WO2012050402A2 - Cell-permeable recombinant parkin protein and a pharmaceutical composition for treating degenerative brain diseases containing the same - Google Patents

Abstract

The present invention relates to: a recombinant parkin protein having cell-permeable properties, wherein a macromolecule transduction domain (MTD) is fused to a degenerative-brain-disease therapeutic protein parkin; and a pharmaceutical composition or the like for treating degenerative brain diseases which contains the cell-permeable recombinant parkin protein as an active ingredient. For the recombinant parkin protein having cell-permeable properties of the present invention, a cell-permeable recombinant protein is produced in which a macromolecule transduction domain is fused to a parkin protein which is a dopamine neuron death suppression factor, and, because the same is endowed with the ability to be transmitted into cells, in use the protein is transmitted into dopamine neurons in in vitro or in vivo environments such that cell death is suppressed, or else there is a dopamine-secretion increasing effect whereby the protein can be used in studies which aim to treat Parkinson's disease, and the protein can be used to advantage as a therapeutic agent for human degenerative brain diseases.

Description

Cell-permeable PARKIN recombinant protein and pharmaceutical composition for treating degenerative brain disease containing the same

The present invention is for the treatment of degenerative brain disease containing Parkin recombinant protein fused with macromolecule transduction domain (MTD) to the degenerative brain disease therapeutic protein Parkin, and the cell permeable Parkin recombinant protein as an active ingredient. It relates to a pharmaceutical composition and the like.

Parkinson's disease was first reported by British physician James Parkinson in 1817, and is the second most common degenerative brain disease after dementia. It is known to affect about%. As the population ages, the number of patients also increases, but there is insufficient development of effective treatments due to insufficient studies. Characteristic clinical symptoms include movements such as bradykinesia, rest tremor, rigidity, flexed posture, freezing of gait, and postural instability. Symptoms, non-motor symptoms such as demen-tia, depression, anxiety, psychotic symptoms, autonomic neuropathy, and sleep disorders, and are characterized by loss of dopaminergic neurons in the middle brain and Lewy body findings. Pathological findings.

Parkinson's disease is reported to be caused by a combination of genetic and environmental factors. In particular, Parkinson's genetic factor is responsible for the high prevalence of Parkinson's genetic factors.

The Parkin gene was first discovered and named in Japanese families with Autosomal Recessive Juvenile Parkinsonism (ARJP), and (Kitada. T., 1998) early-onset Parkinson's disease with autosomal recessive genetics. 50% of the diseases are known to be caused by Parkin gene mutations (Lucking CB, 2000). Normal Parkin functions as an E3 Ubiquitin Ligase, which is important for the ubiquitin-proteasome system, and removes damaged, oxidized, or abnormally structured proteins in the cell to reduce intracellular stress. It serves to reduce. Degeneration of Parkin is known to result in the loss of E3 ubiquitin zygotease function, resulting in the accumulation of intracellular inclusion bodies and abnormal proteins, leading to inhibition of dopamine secretion or death of dopamine neurons (Lam YA, 2000). ).

In addition, Parkin is present in the mitochondrial matrix and is known to be involved in protecting mitochondrial degeneration. Parkin deficient mutant mice have been shown to be highly sensitive to mitochondrial degradation and increased oxidative stress (Greene JC, 2003). Palacino JJ J, 2004). In addition, Parkin gene overexpression has been reported to inhibit the activity of JNK and caspase3 in neurons by 6-OHDA and decrease the activation oxygen (Jiang et al., 2004). In a recent study using fruit flies, Parkin's function was identified, along with another cause of Parkinson's disease, PINK1. Parkinson's dysfunction was a decrease in dopamine secretion due to inactivation, rather than death of dopamine neurons. Dopamine secretion was restored to normal levels and the motor function was restored (Park J. et al. 2006). In addition, it has been reported that Parkinson's disease-related symptoms caused by PINK1, such as mitochondria abnormality, dopamine neuron degeneration, and muscle cell death, can be recovered when Parkin is overexpressed in Drosophila without PINK1 expression. et al., 2006; Yang et al., 2006; Clark et al., 2006).

Many studies have confirmed that Parkin may be used as a therapeutic agent for Parkinson's disease. Parkin-lentivirus was administered to Parkinson's animal models in which neurons were damaged by alpha-synuclein mutations. As a result, 60% inhibition of dopamine-secreting neurons in black matter was suppressed (Bianco et al., 2004), and 50% inhibition of dopamine neuron death by Parkin-lentivirus in Parkinson's disease animal model using 6-OHDA. And allergic disorders were alleviated (Vercammen et al., 2006).

Based on these findings, intracellular high doses of Parkin protein play an important role in destroying inclusion bodies as key enzymes of the ubiquitin-proteasome pathway, maintaining mitochondrial function from oxidative stress, and inhibiting dopamine neuron death. It can be assumed that by inhibiting the dopamine neurons death caused by genetic causes and environmental toxic substances to prevent dopamine neurons degeneration and reactivation. Accordingly, the present inventors have made a thorough study because it is considered that Parkin is of sufficient value as a target material for the development of a therapeutic agent for Parkinson's disease known as degenerative brain disease.

However, unlike small molecules of synthetic compounds or natural products, macromolecules such as proteins, peptides, antibodies, nucleic acids and the like cannot pass through the double lipid membrane structure or plasma membrane and cannot be transferred into cells. In particular, bioactive recombinant proteins, gene therapies, and monoclonal antibodies are unable to pass through the dilipid layer of the plasma membrane of the cell, but also through the blood-brain barrier, an important pathway to the brain. none. Parkin, too, has applied the previously developed macromolecule intracellular transduction technology (MITT) as a way to overcome this problem (JO D.W., 2001).

Accordingly, the present inventors have prepared a Parkin recombinant protein that has been imparted with cell permeability by fusion of a macromolecular delivery domain to Parkin, and the recombinant protein is effectively delivered into neurons not only in vitro but also in an in vivo environment, thereby causing a malfunction of a specific protein or It has been confirmed that the present invention can be used as a therapeutic agent for degenerative brain diseases due to deficiency, and thus has been completed.

An object of the present invention is to impart cell permeability to the treatment factor for degenerative brain disease, and to introduce it into the cell with high efficiency, thereby inhibiting dopamine neuron death or increasing the release of dopamine. It is to provide a recombinant protein.

The present invention provides a cell permeable parkin recombinant protein in which a macromolecular transduction domain (MTD) is fused with a parkin having an amino acid sequence of SEQ ID NO: 1 and an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 to 195. to provide.

In one embodiment of the invention, the recombinant protein is characterized in that it has an amino acid sequence selected from the group consisting of SEQ ID NO: 394 to SEQ ID NO: 403.

In another embodiment of the present invention, the recombinant protein is characterized in that it is prepared using the primers described in the table below.

The present invention also provides a polynucleotide encoding the cell permeable parkin recombinant protein.

In one embodiment of the present invention, the polynucleotide is characterized in that it has a base sequence selected from the group consisting of SEQ ID NO: 409 to SEQ ID NO: 418.

The present invention also provides a recombinant expression vector comprising the polynucleotide.

