc gggcattcta tgtgaacgtg ctgaatgagg 1440 aacagaggaa acgtctgtgt gagaacattg ccggccacct gaaggatgca caaattttca 1500 tccagaagaa agcggtcaag aacttcactg aggtccaccc tgactacggg agccacatcc 1560 aggctcttct ggacaagtac aatgctgaga agcctaagaa tgcgattcac acctttgtgc 1620 agtccggatc tcacttggcg gcaagggaga aggcaaatct gtga 1664 <210> 16 <211> 538 <212> PRT <213> Artificial Sequence <220> <223> oligolysine-catalase fusion protein <400> 16 Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Leu Glu Met Ala Asp Ser Arg   1 5 10 15 Asp Pro Ala Ser Asp Gln Met Gln His Trp Lys Glu Gln Arg Ala Ala              20 25 30 Gln Lys Ala Asp Val Leu Thr Thr Gly Ala Gly Asn Pro Val Gly Asp          35 40 45 Lys Leu Asn Val Ile Thr Val Gly Pro Arg Gly Pro Leu Leu Val Gln      50 55 60 Asp Val Val Phe Thr Asp Glu Met Ala His Phe Asp Arg Glu Arg Ile  65 70 75 80 Pro Glu Arg Val Val His Ala Lys Gly Ala Gly Ala Phe Gly Tyr Phe                  85 90 95 Glu Val Thr His Asp Ile Thr Lys Tyr Ser Lys Ala Lys Val Phe Glu             100 105 110 His Ile Gly Lys Lys Thr Pro Ile Ala Val Arg Phe Ser Thr Val Ala         115 120 125 Gly Glu Ser Gly Ser Ala Asp Thr Val Arg Asp Pro Arg Gly Phe Ala     130 135 140 Val Lys Phe Tyr Thr Glu Asp Gly Asn Trp Asp Leu Val Gly Asn Asn 145 150 155 160 Thr Pro Ile Phe Phe Ile Arg Asp Pro Ile Leu Phe Pro Ser Phe Ile                 165 170 175 His Ser Gln Lys Arg Asn Pro Gln Thr His Leu Lys Asp Pro Asp Met             180 185 190 Val Trp Asp Phe Trp Ser Leu Arg Pro Glu Ser Leu His Gln Val Ser         195 200 205 Phe Leu Phe Ser Asp Arg Gly Ile Pro Asp Gly His Arg His Met Asn     210 215 220 Gly Tyr Gly Ser His Thr Phe Lys Leu Val Asn Ala Asn Gly Glu Ala 225 230 235 240 Val Tyr Cys Lys Phe His Tyr Lys Thr Asp Gln Gly Ile Lys Asn Leu                 245 250 255 Ser Val Glu Asp Ala Ala Arg Leu Ser Gln Glu Asp Pro Asp Tyr Gly             260 265 270 Ile Arg Asp Leu Phe Asn Ale Ile Ala Thr Gly Lys Tyr Pro Ser Trp         275 280 285 Thr Phe Tyr Ile Gln Val Met Thr Phe Asn Gln Ala Glu Thr Phe Pro     290 295 300 Phe Asn Pro Phe Asp Leu Thr Lys Val Trp Pro His Lys Asp Tyr Pro 305 310 315 320 Leu Ile Pro Val Gly Lys Leu Val Leu Asn Arg Asn Pro Val Asn Tyr                 325 330 335 Phe Ala Glu Val Glu Gln Ile Ala Phe Asp Pro Ser Asn Met Pro Pro             340 345 350 Gly Ile Glu Ala Ser Pro Asp Lys Met Leu Gln Gly Arg Leu Phe Ala         355 360 365 Tyr Pro Asp Thr His Arg His Arg Leu Gly Pro Asn Tyr Leu His Ile     370 375 380 Pro Val Asn Cys Pro Tyr Arg Ala Arg Val Ala Asn Tyr Gln Arg Asp 385 390 395 400 Gly Pro Met Cys Met Gln Asp Asn Gln Gly Gly Ala Pro Asn Tyr Tyr                 405 410 415 Pro Asn Ser Phe Gly Ala Pro Glu Gln Gln Pro Ser Ala Leu Glu His             420 425 430 Ser Ile Gln Tyr Ser Gly Glu Val Arg Arg Phe Asn Thr Ala Asn Asp         435 440 445 Asp Asn Val Thr Gln Val Arg Ala Phe Tyr Val Asn Val Leu Asn Glu     450 455 460 Glu Gln Arg Lys Arg Leu Cys Glu Asn Ile Ala Gly His Leu Lys Asp 465 470 475 480 Ala Gln Ile Phe Ile Gln Lys