KR101651330B1 - Methods of TAT-A20 fusion protein with good cell penetration and use thereof - Google Patents

Abstract

The present invention provides a method for producing a TAT-A20 fusion protein, comprising: culturing a transformant expressing a TAT-A20 fusion protein gene fused with a cell permeable TAT protein and an A20 protein; Disrupting the cells of the culture solution obtained above to recover and wash the inclusion bodies; Solubilizing the inclusion bodies obtained above; And a step of dialyzing and refolding the protein obtained in the above step into a buffer solution containing arginine and refolding the TAT-A20 fusion protein, the TAT-A20 fusion protein thus prepared, and the fusion protein comprising the fusion protein To a pharmaceutical composition for the prevention or treatment of inflammatory diseases. A20 fusion protein having excellent inhibitory activity against tumor necrosis factor (TNF) can be produced according to the method of the present invention for producing a TAT-A20 fusion protein, and a pharmaceutical composition containing the same can be used for the treatment of existing inflammatory diseases and rheumatic diseases And can provide a biological agent which is more stable to a large number of patients and has an excellent therapeutic effect.

Description

A20 fusion protein having excellent cell permeability and methods for producing the fusion protein of TAT-A20 fusion protein having excellent cell permeability (Methods of TAT-A20 fusion protein with good cell penetration and use thereof)

The present invention relates to a method for producing a TAT-A20 fusion protein excellent in cell permeability and its use.

Inflammation is a very complex phenomenon between inflammation-related factors and cells in tissues that respond to injury by trauma, infection, post-ischemic, toxic or autoimmune reactions. Usually this process induces recovery and treatment after infection, but if these processes are not done properly, inflammation may cause continuous tissue damage by inflammatory cells or collagenase. In addition, rheumatoid arthritis is a relatively common chronic inflammatory disease, which may lead to disruption of joints despite treatment with antirheumatic agents, immunosuppressants, and corticosteroids, resulting in disability due to joint deformation.

This rheumatoid arthritis is an autoimmune disease that begins by genetic factors and external factors that cause autoantibodies such as autoreactive T cells and rheumatoid factors to cause inflammation in synovial tissue. Autoreactive T cells play a central role in the inflammation of synovial tissues and are known to regulate inflammatory processes through interaction with macrophages, synovial cells, dendritic cells and B cells in synovial tissue. The interaction between these cells involves various cell surface molecules and secretory proteins. Among them, TRANCE / RANK, CD40 / CD40L, and GITR / RANK belong to the tumor necrosis factor (TNF) ligand superfamily and the TNF receptor superfamily, The interaction of GITRL and LT [beta] / LT [beta] R has been reported to play an important role.

The A20 protein is known to function to block tumor necrosis, a major factor causing the inflammatory reaction. The A20 protein (tumor necrosis factor alpha induced protein 3, TNFAIP3) is zinc forceps protein expressed by TNFAIP3 gene (zind finger protein) in, TNF (tumor necrosis factor alpha, TNF) or NF for toll-like receptor -B < / RTI > action. The expression of the TNFAIP3 gene is induced by a tumor necrosis factor and the protein expressed by the gene expresses NF-kappa B activity induced by tumor necrosis factor alpha (TNF) and tumor necrosis factor-mediated cell necrosis (TNF -mediated apoptosis. < / RTI > Thus, TNFAIP3 protein is presumed to play a key role in terminating the inflammatory response by terminating NF-kappa B response induced by tumor necrosis factor alpha (TNF).

The treatment of rheumatoid arthritis, which is currently being used or under development in Korea and abroad, includes nonsteroidal anti-inflammatory drugs (NSAIDs) or antirheumatic drugs, antirheumatic drugs or disease modifying antirheumatic drugs (DMARD) Biologic agents: Antibodies or soluble receptors (such as ENBREL) that inhibit the function of TNF-a, a cytokine, gene therapy: antagonists of IL-1 and TNF- gene therapy for expressing anti-inflammatory cytokines such as? 1 and oral type II collagen immunotherapy. However, since these methods are based on the nonspecific immune control that does not distinguish between the immune phenomena related to the onset and the immunity against the infections, There is a high possibility that the treatment of arthritis is accompanied by a side-effect of decreased immune function.

Therefore, in order to overcome the limitations of existing therapies, it is necessary to understand the pathogenesis of rheumatoid arthritis more precisely and to develop new therapies. Recently, a biological agent that blocks tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), which has been shown to be important prior to the popularity of rheumatoid arthritis, has been reported to be very effective as a therapeutic agent. However, there are still many patients who are not responding to these biological agents and the incomplete response is often necessary, so it is necessary to develop drugs that are more effective or complementary.