In addition, the present invention is a fusion of His-parkin having an amino acid sequence of SEQ ID NO: 389 and macromolecular transduction domain (MTD) having an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 15, 103 and 105 Provides a cell permeable His-parkin recombinant protein.

In one embodiment of the invention, the recombinant protein is characterized in that it has an amino acid sequence selected from the group consisting of SEQ ID NO: 390 to SEQ ID NO: 393.

In another embodiment of the present invention, the recombinant protein is characterized in that it is prepared using the primers described in the table below.

The present invention also provides a polynucleotide encoding the cell-permeable His-parkin recombinant protein.

In one embodiment of the invention, the polynucleotide is characterized in that it has a base sequence selected from the group consisting of SEQ ID NO: 405 to SEQ ID NO: 408.

The present invention also provides a recombinant expression vector comprising the polynucleotide.

The present invention also provides a pharmaceutical composition for treating degenerative brain disease containing the cell permeable parkin recombinant protein as an active ingredient.

In one embodiment of the invention, the degenerative brain disease is characterized in that Parkinson's disease.

Parkin recombination protein having a cell permeability of the present invention is a cell-permeable recombinant protein fused with a macromolecular transport domain to Parkin protein, a dopamine neuronal cell death inhibitory factor, in vitro or in vivo using the protein It can be used for the study of Parkinson's disease through the effect of inhibiting apoptosis by transferring into dopamine neurons in vivo) or increasing dopamine secretion, and is expected to be useful as a therapeutic agent for human degenerative brain diseases. .

1 shows a vector designed for expressing Histidine (His) -tag fusion Parkin recombinant protein.

Figure 2 shows a schematic diagram of the combined MTD and His-tag fusion recombinant human full-length Parkin gene.

Figure 3 shows the SDS-PAGE results of purified His-tag fusion Parkin recombinant protein.

Figure 4 shows the flow cytometry of the His-tag fusion Parkin recombinant protein (HPM01 or HPM13) combined with JO-01 MTD or JO-13 MTD and His-tag fusion Parkin recombinant protein (HP) without MTD. Flow cytometry results are shown.

5 shows the results of observing the visible cell permeability of HPM01 or HPM13 and HP by confocal fluorescence microscopy.

Figure 6 shows the results of comparing the HPM13 and HP mouse blood brain barrier permeability (A. Tissue immunoassay results; B. Analysis using Western blotting).

Figure 7 shows the results of TUNEL analysis showing the protective effect of HPM13 and HP on 6-OHDA induced apoptosis of mouse neurons.

Figure 8 shows the results of quantifying the dopamine secretion effect of HPM13 and HP in mouse dopamine neurons by ELISA.

9 shows His-tag fusion Parkin with JO-10, JO-13, JO-29, JO-78 JO-85, JO-103, JO-130, JO-135, JO-151, JO-174 MTD Shown are vectors designed for expression of recombinant proteins (HPM10, HPM13, HPM29, HPM78, HPM85, HPM103, HPM130, HPM135, HPM151, HPM174).

10 shows His-tag fusion Parkin with JO-10, JO-13, JO-29, JO-78 JO-85, JO-103, JO-130, JO-135, JO-151, JO-174 MTD Expression of recombinant proteins (HPM10, HPM13, HPM29, HPM78, HPM85, HPM103, HPM130, HPM135, HPM151, HPM174) is shown by SDS-PAGE.

Figure 11 shows a vector designed for expression of His-tag non-fusion Parkin recombinant protein.

12 shows a schematic diagram of a combination of MTD and His-tag non-fusion recombinant human full-length Parkin gene.

FIG. 13 shows Parkin recombinant protein HP, HPM13 or unbound PN (His-tag non-fusion Parkin) with JO-10, JO-13, JO-151, JO-174 MTD bound (PNM10, PNM13, PNM151, PNM174) The results of comparing the expression of recombinant proteins are shown.

Figure 14 shows the results of SDS-PAGE analysis after purification of PNM10, PNM13, PNM151, PNM174.

Figure 15 shows the results of comparing the E3 ligase activity of PNM10, PNM13, PNM151, PNM174 with His6-Parkin protein (control).

Figure 16 shows the results of comparing the protective effect of PNM10 against 6-OHDA induced apoptosis of SH-SY5Y neurons.

Figure 17 shows the mouse blood brain barrier permeability results of PNM10 (A. Tissue immunoassay results; B. Analysis using Western blotting).

Figure 18 shows the neurotoxic effect of PNM10 in the MPTP-induced Parkinson's disease animal model (histoimmunochemical labeling results for A. Tyrosin hydroxylase (TH); B. dopamine quantified by ELISA).

The present invention provides a cell permeability through the fusion of Parkin, a dopamine neuronal cell death suppressor, and a macromolecular transduction domain (MTD), thereby introducing a cell-permeable Parkin recombinant protein (CP-Parkin) which introduces Parkin into cells with high efficiency. Provided are polynucleotides that encode them.

A feature of the present invention is to deliver Parkin into cells with high efficiency by converging specific macromolecular delivery domains (hereinafter abbreviated as "MTD") to Parkin, a macromolecule that is not readily introduced into cells. . At this time, the macromolecular delivery domain may be fused only to one end of Parkin, a dopamine neuron cell death inhibitor, or to both ends thereof.

In the present invention, as a macromolecular delivery domain (MTD) that can be fused to Parkin, a dopamine neuronal cell death inhibitor, the Parkin recombinant protein having cell permeability by fusion of Parkin to each of the peptide domains that enables the delivery of macromolecules into cells Developed.

In the present invention, "cell permeable Parkin recombinant protein" includes a macromolecular delivery domain and Parkin, a dopamine neuronal cell death inhibitor, and refers to a covalent complex formed by genetic fusion or chemical bonding thereof. By "genetic fusion" is meant a linear covalent linkage formed through the genetic expression of a DNA sequence encoding a protein.

As a macromolecular delivery domain that can be fused to the neuronal cell death inhibitor Parkin, a polypeptide having a cell permeability including an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 to 195 may be used. The macromolecular delivery domain is a cell permeable polypeptide that can mediate the entry of a polypeptide, protein domain, or biologically active molecule comprising a full length protein into a cell through a cell membrane. The macromolecular delivery domain according to the present invention forms helices in signal peptides consisting of three parts of the N-terminal region, the hydrophobic region and the C-terminal secreted protein cleavage site. It is designed to have a hydrophobic region that confers targeting activity. These macromolecular delivery domains can directly penetrate the cell membrane without damaging the cells, allowing the target protein to move into the cell to exert its desired function.

The macromolecular delivery domains that can be fused to Parkin, a neuronal cell death inhibitor, according to the present invention are shown in Table 1 below.

Table 1 Macromolecular Delivery Domains (MTDs)

In one embodiment of the present invention, Parkin recombinant protein having cell permeability is a macromolecular delivery domain of 12 MTDs (JO-01, JO-10, JO-13, JO-29, JO-78, JO-85, JO- 101, JO-103, JO-130, JO-135, JO-151, and JO-174) are fused to one or both ends of the apoptosis inhibitor Parkin, and easily to one end of this fusion construct. For one purification, the histidine-tag (His-Tag) affinity domain may be fused.