Lys Ala Val Lys Asn Phe Thr Glu Val                 485 490 495 His Pro Asp Tyr Gly Ser His Ile Gln Ala Leu Leu Asp Lys Tyr Asn             500 505 510 Ala Glu Lys Pro Lys Asn Ale Ile His Thr Phe Val Gln Ser Gly Ser         515 520 525 His Leu Ala Ala Arg Glu Lys Ala Asn Leu     530 535 <210> 17 <211> 1664 <212> DNA <213> Artificial Sequence <220> <223> coding sequence for oligoarginine-catalase fusion protein <400> 17 catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagccaaga agaagaagaa 60 gaagaagaag aagactcgag atggctgaca gccgggatcc cgccagcgac cagatgcagc 120 actggaagga gcagcgggcc gcgcagaaag ctgatgtcct gaccactgga gctggtaacc 180 cagtaggaga caaacttaat gttattacag tagggccccg tgggcccctt cttgttcagg 240 atgtggtttt cactgatgaa atggctcatt ttgaccgaga gagaattcct gagagagttg 300 tgcatgctaa aggagcaggg gcctttggct actttgaggt cacacatgac attaccaaat 360 actccaaggc aaaggtattt gagcatattg gaaagaagac tcccatcgca gttcggttct 420 ccactgttgc tggagaatcg ggttcagctg acacagttcg ggaccctcgt gggtttgcag 480 tgaaatttta cacagaagat ggtaactggg atctcgttgg aaataacacc cccattttct 540 tcatcaggga tcccatattg tttccatctt ttatccacag ccaaaagaga aatcctcaga 600 cacatctgaa ggatccggac atggtctggg acttctggag cctacgtcct gagtctctgc 660 atcaggtttc tttcttgttc agtgatcggg ggattccaga tggacatcgc cacatgaatg 720 gatatggatc acatactttc aagctggtta atgcaaatgg ggaggcagtt tattgcaaat 780 tccattataa gactgaccag ggcatcaaaa acctttctgt tgaagatgcg gcgagacttt 840 cccaggaaga tcctgactat ggcatccggg atctttttaa cgccattgcc acaggaaagt 900 acccctcctg gactttttac atccaggtca tgacatttaa tcaggcagaa acttttccat 960 ttaatccatt cgatctcacc aaggtttggc ctcacaagga ctaccctctc atcccagttg 1020 gtaaactggt cttaaaccgg aatccagtta attactttgc tgaggttgaa cagatagcct 1080 tcgacccaag caacatgcca cctggcattg aggccagtcc tgacaaaatg cttcagggcc 1140 gcctttttgc ctatcctgac actcaccgcc atcgcctggg acccaattat cttcatatac 1200 ctgtgaactg tccctaccgt gctcgagtgg ccaactacca gcgtgacggc ccgatgtgca 1260 tgcaggacaa tcagggtggt gctccaaatt actaccccaa cagctttggt gctccggaac 1320 aacagccttc tgccctggag cacagcatcc aatattctgg agaagtgcgg agattcaaca 1380 ctgccaatga tgataacgtt actcaggtgc gggcattcta tgtgaacgtg ctgaatgagg 1440 aacagaggaa acgtctgtgt gagaacattg ccggccacct gaaggatgca caaattttca 1500 tccagaagaa agcggtcaag aacttcactg aggtccaccc tgactacggg agccacatcc 1560 aggctcttct ggacaagtac aatgctgaga agcctaagaa tgcgattcac acctttgtgc 1620 agtccggatc tcacttggcg gcaagggaga aggcaaatct gtga 1664 <210> 18 <211> 538 <212> PRT <213> Artificial Sequence <220> <223> oligoarginine-catalase fusion protein <400> 18 Arg Arg Arg Arg Arg Arg Arg Arg Arg Leu Glu Met Ala Asp Ser Arg   1 5 10 15 Asp Pro Ala Ser Asp Gln Met Gln His Trp Lys Glu Gln Arg Ala Ala              20 25 30 Gln Lys Ala Asp Val Leu Thr Thr Gly Ala Gly Asn Pro Val Gly Asp          35 40 45 Lys Leu Asn Val Ile Thr Val Gly Pro Arg Gly Pro Leu Leu Val Gln      50 55 60 Asp Val Val Phe Thr Asp Glu Met Ala His Phe Asp Arg Glu Arg Ile  65 70 75 80 Pro Glu Arg Val Val His Ala Lys Gly Ala Gly Ala Phe Gly Tyr Phe                  85 