On the other hand, it is unclear whether the A20 protein, which is known to play an important role in the inhibition of tumor necrosis factor activation, is able to translocate through biological membranes and maintain their function because they are coordinated with zinc ions. If the domain is released during migration, zinc ions can be released and lost. Conversely, the folded state will interfere with domain movement. In addition, if the zinc finger domain is exposed to the oxidizing conditions of the extracellular environment, a harmful disulfide bond may be formed between the cysteine residues, resulting in loss of DNA binding activity.

The HIV-1 TAT protein is a transcription factor for viruses that plays an essential role in increasing the replication of viral proteins and DNA. It is expressed in the cytoplasm and then migrates into the nucleus to transfer its RNA. Mutational analysis of TAT showed that TAT contains a large number of acidic groups at the amino terminus, a cysteine-rich cluster with seven cysteines, and arginine and lysine (Positively charged basic region). ≪ / RTI > Acidic groups play a role in further activating the expression of TAT. The cysteine-rich domain plays a role in forming a metal-liked dimer of TAT. The basic domain is involved in the transfer of the TAT protein to the nucleus and its binding to RNA.

TAT proteins are known to have the ability to pass through cell membranes and nuclear membranes without the aid of any receptor or transporter. Many studies have demonstrated that TAT protein is transferred to the cell nucleus when the TAT protein is treated in the culture medium of the cells. The basic domain among the various domains of TAT (including 49th to 57th amino acids, 2 lysines and 6 arginines A total of nine amino acids) are known to be involved in the transduction of proteins. Although the precise mechanism for the transduction of proteins is not well understood, it has been found that the TAT basic domain can transfer covalently linked fusion proteins into all cells.

The transduction capacity of TAT plays an important role in elucidating the mechanism of signal molecules and is suggested as an alternative way to overcome the problems of gene therapy. However, it is not clear whether all the proteins actually carried by the TAT protein are biologically active.

1. Korean Patent Publication No. 10-2013-0037271

1. Christioane Ferran et al., A20 inhibits NF-κB activation in endothelial cells without sensitizing to tumor necrosis factor-mediated apoptosis, blood 1998 91: 2249-2258. 2. Noula Shembade et al., Inhibition of NF-κB signaling by A20 through disruption of ubiquitin enzyme complexes, Science. 2010 February 26; 327 (5969): 1135-1139.

In order to overcome the limitations of the existing therapeutic agents, the present invention aims to understand the mechanism of the disease of inflammatory diseases and rheumatoid arthritis more precisely and to develop new therapies. More specifically, the present invention relates to a biological agent .

According to an aspect of the present invention, there is provided a method for producing a TAT-A20 fusion protein, the method comprising: culturing a transformant expressing a TAT-A20 fusion protein gene fused with a cell permeable TAT protein and an A20 protein; Disrupting the cells of the culture solution obtained above to recover and wash the inclusion bodies; Solubilizing the inclusion bodies obtained above; And a step of dialyzing the protein obtained in the above step into a buffer solution containing arginine and refolding the TAT-A20 fusion protein.

According to one aspect of the present invention, the step of culturing the transformant in the method for producing the present TAT-A20 fusion protein comprises the steps of: i) preparing a DNA sequence encoding a cell permeable TAT protein and an A20 protein, ii) Preparing a recombinant expression vector by introducing the sequence operably fused to the expression vector, iii) transforming the host with the recombinant expression vector, and iv) culturing the resulting transformant to express the DNA sequence .

According to one aspect of the present invention, the process for disrupting cells in the method for producing a TAT-A20 fusion protein of the present invention is characterized by using lysozyme.

According to one aspect of the present invention, the step of solubilizing the TAT-A20 fusion protein of the present invention is characterized by adding a guanidine chloride solution as a denaturant.

According to one aspect of the present invention, the step of refolding the protein in the method for producing a TAT-A20 fusion protein of the present invention comprises separating the protein obtained in the above-mentioned solubilization step by chromatography and then dialyzing the buffer containing arginine .

According to one aspect of the present invention, there is provided a TAT-A20 fusion protein produced by any one of the above-described methods.

According to one aspect of the present invention, the TAT gene of the TAT-A20 fusion protein of the present invention is characterized by being a base sequence encoding the polypeptide of SEQ ID NO: 1 or a base sequence complementary thereto.

According to one aspect of the present invention, the TAT gene of the TAT-A20 fusion protein of the present invention is characterized by being a nucleotide sequence as set forth in SEQ ID NO: 2 or a nucleotide sequence complementary thereto.

According to one aspect of the present invention, the A20 gene of the TAT-A20 fusion protein of the present invention is characterized by being a base sequence encoding the polypeptide of SEQ ID NO: 3 or a base sequence complementary thereto.

According to one aspect of the present invention, the A20 gene of the TAT-A20 fusion protein of the present invention is characterized by being a base sequence as set forth in SEQ ID NO: 4 or a base sequence complementary thereto.

According to one aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating an inflammatory disease comprising TAT-A20 fusion protein produced by any one of the above-mentioned methods as an active ingredient.