In one embodiment of the present invention, the 12 types of MTD (JO-01, JO-10, JO-13, JO-29, JO-78, JO-85, JO-101, JO-103, JO-130, Full-length forms can be devised as Parkin recombinant proteins using any one of JO-135, JO-151 and JO-174.

As used herein, the term “full length form” refers to a form comprising an intact amino acid sequence that does not include the deletion, addition, insertion or substitution of one or more amino acid residues in the amino acid sequence set forth in SEQ ID NO: 1 of the apoptosis inhibitor Parkin. it means. However, not only full-length Parkin, but also Parkin derivatives containing various modifications by deletion, addition, insertion or substitution of one or more amino acid residues in its amino acid sequence within the range not impairing the killing inhibitory effect of Parkin's dopamine secreting cells It can be used in the present invention.

In the present invention, as a control of the cell permeable Parkin recombinant protein Parkin (PN) that is not fused MTD can be used. The control protein has an amino acid sequence of SEQ ID NO: 1, which may be encoded by a polynucleotide having a nucleotide sequence of SEQ ID NO: 2.

In addition, in the present invention, His-Parkin (HP) in which a histitin label is fused to one end of the cell permeable Parkin recombinant protein is not fused to MTD. The control protein has an amino acid sequence of SEQ ID NO: 389, which may be encoded by a polynucleotide having a nucleotide sequence of SEQ ID NO: 404.

The present invention also provides a recombinant expression vector comprising a polynucleotide encoding the cell-permeable Parkin recombinant protein.

As used herein, a "recombinant expression vector" refers to a gene construct that is capable of expressing a protein of interest or RNA of interest in a suitable host cell and comprises an essential regulatory element operably linked to express the gene insert.

As used herein, the term "operably linked" refers to a functional linkage of a nucleic acid expression control sequence and a nucleic acid sequence encoding a protein or RNA of interest to perform a general function. For example, a promoter and a nucleic acid sequence encoding a protein or RNA may be operably linked to affect expression of the nucleic acid sequence encoding. Operative linkage with recombinant expression vectors can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation uses enzymes commonly known in the art.

Expression vectors usable in the present invention include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, viral vectors, and the like. Suitable expression vectors include membrane targeting or in addition to expression control sequences such as promoters, operators, initiation codons, termination codons, polyadenylation signals, and enhancers. It may be prepared in various ways according to the purpose, including a signal sequence (leader sequence) or a signal sequence for secretion. The promoter of the expression vector may be constitutive or inducible. In addition, the expression vector includes a selection marker for selecting a host cell containing the vector, and in the case of an expression vector capable of replication, includes a replication origin.

In one embodiment of the present invention, His-Tag is expressed by artificially including six histidine-tags in the N-terminal region of the cell-permeable Parkin recombinant protein for the purpose of facilitating purification of the protein. The nucleotide of the present invention can be cloned into a pET-28a (+) vector having a sequence (Novagen, USA).

The present invention also provides a pharmaceutical composition comprising the cell permeable parkin recombinant protein as an active ingredient. The composition may further comprise a pharmaceutically acceptable carrier, such as a carrier for oral administration or a carrier for parenteral administration. Carriers for oral administration include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid and the like. For oral administration, the recombinant protein according to the invention can be mixed with excipients and used in the form of intake tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups and wafers. In addition, carriers for parenteral administration include water, suitable oils, saline, aqueous glucose and glycols, and the like, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-parabens and chlorobutanol. Other pharmaceutically acceptable carriers may be used by reference to those described in the following references (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

The compositions according to the invention can be formulated in a variety of parenteral or oral dosage forms. Representative of parenteral formulations are injectable formulations, preferably aqueous isotonic solutions or suspensions. Injectable formulations may be prepared according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. For example, each component may be formulated for injection by dissolving in saline or buffer. In addition, oral dosage forms include, for example, tablets and capsules, which include diluents (e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine) and glidants (in addition to the active ingredients). Such as silica, talc, stearic acid and its magnesium or calcium salts and / or polyethylene glycols). The tablets may comprise binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine, optionally starch, agar, alginic acid or Disintegrants such as sodium salts, absorbents, colorants, flavors and / or sweeteners may be further included. The formulations may be prepared by conventional mixing, granulating or coating methods.

Compositions of the present invention may further comprise auxiliaries such as preservatives, hydrating agents, emulsifiers, salts for regulating osmotic pressure and / or buffers and other therapeutically useful substances, and may be formulated according to conventional methods.

In addition, the route of administration of the composition according to the present invention may be administered to humans and animals orally or parenterally, such as intravenous, subcutaneous, intranasal or intraperitoneal. Oral administration also includes sublingual application. Parenteral administration includes injection and drip methods such as subcutaneous injection, intramuscular injection and intravenous injection.

In the composition of the present invention, the total effective amount of the recombinant protein of the present invention may be administered to a patient in a single dose, and the fractionated treatment protocol in which multiple doses are administered for a long time. It may also be administered by. The composition of the present invention may vary the content of the active ingredient depending on the extent of the disease, but can be repeatedly administered several times a day at an effective dosage of 5 to 20 mg once a single administration based on adults. However, the effective dose of the recombinant protein may be determined in consideration of various factors such as the age, weight, health condition, sex, severity of the disease, diet and excretion rate, as well as the route and frequency of treatment of the drug. . In view of this, one of ordinary skill in the art would then be able to determine the appropriate effective dosage for the particular use of the recombinant protein as a cancer metastasis inhibitor. The composition according to the present invention is not particularly limited to the formulation, route of administration and method of administration as long as the effect of the present invention is shown.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention more specifically, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention.

EXAMPLE

Example 1 Preparation of Histidine (His) -tag Fusion Cell Permeable Parkin Recombinant Protein Expression Vector

In order to facilitate protein purification, first, an expression vector was produced to produce cell-permeable Parkin recombinant protein fused with His-tag. 4 randomly selected JO-01, JO-13, JO-101, JO-103 (SEQ. ID. NO .: 3, 15, 103, 105) MTD at the full-length Parkin gene C-terminus Amplified the gene.