90 95 Glu Val Thr His Asp Ile Thr Lys Tyr Ser Lys Ala Lys Val Phe Glu             100 105 110 His Ile Gly Lys Lys Thr Pro Ile Ala Val Arg Phe Ser Thr Val Ala         115 120 125 Gly Glu Ser Gly Ser Ala Asp Thr Val Arg Asp Pro Arg Gly Phe Ala     130 135 140 Val Lys Phe Tyr Thr Glu Asp Gly Asn Trp Asp Leu Val Gly Asn Asn 145 150 155 160 Thr Pro Ile Phe Phe Ile Arg Asp Pro Ile Leu Phe Pro Ser Phe Ile                 165 170 175 His Ser Gln Lys Arg Asn Pro Gln Thr His Leu Lys Asp Pro Asp Met             180 185 190 Val Trp Asp Phe Trp Ser Leu Arg Pro Glu Ser Leu His Gln Val Ser         195 200 205 Phe Leu Phe Ser Asp Arg Gly Ile Pro Asp Gly His Arg His Met Asn     210 215 220 Gly Tyr Gly Ser His Thr Phe Lys Leu Val Asn Ala Asn Gly Glu Ala 225 230 235 240 Val Tyr Cys Lys Phe His Tyr Lys Thr Asp Gln Gly Ile Lys Asn Leu                 245 250 255 Ser Val Glu Asp Ala Ala Arg Leu Ser Gln Glu Asp Pro Asp Tyr Gly             260 265 270 Ile Arg Asp Leu Phe Asn Ale Ile Ala Thr Gly Lys Tyr Pro Ser Trp         275 280 285 Thr Phe Tyr Ile Gln Val Met Thr Phe Asn Gln Ala Glu Thr Phe Pro     290 295 300 Phe Asn Pro Phe Asp Leu Thr Lys Val Trp Pro His Lys Asp Tyr Pro 305 310 315 320 Leu Ile Pro Val Gly Lys Leu Val Leu Asn Arg Asn Pro Val Asn Tyr                 325 330 335 Phe Ala Glu Val Glu Gln Ile Ala Phe Asp Pro Ser Asn Met Pro Pro             340 345 350 Gly Ile Glu Ala Ser Pro Asp Lys Met Leu Gln Gly Arg Leu Phe Ala         355 360 365 Tyr Pro Asp Thr His Arg His Arg Leu Gly Pro Asn Tyr Leu His Ile     370 375 380 Pro Val Asn Cys Pro Tyr Arg Ala Arg Val Ala Asn Tyr Gln Arg Asp 385 390 395 400 Gly Pro Met Cys Met Gln Asp Asn Gln Gly Gly Ala Pro Asn Tyr Tyr                 405 410 415 Pro Asn Ser Phe Gly Ala Pro Glu Gln Gln Pro Ser Ala Leu Glu His             420 425 430 Ser Ile Gln Tyr Ser Gly Glu Val Arg Arg Phe Asn Thr Ala Asn Asp         435 440 445 Asp Asn Val Thr Gln Val Arg Ala Phe Tyr Val Asn Val Leu Asn Glu     450 455 460 Glu Gln Arg Lys Arg Leu Cys Glu Asn Ile Ala Gly His Leu Lys Asp 465 470 475 480 Ala Gln Ile Phe Ile Gln Lys Lys Ala Val Lys Asn Phe Thr Glu Val                 485 490 495 His Pro Asp Tyr Gly Ser His Ile Gln Ala Leu Leu Asp Lys Tyr Asn             500 505 510 Ala Glu Lys Pro Lys Asn Ale Ile His Thr Phe Val Gln Ser Gly Ser         515 520 525 His Leu Ala Ala Arg Glu Lys Ala Asn Leu     530 535

Claims (3)

A pharmaceutical composition for the prevention and treatment of Parkinson's disease comprising a catalase fusion protein having a protein transport domain covalently bonded to one or more of the N-terminal and C-terminal of human catalase protein.
The method according to claim 1,
Wherein the protein transport domain is a PEP-1 peptide, wherein the protein transport domain is a PEP-1 peptide.
The method according to claim 1,
Wherein the fusion protein is SEQ ID NO. 16. 16. A pharmaceutical composition for preventing and treating Parkinson's disease comprising the fusion protein of &lt; RTI ID = 0.0 &gt;

Images