A20 fusion protein having excellent inhibitory activity against tumor necrosis factor (TNF) can be produced according to the method of the present invention for producing a TAT-A20 fusion protein, and a pharmaceutical composition containing the same can be used for the treatment of existing inflammatory diseases and rheumatic diseases And can provide a biological agent which is more stable to a large number of patients and has an excellent therapeutic effect.

Figure 1 is a diagram of a recombinant vector constructed to express a TAT-A20 fusion protein according to the present invention.
2 is a schematic diagram of a TAT-A20 fusion protein according to the present invention. A20 is a control group that does not fuse with Tta.
FIG. 3 shows the results obtained by introducing a vector constructed to express a TAT-A20 fusion protein according to the present invention into a host cell and confirming that the TAT-A20 fusion protein was synthesized. (Estimated size of the fusion protein: 94 kD, )
FIG. 4 is a graph showing the presence of the TAT-A20 fusion protein according to the present invention during the purification process.
FIG. 5 shows experimental results comparing the amount of fusion protein produced depending on the kind of the refolding buffer in the process of producing the TAT-A20 fusion protein.
FIG. 6 shows the results of separation using Superdex 200 chromatography in the course of preparing the TAT-A20 fusion protein.
FIG. 7 shows the result of confirming the TAT-A20 fusion protein prepared through the expression and purification process according to the present invention (the expected size of the fusion protein is 94 kD, see the red arrow)
FIG. 8 shows the results of confirming inhibition of NF-κB activation by adding TAT-A20 fusion protein according to the present invention to cells at different concentrations.
FIG. 9 shows results of inhibition of activation of NF-κB after addition of TAT-A20 fusion protein according to the present invention to cells.
FIG. 10 shows the results of confirming the effect of the TAT-A20 fusion protein according to the present invention on the NF-κB signal pathway by adding TNF to the cells after concentration. NE (nuclear extract): nuclear extract, Nucln: nucleolin.
FIG. 11 shows the results of confirming that the TAT-A20 fusion protein (fluorescent label) according to the present invention was introduced into cells.

Before describing the specific contents of the present invention, the meanings used in this specification will be described.

As used herein, " polypeptide " refers to a polymer of three or more amino acids linked by peptide bonds. The term "protein" can include one or more polypeptide chains, including polypeptides and peptides. The protein or polypeptide may also comprise one or more modifications, for example, natural modifications or artificial modifications. The term "domain" refers to a functional unit within a polypeptide. The tertiary structure of the domain may be folded or loose.

Also, the polypeptide is interpreted to include an amino acid sequence that exhibits substantial identity to the amino acid sequence of interest. The above-mentioned substantial identity is determined by aligning the amino acid sequence of the present invention with any other sequence as much as possible, and analyzing the aligned sequence using the algorithm commonly used in the art, at least 60% Homology, more preferably at least 80% homology, most preferably at least 90% homology.

For example, the polypeptide may comprise at least about 60%, at least 80%, at least 90%, at least 95%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4% , 99.5% or more, or 99.9% or more identity with the amino acid sequence of SEQ ID NO: 2. In general, the higher the percent identity, the more preferable.

Also, the polypeptides having the above identity include polypeptides associated with the beta-adipate pathway, including amino acid sequences in which one or more amino acid residues are deleted, substituted, inserted, and / or added in the polypeptide of the specific amino acid sequence described do. In general, the smaller the number of elimination, substitution, insertion, and / or addition is, the more preferable.

As used herein, the term "polynucleotide" encompasses both DNA (gDNA and cDNA) and RNA molecules, and nucleotides that are basic constituent units in nucleic acid molecules include not only natural nucleotides, It also includes analogues.

The polynucleotide of the present invention is not limited to a nucleic acid molecule encoding the above-described specific amino acid sequence (polypeptide), and may be a polynucleotide having an amino acid sequence exhibiting substantial identity to a specific amino acid sequence as described above or a poly Is interpreted to include nucleic acid molecules encoding the peptides. The above substantial identity is determined by aligning the amino acid sequence of the present invention with any other sequence to the greatest correspondence and analyzing the aligned sequence using algorithms commonly used in the art to obtain a homology of at least 60% , More preferably at least 80% homology, and most preferably at least 90% homology.

The polypeptide having the corresponding function includes, for example, a polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, and / or added. Such a polypeptide comprises an amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted, and / or added, as described above, and includes polypeptides related to 3-hydroxypropionic acid synthesis. , The number of insertions and / or additions is preferably small. In addition, the polypeptide includes a polypeptide having an amino acid sequence having about 60% or more identity with the specific amino acid sequence described above as described above, and the higher the identity, the more preferable.