Table 2 primer set

SEQ. SEQ ID NO. ID. NO .: 424 (forward) and SEQ. ID. NO .: Human full length Parkin gene using 425 (reverse) primer pair, SEQ. ID. NO .: 424 (forward) and SEQ. ID. NO .: 426 (reverse) primer pair was used for the human full-length Parkin-MTD ₀₁ gene, SEQ. ID. NO .: 424 (forward) and SEQ. ID. NO .: 427 (reverse) primer pair was used for the human full-length Parkin-MTD ₁₃ gene, SEQ. ID. NO .: 424 (forward) and SEQ. ID. NO .: 428 (reverse) primer pair was used for the full length Parkin-MTD ₁₀₁ gene, SEQ. ID. NO .: 424 (forward) and SEQ. ID. Human full-length Parkin-MTD ₁₀₃ gene was amplified using polymerase chain reaction (PCR) using NO .: 429 (reverse) primer pair. The polymerase chain reaction product obtained above was cloned into the pGEM-Tease vector, and then subcloned to the NdeI position of PET-28 (+) a (Novagen), which is a His-tag expression vector, at the N-terminus. His-tag allows the MT-D gene to be fused at the C-terminus and ultimately His-Parkin-MTD ₀₁ (HPM ₁ ), His-Parkin-MTD ₁₃ (HPM ₁₃ ), His-Parkin-MTD ₁₀₁ (HPM ₁₀₁ ), His-Parkin-MTD ₁₀₃ (HPM ₁₀₃ ) Parkin recombinant protein was prepared to express, and the results are shown in Figures 1 and 2. CDNA base sequence for the protein expression is SEQ. ID. NO.:404 (His-Parkin), SEQ. ID. NO .: 405 (His-Parkin-MTD ₀₁ ), SEQ. ID. NO .: 406 (His-Parkin-MTD ₁₃ ), SEQ. ID. NO .: 407 (His-Parkin-MTD ₁₀₁ ), SEQ. ID. NO .: 408 (His-Parkin-MTD ₁₀₃ ), and the amino acid sequence of the expressed His-tag Parkin recombinant protein is shown in SEQ. ID. NO .: 389 (His-Parkin), SEQ. ID. NO .: 390 (His-Parkin-MTD ₀₁ ), SEQ. ID. NO .: 391 (His-Parkin-MTD ₁₃ ), SEQ. ID. NO .: 392 (His-Parkin-MTD ₁₀₁ ), SEQ. ID. NO .: 393 (His-Parkin-MTD ₁₀₃ ).

Example 2. Overexpression and Purification of His-tag Fusion Cell Permeable Parkin Recombinant Protein

E. coli BL21 Codon Plus (DE3) (Invitrogen) with a LacI promoter was used to express the cell permeable Parkin recombinant protein, and after transformation, E. coli was 50 ug / mL kanamycine in 500 mL LB. After inoculation into the added culture, when E. coli reached 0.5 at A ₆₀₀ , it was overexpressed with 0.5 mM IPTG (Isopropyl β-D-1-thiogalactopyranoside) for 2 hours. All four cell-permeable Parkin recombinant proteins were expressed as inclusion bodies. Among them, Parkin recombinant proteins (HPM ₁ , HPM ₁₃ ) and JO-01 and JO-13 MTD were combined in consideration of their expression and physical properties. Purification was performed by selecting Parkin recombinant protein (HP) that did not bind MTD. To purify this, 8 M urea, a potent denaturant, was used to loosen the protein structure. First, the E. coli harvested by centrifugation of the culture was suspended in 20 mL lysis buffer (100 mM NaH ₂ PO ₄ , 10 mM Tris-HCl, 8 M urea, pH 8.0), which was then equipped with a microtip. E. coli was crushed by repeating the 30 second-ON and 10 second-OFF conditions for 50 minutes on ice using an ultrasonic crusher. The supernatant was collected by centrifugation at 3,000 rpm at 4 ° C. for 25 minutes and combined with Ni ²⁺ -NTA agarose resin, followed by centrifugation at 1,000 rpm for 5 minutes at 4 ° C. Removed. To remove the nonspecific adsorbate, it was washed five times with 100 mM NaH ₂ PO ₄ , 10 mM Tris-HCl, 8 M urea, pH 6.3 buffer. Finally, fractions containing recombinant Parkin protein were collected with resin 2 times elution buffer (100 mM NaH ₂ PO ₄ , 10 mM Tris-HCl, 8 M urea, pH 4.5) to confirm purification by 10% SDS-PAGE. The results are shown in FIG. 3. As shown in Figure 3, it can be seen that the cell-permeable Parkin recombinant protein HP, HPM ₁ , HPM ₁₃ each was purified in a single band, the final protein purified amount was obtained at 9 mg / L.

Example 3 Refolding of His-tag Fusion Cell Permeable Parkin Recombinant Protein

Purified His-tag fusion cell permeable recombinant Parkin protein is denatured by urea to form a released structure and must be refolded. Thus, purified recombinant protein was refolded in buffer (0.55 M Guanidine-HCl, 0.44 M L-arginine, 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 100 mM NDSB, 2 mM oxidized glutathione, 0.2 mM reduction). Urea was removed by dialysis at 4 ° C. for 24 hours using glutathione), and cell permeability and in vitro functional tests were performed on stable water-soluble cell permeable Parkin recombinant protein (HPM ₁ , HPM ₁₃ , HP).

Example 4 In Vitro of His-tag Fusion Cell Permeable Parkin Protein ( in vitro Cell permeability test

To confirm in vitro cell permeability, purified Parkin recombinant protein was dialyzed in Dulbecco's Modified Eagle Medium (DMEM), a cell culture medium, for 4 hours at 4 ° C., followed by the fluorescent material FITC (fluorescein-5-isothiocyanate, Molecular Probe). 333 ug was used to bind the protein while shaking for 1 hour at room temperature with little light. Cell permeable recombinant Parkin protein labeled with FITC fluorescence was dialyzed in DMEM medium at 4 ° C. for 1 day to remove unlabeled FITC.

In order to confirm the cell permeability of the cell-permeable Parkin recombinant protein, 10 μM HPM ₁ , HPM ₁₃ , HP were injected into NIH3T3 cells (Korea Cell Line Bank, Seoul, Korea) derived from fibroblast connective tissue cells of mice. Of recombinant protein was treated and incubated for 1 hour, followed by flow cytometry. Culture conditions were incubated in DMEM medium containing 500 mg / mL of 10% FBS (fetal bovine serum) and 5% penicillin / streptomycin (penicillin / streptomycin). After 1 hour, trypsin was treated to remove free FITC, washed three times with cold PBS, and cell permeability was measured with a flow cytometer equipped with FACS Calibur (Beckton-Dickinson) CellQuesPro software. The results are shown in FIG. 4.

As shown in FIG. 4, the cell permeable Parkin recombinant protein (HPM ₁ , HPM ₁₃ ) in which JO-01 MTD and JO-13 MTD are combined is compared to the cell permeable Parkin recombinant protein (HP) without MTD binding. It can be seen that the permeability is high.

In order to observe cell permeability by confocal fluorescence microscopy, purified HPM ₁ , HPM13, HP Parkin recombinant protein was treated with NIH3T3 cells (Korea Cell Line Bank, Seoul, Korea) at a concentration of 10 uM and incubated at 37 ° C. for 1 hour. . NIH3T3 cells were cultured in DMEM medium containing 10% FBS, 5% penicillin and streptomycin. After incubation, in order to preserve the fluorescent label of the recombinant protein, 10 uL of mounting media was deposited on the slide, and after 15 minutes, it was observed under a confocal fluorescence microscope (Nikon). Cells treated with the recombinant protein could be distinguished and observed for protein permeability in cytosol by staining nuclei with propidium idodide (PI) in order to facilitate differentiation of intracellular delivery sites of MTD fusion proteins. The shape of the cells was confirmed using a filter and the results are shown in FIG. 5. As shown in FIG. 5, MTD-coupled cell permeable Parkin recombinant protein (HPM ₁ , HPM ₁₃ ) has a significantly higher cell permeability compared to Parkin recombinant protein (HP) without MTD-binding. have.

Example 5 Brain-blood barrier permeability test of His-tag fusion cell permeable Parkin protein

In order to confirm blood brain barrier permeability using immunohistochemistry, Parkin recombinant protein (HPM ₁₃ ) with 200 µg MTD and Parkin recombinant protein with MTD was synthesized in 11-week-old Balb / c mice. 2 hours and 4 hours after subcutaneous injection of HP), the mouse brain was extracted. After fixing the tissue in 4% paraformaldehyde for more than 24 hours, the slide was prepared by frozen sectioning the tissue to 5㎛ thickness. After fixing the sections for 10 minutes using cold acetone, the sections were washed with PBS buffer for 5 minutes and the frozen sections were reacted with 0.2% hydrogen peroxide for 30 minutes. Sections were washed in PBS buffer solution for 5 minutes, then blocked in PBS buffer solution containing 3% normal horse serum for 30 minutes, and then Anti-His mAb (1: 500) and Anti-Parkin mAb (1: 500) The reaction was reacted overnight at 4 ° C. After washing the sections in PBS buffer solution for 5 minutes, reacted with biotin fusion anti-mouse antibody (1: 200, Zymed) for 1 hour, followed by reaction for 30 minutes using ABC kit (Vectastain), Sections were washed in PBS buffer for 5 minutes. After color reaction with DAB (3,3'-Diaminobenzidine) solution, staining was observed under an optical microscope, and the results are shown in FIG. 6A.

As shown in FIG. 6A, positive immunostaining was observed in the brains of the Parkin recombinant protein HPM ₁₃ fused with JO-13 MTD, whereas it was not observed in the mice brains of HP, the Parkin recombinant protein, to which MTD was not bound. Did. From the above, it can be seen that Parkin recombinant protein coupled with MTD has blood brain barrier permeability.

Next, Western blotting using brain crushing fluid was performed to confirm blood brain barrier permeability. Balb / c mice (8 weeks old, females) were injected subcutaneously with 200 μg protein and brain was extracted 2 hours later. After removing residual blood flow with PBS buffer, brain with RIPA buffer (50 mM Tris-HCl, pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, protease inhibitor) Was homogenized. After centrifugation at 13,000 rpm for 10 minutes at 4 ° C, the supernatant was taken, followed by 10% SDS-PAGE. For Western blotting analysis, Parkin antibody (mouse mAb Parkin, 1/2000) and GAPDH (mouse monoclonal GAPDH, 1/5000) were used as primary antibodies, and Goat-anti-mouse IgG-HRP was used as secondary antibody. It was. After blocking for 1 hour at 4 ° C. with 5% skim milk, primary antibodies were added and reacted overnight at 4 ° C. After washing with TBS-T (10 mM Tris-HCl, pH 8.0, 50 mM NaCl, 0.05% tween-20), the secondary antibody was treated for 1 hour, washed with TBS-T, and then ECL for chemiluminescence detection. Observed and analyzed using (Enhanced Chemiluminescence), the results are shown in Figure 6B.

As shown in FIG. 6B, it can be seen that ParkM antibody positive band was observed to be about twice as thick as HPM ₁₃ compared with HP, indicating that MTD-coupled Parkin recombinant protein has blood-brain barrier permeability.

Example 6 In Vitro of His-tag Fusion Cell Permeable Parkin Protein ( in vitro ) Neuronal cell death (Apoptosis) protective effect test

In order to confirm the in vitro function of the cell permeable Parkin recombinant protein, TUNEL assay was performed to confirm the protective effect against neuronal cell death by the neurotoxin 6-hydroxydopamine (6-OHDA). CATHa, a mouse dopamine producing neuron, was cultured in DMEM media containing 10% fetal bovine serum, 5% penicillin and streptomycin, and treated with 6-OHDA at a concentration of 50 uM for 30 minutes. Afterward, JO-13 MTD-coupled Parkin recombinant protein (HPM ₁₃ ) and MTD-coupled Parkin recombinant protein (HP) were each added 2.5 uM and incubated at 37 ° C. for 2 hours and 30 minutes. After the culture was removed, the terminal dUTP nick-end labeling (TUNEL) analysis was performed using an In situ cell death detection kit (Roche), and then observed with a confocal fluorescence microscope (Nikon), and a Glomax (Amersham) instrument. Fluorescence intensity was measured, and the results are shown in FIG. 7.

As shown in FIG. 7, the 6-OHDA-only group was observed to kill a large number of cells, whereas HPM ₁₃ , a Parkin recombinant protein with JO-13 MTD, was observed to kill dopamine neurons by 6-OHDA. Was inhibited by 85%, and a very small number of apoptosis was observed in fluorescence microscopy. On the other hand, similar results to the group treated with 6-OHDA were observed by HP, a Parkin recombinant protein without MTD binding.

From the above, it can be seen that the MTD-coupled cell permeable Parkin recombinant protein is transferred into the dopamine neurons to show the dopamine neuron protective effect.

Example 7 In Vitro Dopamine Secretion Testing of His-tag Fusion Cell Permeable Parkin Protein

In order to confirm the in vitro dopamine secretion effect of the cell-permeable Parkin recombinant protein, mouse dopamine neurons CATHa (Korea Cell Line Bank, Seoul, South Korea) was used. CATHa cells were cultured in DMEM medium containing 10% FBS, 5% penicillin / streptomycin, and dopamine quantification was performed according to the method of GenWay from dopamine ELISA supplier. After 24 h of tyrosine treatment to CATHa cells, HPM ₁₃ recombinant protein fused with JO-13 MTD and recombinant HP protein without MTD were administered to dopamine neuron culture at 2.5 uM, respectively. After 1, 3, 5 hours, the dopamine concentration in the medium was quantified by ELISA (GenWay), and the results are shown in FIG. 8. As shown in FIG. 8, after 3 hours, the dopamine content was increased by 366% or more in the culture solution in which HPM ₁₃ was administered compared to HP protein, and after 5 hours, HPM ₁₃ was dopamine compared to Parkin protein without MTD fusion. The content was significantly increased. Therefore, it can be seen that the HPM ₁₃ recombinant protein fused with MTD is transferred into dopamine neurons to increase dopamine secretion ability.

Example 8 Expression Vector Preparation for Optimal MTD Selection

To select the optimal MTD for non-clinical testing by pre-investigating the correlation between Parkin recombinant protein and physical properties, 10 candidate MTDs (JO-10, JO-13, JO-29, JO-78, JO-85, JO-103, JO-130, JO-135, JO-151, JO-174) (SEQ.ID.NO .: 205, 208, 224, 273, 280, 298, 325, 330, 346, 369) was constructed with a recombinant protein expression vector fused with a human full-length Parkin gene.