The term " complementary "or" complementarity ", as used herein, refers to the ability of purine and pyrimidine nucleotides to bind through hydrogen bonding to form double stranded polynucleotides, including partially complementary. The following base pairs are associated with complementarity: guanine and cytosine; Adenine and thymine; And adenine and uracil. "Complementary" applies substantially to all base pairs in which the above-mentioned relationship includes two single-stranded polynucleotides across the molecule of the full length. "Partially complementary" means that a portion of one of the molecules remains as a single strand because the length of one of the two single-stranded polynucleotides is short.

The term "natural" as used herein refers to a sequence (e.g., nucleic acid or amino acid sequence) present in a cell of a natural organism, i.e., an organism that is not modified by molecular biological techniques. For example, transgenic mice are not naturally occurring organisms, but highly inbred mice that have not been modified by molecular biology techniques are considered natural. When describing a sequence, the term "viral" refers to a naturally occurring virus, i.e., a sequence of a virus that has not been modified by molecular biological techniques.

The term "exogenous " as used herein generally does not occur naturally in wild-type cells or organisms, but typically refers to molecular biology techniques, that is, polynucleotide sequences or polypeptides that are introduced into cells by engineering processes to produce recombinant microorganisms .

The term "recombinant" microorganism as used herein typically includes one or more foreign nucleotide sequences, e.g., in a plasmid or vector.

The term "vector" as used herein refers to any nucleic acid that is inserted into a host cell, recombined with the host cell genome and inserted into it, or comprising a competent nucleotide sequence that replicates spontaneously as an episome. Such vectors include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, and viral vectors.

As used herein, the term "transformation or transfection" refers to the process by which extracellular DNA enters a host cell in the absence and presence of entities. "Transfected cell" refers to a cell in which extracellular DNA is introduced into a cell and contains extracellular DNA. DNA can be introduced into the cell and the nucleic acid can be inserted into the chromosome or replicated as an extrachromosomal material.

The term "host cell" as used herein includes individual cells or cell cultures that may or may not be receptors of any recombinant vector (s) or isolated polynucleotide of the present invention. The host cell may be a progeny of a single host cell, and the progeny may not be completely identical to the original parent cell (either in form or in a total DNA image) due to natural, accidental or artificial mutations and / or alterations. Host cells include cells transfected, transformed, or infected with a recombinant vector or polynucleotide of the invention in vivo or in vitro.

The term "fusion ", as used herein, refers to a single polypeptide chain comprising fused components. Exemplary fusion proteins are DNA binding proteins and protein transfer domains. Fused components need not be linked directly. For example, other sequences (e. G., Linkers or functional domains) may be located between fused elements

The term "cell permeable domain" as used herein refers to an amino acid sequence that can pass through a biological membrane, particularly a cell membrane. When attached to a heterologous polypeptide, the PTD enhances the migration of the heterologous polypeptide across the biological membrane. The term "cell permeable domain" does not refer to a nuclear localization signal that facilitates transport of the compound through the pores in the nuclear envelope. Proteins entering the nucleus do not pass through the actual membrane bilayer.

As used herein, "treating" or "treating" refers to treating a disease, symptom or prognosis of a disease, or treating, reducing, alleviating, altering, curing, And administering or administering a TAT-A20 protein to enter and administer the subject, e.g., a TAT-A20 protein to regulate gene expression in a patient's cell, or to administer or administer a drug to a cell, such as a tissue or cell line, In which the patient refers to a subject having an inflammatory disease or rheumatism, or showing symptoms or prognosis of the disease.

In order to accomplish the above object, the present invention provides a method for producing a TAT-A20 fusion protein, comprising: culturing a transformant expressing a TAT-A20 fusion protein gene fused with a cell permeable TAT protein and an A20 protein; Disrupting the cells of the culture solution obtained above to recover and wash the inclusion bodies; Solubilizing the inclusion bodies obtained above; And a step of dialyzing and refolding the protein obtained in the above step to a buffer containing arginine and refolding the TAT-A20 fusion protein, and the TAT-A20 fusion protein and the inflammatory disease comprising the same To provide a pharmaceutical composition. BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the drawings.

The present invention provides a method for producing a TAT-A20 fusion protein, comprising: culturing a transformant expressing a TAT-A20 fusion protein gene fused with a cell permeable TAT protein and an A20 protein; Disrupting the cells of the culture solution obtained above to recover and wash the inclusion bodies; Solubilizing the inclusion bodies obtained above; And a step of dialyzing and refolding the protein obtained above in a buffer containing arginine, and a method for producing the TAT-A20 fusion protein.

The step of culturing the transformant in the method for producing a TAT-A20 fusion protein comprises the steps of: i) preparing a DNA sequence encoding a cell-permeable TAT protein and an A20 protein; ii) To produce a recombinant expression vector, iii) transforming the host with the recombinant expression vector, and iv) culturing the resulting transformant to express the DNA sequence.