Table 3 Primer set

SEQ. SEQ ID NO. Described in Table 3 above. ID. NO .: 430 (forward) and SEQ. ID. NO .: 431 (reverse) primer pair was used for the full length Parkin gene, SEQ. ID. NO .: 430 (forward) and SEQ. ID. NO .: 441 (reverse) primer pair was used for the full length Parkin-MTD ₁₀ gene, SEQ. ID. NO .: 430 (forward) and SEQ. ID. NO .: 444 (reverse) primer pair was used for the full length Parkin-MTD ₁₃ gene, SEQ. ID. NO .: 430 (forward) and SEQ. ID. NO .: Human full-length Parkin-MTD ₂₉ gene using 460 (reverse) primer pair, SEQ. ID. NO .: 430 (forward) and SEQ. ID. NO .: 509 (reverse) primer pair was used for the human full-length Parkin-MTD ₇₈ gene, SEQ. ID. NO .: 430 (forward) and SEQ. ID. NO .: human full-length Parkin-MTD ₈₅ gene using 516 (reverse) primer pair, SEQ. ID. NO .: 430 (forward) and SEQ. ID. NO .: human full-length Parkin-MTD ₁₀₃ gene using 534 (reverse) primer pair, SEQ. ID. NO .: 430 (forward) and SEQ. ID. NO .: 561 (reverse) primer pair was used for the human full-length Parkin-MTD ₁₃₀ gene, SEQ. ID. NO .: 430 (forward) and SEQ. ID. NO .: 566 (reverse) primer pair was used for the human full-length Parkin-MTD ₁₃₅ gene, SEQ. ID. NO .: 430 (forward) and SEQ. ID. NO .: 582 (reverse) primer pair was used for the human full-length Parkin-MTD ₁₅₁ gene, SEQ. ID. NO .: 430 (forward) and SEQ. ID. Human full-length Parkin-MTD ₁₇₄ gene was amplified using a polymerase chain reaction (PCR) method using a NO .: 605 (reverse) primer pair. The polymerase chain reaction product obtained above was cloned into the pGEM-Tease vector, and then subcloned into the BamHI and HindIII positions of the expression vector pET-28a (+) (Novagen). -At the end, each MTD gene was designed to be fused, which is shown in FIG. 9. The cDNA base sequence of the cloned gene is shown in SEQ. ID. NO .: 2 (Parkin), SEQ. ID. NO .: 409 (Parkin-MTD ₁₀ ), SEQ. ID. NO .: 410 (Parkin-MTD ₁₃ ), SEQ. ID. NO .: 411 (Parkin-MTD ₂₉ ), SEQ. ID. NO .: 412 (Parkin-MTD ₇₈ ), SEQ. ID. NO .: 413 (Parkin-MTD ₈₅ ), SEQ. ID. NO .: 414 (Parkin-MTD ₁₀₃ ), SEQ. ID. NO .: 415 (Parkin-MTD ₁₃₀ ), SEQ. ID. NO .: 416 (Parkin-MTD ₁₃₅ ), SEQ. ID. NO .: 417 (Parkin-MTD ₁₅₁ ), SEQ. ID. NO .: 418 (Parkin-MTD ₁₇₄ ), and the amino acid sequence of the expressed Parkin recombinant protein is shown in SEQ. ID. NO .: 1 (Parkin), SEQ. ID. NO .: 394 (Parkin-MTD ₁₀ ), SEQ. ID. NO .: 395 (Parkin-MTD ₁₃ ), SEQ. ID. NO .: 396 (Parkin-MTD ₂₉ ), SEQ. ID. NO .: 397 (Parkin-MTD ₇₈ ), SEQ. ID. NO .: 398 (Parkin-MTD ₈₅ ), SEQ. ID. NO .: 399 (Parkin-MTD ₁₀₃ ), SEQ. ID. NO .: 400 (Parkin-MTD ₁₃₀ ), SEQ. ID. NO .: 401 (Parkin-MTD ₁₃₅ ), SEQ. ID. NO .: 402 (Parkin-MTD ₁₅₁ ), SEQ. ID. NO .: 403 (Parkin-MTD ₁₇₄ ). Ultimately Parkin-MTD ₁₀ (HPM ₁₀ ), Parkin-MTD ₁₃ (HPM ₁₃ ), Parkin-MTD ₂₉ (HPM ₂₉ ), Parkin-MTD ₇₈ (HPM ₇₈ ), Parkin-MTD ₈₅ (HPM ₈₅ ), Parkin-MTD ₁₀₃ (HPM ₁₀₃ ), Parkin-MTD ₁₃₀ (HPM ₁₃₀ ), Parkin-MTD ₁₃₅ (HPM ₁₃₅ ), Parkin-MTD ₁₅₁ (HPM ₁₅₁ ), Parkin-MTD ₁₇₄ (HPM ₁₇₄ ) recombinant proteins were prepared and expressed. The results are shown in FIG. As shown in FIG. 10, the soluble yield of the Parkin recombinant protein (HPM ₁₀ , HPM ₁₃ , HPM ₁₅₁ , HPM ₁₇₄ ) to which JO-10, JO-13, JO-151, and JO-174 MTD were combined was high. Cell permeability was highly evaluated through preliminary investigation, and the four MTDs were selected as the optimal MTD.

Example 9 Codon Optimization and His-tag Non-fusion Cell Permeable Parkin Recombinant Protein Expression Vector Preparation

In order to proceed with the non-clinical test, first, an expression vector was prepared for the production of His-tag non-fusion cell-permeable Parkin recombinant protein in which JO-10, JO-13, JO-151, and JO-174 MTD were combined in E. coli . Codon Optimization Service was commissioned from GenScript (USA) to produce codon optimized genes cloned at the Nco I and Hind III positions in the pUC57 vector. The reason for using parkin optimized cDNA (parkin optimized cDNA) is to facilitate the expression of Parkin in the expression system, and to increase the physical properties.

Specifically, pUC57 vector was treated with HindIII restriction enzyme with NcoI, and each of the optimized genes was subcloned into NcoI and HindIII positions of pET-28a (+) (Novagen), and His-tag was removed at the N-terminus, and C -At the end, each MTD gene is designed to be fused, and is shown in FIGS. 11 and 12. Ultimately, it was prepared to express Parkin-MTD ₁₀ (PNM ₁₀ ), Parkin-MTD ₁₃ (PNM ₁₃ ), Parkin-MTD ₁₅₁ (PNM ₁₅₁ ), Parkin-MTD ₁₇₄ (PNM ₁₇₄ ) recombinant proteins (FIGS. 11 and 12). Reference). To confirm this, the code optimized Parkin DNA was overexpressed in transformed BL21 codon plus (DE3) Escherichia coli, and the results are shown in FIG.

As shown in Figure 13 it can be seen that the expression of the codon-optimized Parkin recombinant protein is increased 4 ~ 10 times compared to the His-tag Parkin recombinant protein, HPM ₁₃ .

The cDNA base sequence of the cloned gene is shown in SEQ. ID. NO .: 419 (PN), SEQ. ID. NO .: 420 (PNM ₁₀ ), SEQ. ID. NO .: 421 (PNM ₁₃ ), SEQ. ID. NO .: 422 (PNM ₁₅₁ ), SEQ. ID. NO .: 423 (PNM ₁₇₄ ).