In preparing the TAT-A20 fusion protein of the present invention, i) a step of preparing a DNA sequence encoding the cell-permeable TAT protein and the A20 protein may be performed. The DNA sequence coding for the cell permeable TAT protein and the A20 protein can be prepared by various methods known in the art such as chemical synthesis and RT-PCR. Preferably, the DNA of the target protein is used as a template, It is preferable to prepare by PCR using a primer that can be used. Examples of the primers for amplifying the target gene include, for example, cloning and restriction enzyme digestion of appropriate sequences, phosphotriester methods such as Narang and the like, diethylphosphoramidate methods such as Beaucage and U.S. Patent No. 4,458,066 Including but not limited to direct chemical synthesis methods such as the solid support method of the invention.

The DNA sequence encoding the TAT protein may be a nucleotide sequence encoding the polypeptide of SEQ ID NO: 1 or a complementary nucleotide sequence thereof, which may also be the nucleotide sequence of SEQ ID NO: 2 or a complementary base sequence thereof. Preferably, the polypeptide known to exhibit cellular permeability in the TAT protein is YGRKKRRQRRR.

In addition, the polypeptide sequence encoding the A20 protein may be SEQ ID NO: 3, and the nucleotide sequence of A20 encoding the same may be SEQ ID NO: 4.

Next, ii) the DNA sequence may be introduced into the expression vector so as to be operatively fused to prepare a recombinant expression vector.

For such fusion, TAT DNA can be linked to A20 DNA using, for example, a flexible linker. The flexible linker may comprise one or more glycine residues to impart free rotation. For example, TAT DNA can be spaced 10, 20, or 50 amino acids away from A20 DNA.

The usability and design of the available linkers is well known in the art. Particularly useful linkers are peptide linkers that are encoded by nucleic acids. Thus, TAT, a peptide linker, and a synthetic gene encoding A20 can be produced. This design can be repeated to produce large scale synthetic multiple TAT-A20 fusion proteins. For practice utilizing zinc finger domains, peptides that are found naturally among zinc finger can be used as linkers to link fingers together. Typically such a naturally occurring linker is Thr-Gly- (Glu-Gln) - (Lys-Arg) -Pro- (Tyr-Phe).

Additional peptide linkers that form tertiary structures of random coils, alpha-helices or beta-wrinkles may be used. Polypeptides that form suitable flexible linkers are well known in the art. Flexible linkers typically include glycine because glycine is the only amino acid with a rotational degree of freedom lacking a side chain. Serine or threonine can be inserted into the linker to increase hydrophilicity. In addition, amino acids that can interact with the phosphate backbone of DNA to increase binding affinity can be used.

A substitute for a peptide linker is a linker that uses a different type of chemical linkage. Thus, TAT DNA and A20 DNA can be linked by synthetic non-peptide linkers. A polypeptide comprising TAT can be conjugated to a polypeptide comprising A20 in a synthesis reaction. Homo-or heterobifunctional cross linkers can be used. In one embodiment, the synthetic linker is cleaved within the cell. For example, the synthetic linker includes a reducing thiol linkage. Examples of synthetic linkers include BM [PEO] 3 (1,8-bis-maleimidotriethylene glycol), OCOES (bis [2- (succinimidooxycarbonyloxy) ethyl] sulfone), and DSG Glutarate).

TAT DNA can be located at the N- or C- terminus on the basis of A20 DNA. Positioning at the N- or C-terminus for a particular DNA does not require it to be contiguous to that particular DNA. For example, TAT DNA at the N-terminus for A20 DNA can be separated from A20 DNA by a spacer and / or other type of domain. TAT DNA can be chemically synthesized and chemically conjugated to separately prepared A20 DNA in the presence or absence of linker peptide. It may also comprise a plurality of transmembrane domains, for example, different types of cell permeable domains or two or more copies of the same TAT DNA.

Standard recombinant nucleic acid methods can be used to express the TAT-A20 fusion proteins of the invention. In one embodiment, a nucleic acid sequence encoding a TAT-A20 fusion protein of the invention can be cloned into a nucleic acid expression vector, for example, with appropriate signals and processing sequences and control sequences for transcription and translation.

A recombinant expression vector for a TAT-A20 fusion protein of the invention may comprise a regulatory sequence comprising, for example, a promoter operably linked to a sequence encoding a TAT-A20 fusion protein of the invention. Non-limiting examples of inducible promoters that may be used include, but are not limited to, steroid-hormone responsive promoters (e.g., ecdysone-responsive, estrogen-responsive, and glutacorticoid-reactive promoters), tetracycline "Tet-on" -Off "systems, and metal-reactive promoters. The construct may be introduced into an appropriate host cell, for example, a bacterial cell, a yeast cell, an insect cell, or a tissue culture cell. This construct may also be introduced into embryonic stem cells to produce a transgenic organism as a model subject. A large number of suitable vectors and promoters are known to those skilled in the art and are commercially available to produce the recombinant constructs of the present invention.

Known methods can be used to construct vectors comprising polynucleotides of the invention and appropriate transcription / translation control signals. These methods include in vitro recombinant DNA technology, synthetic techniques, and in vivo recombination / genetic recombination. See, for example, Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory, NY (2001), and Ausubel et al., Current Protocols in Molecular Biology (Greene Publishing Associates and Wiley Interscience, NY 1989).

Next, iii) transforming the host with the recombinant expression vector may be performed.

A wide variety of mononuclear cell host cells can be used to express the DNA sequence of the TAT-A20 fusion protein according to the invention. These hosts include this. Known eukaryotic and prokaryotic hosts such as E. coli, Pseudomonas, Bacillus, Streptomyces, fungi and yeast, insect cells such as Spodoptera frugiperda (SF9), animal cells such as CHO and mouse cells, COS 1, African green monkey cells such as COS 7, BSC 1, BSC 40 and BMT 10, and tissue cultured human cells and plant cells. Preferred host organisms include this. Coli and insect cells.

Transformation or transfection into a host cell in the present invention includes any method of introducing the nucleic acid into an organism, cell, tissue or organ, and may be carried out by selecting a suitable standard technique depending on the host cell, as is known in the art have. Such methods include electroporation, protoplast fusion, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, Agrobacterium mediated transformation, PEG, dextran sulfate, lipofectamine and dry / Methods of conversion, and the like.

Next, iv) culturing the resulting transformant to express the DNA sequence may be performed. The transformed host cells according to the invention are cultured in a nutrient medium suitable for the production of the fusion protein using known techniques. For example, cells may be cultured by small or large scale fermentation, shake flask culture in laboratory or industrial fermentors conducted under conditions permitting the appropriate medium and polypeptide to be expressed and / or isolated. The cultivation takes place in a suitable nutrient medium containing carbon, nitrogen source and inorganic salts using known techniques. Suitable media can be obtained from commercial suppliers or can be prepared according to the ingredients and composition ratios described in publications such as, for example, catalogs of the American Type Culture Collection. If the fusion protein is secreted directly into the medium, it can be isolated directly from the medium and, if not secreted, can be isolated from the cell lysate.

According to a specific embodiment of the present invention, the cDNA of TAT-A20 was cloned into a pRSET vector and expressed in E. coli to produce a recombinant protein.

In the present invention, the next step of the method for producing the TAT-A20 fusion protein is a step of disrupting the cells of the culture solution obtained above and recovering and washing the inclusion bodies. In this step, a cell pellet is obtained by centrifugation or filtration, and an eluent containing cell lysate is obtained by removing media components containing ruptured cell walls or cell membranes. Specifically, cell pellets may be prepared by methods well known in the art, such as the use of alkalis, surfactants (such as CHAPS and SDS), organic solvents and enzymes (such as lysozyme), sonicatino and high pressure mills , Periodic high temperature, pressure, refrigeration and thaw cycles. The cell lysate thus obtained can be fractionated to remove the minimum area of the biomass in the cell lysate to obtain a clean eluate. For example, the cell lysate can be obtained by centrifugation, To obtain a clean eluent and, if necessary, can be subjected to further purification steps known in the art. For example, lysogenase may be used for lysogenesis of the cells, and E. coli is broken using lysozyme, and the inclusion bodies are recovered by centrifugation and washed several times with a buffer containing detergent to remove impurities.

The next step is to solubilize the inclusion bodies obtained above. Conventional methods known to those skilled in the art may be used for solubilization, and for example, a denaturant may be used. For example, the step of solubilizing may be by adding a solution of guanidine chloride, which is a denaturant. The concentration of the guanidine chloride solution may be 2 to 15M, preferably 8M.

The next step is characterized by the step of dialyzing the protein obtained above into a buffer containing arginine. When dialyzed and refolded in a buffer containing arginine, the purification rate of the TAT-A20 fusion protein is excellent. The arginine contained in the buffer solution may be L-arginine at a concentration of 10 mM or more and 10 M or less, preferably 100 mM to 1 M, and most preferably 440 mM. The buffer may also include conventional buffer components used in the art, for example, tris, sodium chloride, potassium chloride. The acidity of the buffer solution is about 7 to 8, of which 8.2 is excellent.

For example, the protein obtained in the above-mentioned solubilization step may be separated by chromatography and then dialyzed into a buffer solution containing arginine. The separation step used in the present invention may be any separation step conventionally used for the separation of proteins. The separation step may be selected from the group consisting of reversed phase chromatography (e. G., HPLC), size-exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography, affinity chromatography and electrophoresis such as capillary electrophoresis ≪ / RTI > More specifically, the chromatography may be size exclusion chromatography using Superdex 200.

According to another aspect of the present invention, there is provided a TAT-A20 fusion protein produced by the above-mentioned method for producing a TAT-A20 fusion protein.