Example 10 Purification of Histidine-tag Non-fusion Cell Permeable Parkin Recombinant Protein

In order to select the optimal E. coli strain expressing the cell-permeable Parkin recombinant protein was transformed using E. coli BL21 Codon Plus (DE3) with a LacI promoter, E. coli 50 ug / mL kanamycine, 500 uM in 500 mL LB When inoculated into the culture medium to which ZnCl ₂ was added and reached 0.5 to 0.6 at A ₆₀₀ , overexpression was performed by adding 0.5 mM IPTG for 3 hours.

The overexpressed E. coli was harvested by centrifugation, and then repeated 10 sec-on and 20 sec-OFF conditions on ice for 30 minutes, followed by centrifugation to harvest inclusion bodies, 50 mM Tris-HCl (pH 8.0), Washed three times with 100 mM NaCl, 0.1% Triton X-100 buffer. Unpack the suture with 50 mM Tris-HCl (pH 10.0), 8 M urea buffer, and then directly administer to 30 mM Sodium phosphate (pH 8.0), 0.02% Tween-20 buffer at 4 ° C. Stir for 48 hours and refold.

The refolded Parkin protein was centrifuged at 9,000 rpm for 30 minutes, followed by Q-sepharose anion exchange resin column chromatography equipped with AKTA purifier for purification. 5 mM column volume was passed through 30 mM Sodium phosphate (pH 8.0) and 30 mM NaCl buffer to remove unbound proteins.

Concentration gradient was given with 30 mM Sodium phosphate (pH 8.0), 1 M NaCl pH 8.0 buffer solution, and 20% column volume was eluted to elute Parkin recombinant protein, and the results are shown in FIG. 14.

As shown in FIG. 14, PN, PNM ₁₀ , PNM ₁₃ , PNM ₁₅₁ , and PNM ₁₇₄ were all identified as a single band of 53 kDa on SDS-PAGE, indicating that they were purified by Q-sepharose chromatography.

Example 11 Biochemical Functionality (E3 Conjugation Enzyme Activity) of His-tag Non-Fusable Parkin Recombinant Protein

In order to confirm the in vitro biochemical functionalities of the purified His-tag non-fusion Parkin recombinant protein, the self-ubiquitination reaction of the E3 conjugate enzyme Parkin protein was confirmed.

1 ug of purified Parkin recombinant protein (PN, PNM ₁₀ , PNM ₁₃ , PNM ₁₅₁ , PNM ₁₇₄ ) and 1 ug His ₆ -Parkin protein (Boston Biochem) were prepared using 1 uM E1, 50 uM E2, 1 from Boston Biochem. The mixture was mixed with mM His6-Ubiquitin, 10 mM Mg-ATP, and reaction buffer solution (50 mM HEPES, 0.5 M NaCl, 10 mM DTT) for 1 hour at 37 ° C. Nm23 protein purified as a negative control was used. After adding 4x sample buffer to boil to terminate the reaction, 10% SDS-PAGE was performed. Western blotting was performed using an ubiquitin antibody (Enzo life science) at 1: 1,000, and the Parkin recombinant protein to which ubiquitin was conjugated was confirmed, and the results are shown in FIG. 15.

As shown in FIG. 15, all four Parkin recombinant proteins bound to JO-10, JO-13, JO-151, and JO-174 MTD showed E3 conjugation enzyme activity. It showed similar activity to (His ₆ -Parkin), and PNM ₁₀ showed many polyubiquitinations, and most of the large molecular weight proteins were detected, indicating high E3 ligase activity. On the other hand, in the case of PNM ₁₃ , PNM ₁₅ and PNM ₁₇₄ , relatively less ubiquitin conjugated Parkin protein was observed than PNM ₁₀ . Therefore, PNM ₁₀ was refolded into the most stable form in terms of physical properties and structure, which had inherent E3 ligase activity and similar structure to the intrinsic Parkin protein.

Example 12. In Vitro Protection of In Vitro Neuronal Apoptosis of His-tag Unfused Parkin Recombinant Protein

To confirm the in vitro function of the cell-permeable Parkin recombinant protein, TUNEL was performed to confirm the protective effect against neuronal cell death by the neurotoxin 6-OHDA. Human brain cancer dopamine producing neurons SH-SY5Y cells (Korea Cell Line Bank, Seoul, Korea) were cultured in DMEM medium containing 10% fetal bovine serum, 5% penicillin and streptomycin, and 6-OHDA at 100 uM concentration. After 30 minutes of treatment, 2.5 uM of cell-permeable Parkin recombinant protein was added and incubated at 37 ° C. for 2 hours and 30 minutes. After the culture was removed, the terminal dUTP nick-end labeling (TUNEL) analysis was performed using an In situ cell death detection kit (Roche), and then observed with a confocal fluorescence microscope (Nikon), and a Glomax (Amersham) instrument. Fluorescence intensity was measured, and the results are shown in FIG. 16.

As shown in FIG. 16, while SH-SY5Y cells were killed by treatment with 6-OHDA and showed 38% survival rate, when PNM ₁₀ recombinant protein was simultaneously administered, cell survival rate increased to 72%. From the above, it can be seen that the MTD-binding cell permeable Parkin recombinant protein is transferred into cells, thereby protecting the cells from apoptosis by 6-OHDA.

Example 13.Brain-blood barrier permeability test of His-tag non-fusion Parkin recombinant protein

In order to confirm blood brain barrier permeability by immunohistochemical labeling method, PNM ₁₀ cell permeable Parkin recombinant protein having the highest physical properties and E3 ligase activity was selected and subcutaneous injection to confirm blood brain barrier permeability after PNM ₁₀ protein present in brain Immunohistochemical labeling and Western blotting were performed. Dose subcutaneous injection of 200 μg of PNM ₁₀ recombinant protein three times at 2 hour intervals subcutaneously in the dorsal subcutaneous of 8-week-old C57BL / 6 mice (Orient Bio Co.), followed by anesthesia with 2.5% avertin 2 hours after the last injection, followed by PBS buffer The solution was perfused through the heart using a pump to remove blood. Mouse brains were extracted, and half of the brains were placed in 4% Paraformaldehyde solution and fixed at 4 ° C. for at least 24 hours, and immunohistochemical labeling was performed. The other half of the brains were prepared with Western blotting.

Specifically, a fixed brain tissue was put in the OCT compound to make a block, the tissue was frozen in 20㎛ thickness section and stored in PBS buffer solution. After washing three times with PBS buffer 10 minutes each, 3% hydrogen peroxide solution was allowed to stand at room temperature for 30 minutes. After washing three times with PBS for 10 minutes, it was left in 4% normal goat serum and 4% BSA mixture for 1 hour. After removing the mixed solution, the MTD-10 antibody was diluted 1: 1,000 and reacted overnight at 4 ° C. After washing three times with PBS buffer for 10 minutes, the biotinylated anti-Rabbit was diluted 1: 500 and reacted at room temperature for 2 hours. After washing 3 times with 1xPBS buffer three times each 10 minutes and reacted with Avidin-Biotin Complex (vector) at room temperature for 1 hour. After washing three times with PBS buffer three times for 10 minutes, after the color reaction with DAB (3,3'-Diaminobenzidine) solution was observed under a microscope, the results are shown in Figure 17A.