According to still another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating an inflammatory disease comprising TAT-A20 fusion protein produced by any one of the above-mentioned methods as an active ingredient.

The pharmaceutical composition of the present invention further comprises a pharmaceutically acceptable carrier for the TAT-A20 protein prepared above. "Pharmaceutically acceptable carrier" includes any or all of the solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for the administration (e. G., Injection or infusion) of intravenous, intramuscular, subcutaneous, parenteral, vertebral, or epithelial. Depending on the route of administration, the active substance may be coated in a material to protect the substance from the action of an acid or other natural environment that may inactivate the substance.

"Pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the source material and does not impart any undesirable toxicological effects. Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids derived from hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, trivalent phosphoric acid and the like, as well as aliphatic mono- and dicarboxylic acids, alkanoic acids substituted with phenyl, hydroxyalkanoic acids, , Salts derived from non-toxic organic acids such as aliphatic and aromatic sulfonic acids, and the like. Base addition salts include those derived from alkaline earth metals such as sodium, potassium, magnesium and calcium, as well as those derived from N, N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, Min, procaine, and the like.

The pharmaceutical composition may be in various forms. These include, for example, liquid, semi-solid and solid formulations such as liquid solutions (e.g., injectable or infusible solutions), dispersing or suspending agents, tablets, pills, powders, liposomes and suppositories. The formulations will vary depending upon the method of administration desired and the therapeutic application. The composition may, for example, contain an anionic or negatively charged carrier to stabilize the composition. For example, the composition may comprise small fragments of poly A-poly T DNA double helix.

Conventional compositions are in the form of injectable or infusible solutions, such as compositions similar to those used to administer the antibody to the human body. There are parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular) administration by conventional administration methods. In one embodiment, the TAT-A20 fusion protein is administered by intravenous infusion or injection. In other embodiments, the TAT-A20 fusion protein is administered by intramuscular or subcutaneous injection. Exemplary routes of administration include oral administration, epidermal tissue (e.g., skin) or mucosal (e.g., eye) administration, or inhalation. Other routes of administration may also be used.

For various applications, the route / mode of administration is intravenous or infusion, but the TAT-A20 fusion protein can be administered in a variety of well known ways. For example, for therapeutic applications, the TAT-A20 fusion protein may be administered at a rate of 30, 20, 10, 5, 3, 1, or 0.1 mg / 25 mg / m < 2 >, or 0.5 to 15 mg / m < 2 > The route and / or method of administration may vary depending on the outcome expected. In certain embodiments, the active ingredient may be formulated with a carrier, for example, a controlled release formulation, i.e., an implant, a microcapsule delivery system, which is capable of protecting the component from rapid release. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters and polylactic acid may be used.

The pharmaceutical compositions may be administered in a known medical device. For example, the pharmaceutical composition may be administered in a needle-free subcutaneous injection device, such as the device disclosed in U.S. Patent No. 5,399,163. Useful implants and modules well known in the present invention include implantable micro-infusion pumps (US Pat. No. 4,487,603) for dispersing drugs at sustained-release rates; A therapeutic device for administering a drug through the skin (U.S. Patent No. 4,486,194); Drug infusion pumps (4,447, 233) for delivering medicines at the correct infusion rate; Variable flow rate implantable injection devices for continuous drug delivery (US 4,447,224); An osmotic drug delivery system with multiple chamber fractions (U.S. 4,439,196); And an osmotic drug delivery system (4,475,196). Many other implants, delivery systems, and modules are also well known.

A pharmaceutical composition may comprise a "therapeutically effective amount" or "prophylactically effective amount" of a TAT-A20 fusion protein. A "therapeutically effective amount" refers to an amount effective to achieve the desired treatment outcome at the dose and in the required time period. The therapeutically effective amount of the composition may vary depending on the stage of the individual's disease, age, sex, and body weight, and on the ability of the TAT-A20 fusion protein to elicit the desired response in the individual. A therapeutically effective amount also provides a therapeutically beneficial effect and is usually a greater amount of beneficial effect than any toxic or deleterious effect of the composition.

A "prophylactically effective amount" refers to an amount that is effective to achieve the desired prophylactic result over the course of the dose and the required time period. Typically, since prophylactic administration is used prior to or at an early stage of the disease, the prophylactically effective amount will be less than the therapeutically effective amount.

Hereinafter, the present invention will be described in more detail with reference to Examples. It should be understood, however, that these examples are for illustrative purposes only and are not to be construed as limiting the scope of the present invention.

[ Example 1 ] The fusion protein  Expression, expression, purification of transformants

1) Production

The A20 protein and the cytotoxic TAT-A20 protein used in the study were designed to include the polyhistidine tag (His, HHHHHH) used for protein separation and the hemagglutinin tag (HA, YPYDVPDYA) used for detection (Fig. 1).