As shown in FIG. 17A, immunohistochemically stained neurons against MTD-10 antibody were observed in the brains of mice injected with PNM ₁₀ cell permeable Parkin recombinant protein. From the above, it can be seen that the MTD-coupled cell permeable Parkin recombinant protein was transmitted through the blood brain barrier and transferred into the brain neurons.

Next, in order to perform western blotting analysis, the tissue was weighed and RIPA buffer (Sigma) was added at 0.1 g / mL, followed by crushing the tissue with a homogenizer and standing on ice for 30 minutes. Prepared. After centrifugation at 10,000 rpm for 10 minutes at 4 ° C, the supernatant was quantified using the Bradford quantitative method, and 100 ug of each protein sample was loaded on a 10% SDS-PAGE gel. After transferring the gel to the nitrocellulose membrane, it was blocked for 1 hour at room temperature with 5% skim milk. Parkin antibody (Millipore) and actin (Santacruz) were diluted 1: 1,000 and reacted at room temperature for 90 minutes. After washing three times with TBS-T buffer for 10 minutes, Anti-mouse-HRP (Santacruz) secondary antibody was diluted 1: 1,000 and reacted at room temperature for 50 minutes. After washing three times with TBS-T buffer for 10 minutes, ECL (Amersham) solution was allowed to react with the membrane for 1 minute, followed by LAS-4000 (Fujifilm Life Science) image analyzer. Was detected and the result is shown in FIG. 17B.

As shown in FIG. 17B, a high molecular weight PNM ₁₀ (about 53 kDa) protein was detected along with the endogenous Parkin protein (52 kDa) in brain tissue lysate. From the above, it can be seen that the human full-length cell permeable Parkin recombinant protein (PNM ₁₀ ) fused with MTD has blood-brain barrier permeability.

Example 14 Using Parkinson's Disease Animal Model in vivo  Efficacy test

Parkinson's disease animal models using MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) are well known and cause dopamine neuronal death in the brain and striatum of the brain. Therefore, to establish a Parkinson's disease animal model by intraperitoneal injection of MPTP in C57BL / 6 mice, to determine the effect of protecting the death of dopamine neurons by PNM ₁₀ cell permeable Parkin recombinant protein, marker enzyme of dopamine neurons Dopamine in brain tissues was quantitatively analyzed by immunohistochemical labeling and ELISA using antibodies against tyrosine hydroxylase.

First, to establish an animal model of Parkinson's disease, MPTP was dissolved in 0.9% NaCl solution and injected at 15 mg / kg intraperitoneally three times per day for 2 days. After the last injection, anesthesia was performed with 2.5% avertin on day 7, and then PBS buffer containing 1 mM EDTA and 4 mM metabisulfite was perfused through the heart using a pump to remove blood, and then the brains of the mice were extracted. Half was fixed in 4% Paraformaldehyde solution for more than 24 hours at 4 ℃, the other half was used for quantitative dopamine analysis.

The immobilized brain tissue was placed in an OCT compound to make a block, and the tissue was frozen in 20 μm thickness and stored in PBS buffer. After washing three times with PBS buffer for 10 minutes each, 3% hydrogen peroxide solution was left at room temperature for 30 minutes. After washing three times with PBS for 10 minutes, it was left in 4% normal goat serum and 4% BSA mixture for 1 hour. After removing the mixture, the tyrosin hydroxylase antibody (Millipore) was diluted 1: 1,000 and reacted at 4 ° C. overnight. After washing three times with PBS buffer for 10 minutes, the biotinylated anti-Rabbit was diluted 1: 500 and reacted at room temperature for 2 hours. After washing three times with PBS buffer three times each 10 minutes and reacted with Avidin-Biotin Complex (Vector) for 1 hour at room temperature. After washing three times with PBS buffer for 10 minutes, after the color reaction with DAB (3,3'-Diaminobenzidine) solution, and observed under an optical microscope and the results are shown in Figure 18.

As shown in FIG. 18A, a large number of dopamine neurons were observed in the melanoma and striatum of mice treated with PNM ₁₀ recombinant protein, compared to the group administered with MPTP alone. It can be seen that there is a neuronal protective function transmitted by intracellular neurotoxic substances. In addition, the mouse brain striatum was isolated and quantitatively compared to the dopamine content according to the product manual using ELISA kit (LDN), the results are shown in Figure 18B.

As shown in FIG. 18B, the dopamine content was increased by 40% in the PNM ₁₀ cell permeable Parkin recombinant protein treated group compared to the MPTP treated group. Therefore, MTD-coupled cell-permeable Parkin recombinant protein increases dopamine secretion by penetrating the blood brain barrier and protecting dopamine neuron death by MPTP, a neurotoxic substance, through the blood brain barrier in the Parkinson's disease animal model. Able to know.

From the above, MTD-bound His-tag fusion or non-fusion Parkin recombinant protein is expected to be very likely to develop new drugs as a drug that can treat degenerative brain diseases as well as Parkinson's disease.

The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above are to be understood in all respects as illustrative and not restrictive.

The cell-permeable Parkin recombination protein according to the present invention is designed to be transferred into cells, and the recombinant protein of the present invention can be used for the purpose of treating Parkinson's disease, and ultimately, can be useful for developing a therapeutic agent for degenerative brain disease in humans. It is expected to be.

Claims (14)

1. A cell permeable parkin recombinant protein in which a macromolecular transduction domain (MTD) is fused with a parkin having an amino acid sequence of SEQ ID NO: 1 and an amino acid sequence selected from the group consisting of SEQ ID NO: 3 to SEQ ID NO: 195.
2. The cell permeable parkin recombinant protein of claim 1, wherein the recombinant protein has an amino acid sequence selected from the group consisting of SEQ ID NOs: 394 and 403. 3.
3. The cell permeable parkin recombinant protein according to claim 2, wherein the recombinant protein is prepared using the primers described in the following table.

   
4. A polynucleotide encoding the cell permeable parkin recombinant protein of claim 2.
5. The polynucleotide of claim 4, wherein the polynucleotide has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 409 to 418.
6. Recombinant expression vector comprising the polynucleotide of claim 4.
7. Cell-permeable His-parkin fused with His-parkin having an amino acid sequence of SEQ ID NO: 389 and a macromolecular transduction domain (MTD) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 15, 103, and 105 Recombinant protein.
8. 8. The cell-permeable His-parkin recombinant protein according to claim 7, wherein the recombinant protein has an amino acid sequence selected from the group consisting of SEQ ID NOs: 390 to 393.
9. The cell-permeable His-parkin recombinant protein according to claim 8, wherein the recombinant protein is prepared using the primers described in the following table.

   
10. A polynucleotide encoding the cell permeable His-parkin recombinant protein of claim 8.
11. The polynucleotide of claim 10, wherein the polynucleotide has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 405 and 408.
12. Recombinant expression vector comprising the polynucleotide of claim 10.
13. Regression containing the cell permeable parkin recombinant protein of claim 1 as an active ingredient

    Pharmaceutical composition for treating sexual brain disease.
14. The pharmaceutical composition of claim 13, wherein the degenerative brain disease is Parkinson's disease.

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