TAT-A20 was separately cloned to have the cytotoxic sequence (YGRKKRRQRRR) of the HIV-1 TAT protein (Fig. 2). More specifically, the A20 cDNA was cloned into the bacterial expression vector pRSET to prepare an expression vector for A20. The pRSET plasmid (Invitrogen, Carlsbad, USA) contains the T7 promoter and the 6xHis tag. We used the Tat sequence (YGRKKRRGRRR) of the HIV-1 transactivator for cell membrane permeation and the hemagglutinin (HA) tag sequence (YPYDVPDYA ) Was synthesized and inserted into pRSET (Supplementary Table 1, Tat-HA). This sequence has BamHI on the 5 'side, a SacI and BglII restriction site on the 3' side, and an XhoI cleavage site between the Tat and HA sequences. Both ends of the Tat-HA oligo DNA amplified by PCR were cut with BamHI and BglII to create a junction site and ligated with the same restriction enzyme-cut pRSET vector. This was transplanted into E. coli DH5a to obtain a recombinant clone of pRSET-His-Tat-HA. In order to obtain A20 cDNA, THP-1 cells, a human monocyte cell line, were stimulated with PMA / ionomycin and total RNA was isolated from the cell lysate. The whole cDNA was prepared using reverse transcriptase, oligo-dT and random primer, and amplified by A20 cDNA having 790 amino acid sequence using the primer pair shown in Table 1 with A20 specific sequence. For amplification, 1 unit of DNA polymerase (Takara, PrimeSTAR HS), 2.5 mM of dNTP, 0.2 μM of each primer, 0.1 μg of cDNA, 1x enzyme buffer was denatured at 98 ° C for 5 minutes, Deg.] C for 10 seconds, 65 [deg.] C for 15 seconds, and 72 [deg.] C for 1 minute) and 72 [deg.] C for 3 minutes. The primer used for amplification was designed to have a BglII site at the 5 'position and an EcoRI restriction site at the 3' position. The DNA amplified by PCR was digested with two enzymes to make a junction site, and pRSET-His- Tat-HA vector. This was transplanted into E. coli DH5a to obtain a recombinant clone of pRSET-His-Tat-HA-A20. (PRSET-His-HA-A20) for the expression of A20 without the Tat sequence for use as a control in the experiment was constructed by replacing the pRSET-His-Tat-HA-A20 plasmid with the BamHI And XhoI restriction enzyme sites were cut with each enzyme and inserted into an oligonucleotide linker (Table 1). It was confirmed that each of the synthesized plasmid vectors was correctly cloned through DNA sequencing.

Primer sequence name order Tat-HA (sense) 5'-CGC GGA TCC GGT TAC GGT CGT AAA AAA CGT CGT CAG CGT CGT CGT GGT CTC GAG TAC CCG TAC GAC GTT CCG GAC TAC GCT GAG CTC AAG ATC TGC GCG-3 ' Tat-HA (anti-sense) 5'-CGC GCA GAT CTT GAG CTC-3 ' A20 (sense) 5'-GCC AGA TCT GCA TGG CTG AAC AAG TCC TT-3 ' A20 (anti-sense) 5'-GCG GAA TTC TTA GCC ATA CAT CTG CTT-3 ' BamHI / XhoI linker 5'-GA TTC GGT GGT C-3 ', 5'-TC GAG ACC ACC G-3'

2) Expression

The protein obtained by the culture was confirmed by electrophoresis to synthesize the fusion protein. The expected size of the fusion protein is 94 kD (Figure 3).

3) Refining

The culture broth obtained above was broke with Escherichia coli using lysozyme, and the inclusion body was recovered by centrifugation and washed several times with a buffer containing detergent to remove impurities.

The recombinant protein was solubilized by adding 8 M guanidine hydrochloride solution as a denaturant to the separated inclusion bodies. And electrophoresis was performed to confirm protein solubilization (FIG. 4).

The solubilized protein was then further separated (size exclusion chromatography) using Superdex 200 chromatography and dialyzed in buffer to refold the protein. To compare the purification purity according to the type of buffer, the following buffers were added and dialyzed.

division Arginine
(L-arginine) Guanidine
(guadine) Tris
(Tris) Sodium chloride
(NaCl) Potassium chloride
(KCl) EDTA Glutathione (reduced form) Glutathione (oxidized form) pH Buffer 1 - - 50 mM 20mM 0.8 mM 1 mM 2 mM 1 mM 8.2 Buffer 2 400 mM - 50 mM 20mM 0.8 mM 1 mM 2 mM 1 mM 8.2 Buffer 3 800mM - 50 mM 20mM 0.8 mM 1 mM 2 mM 1 mM 8.2 Buffer 4 - 500 mM 50 mM 20mM 0.8 mM 1 mM 2 mM 1 mM 8.2 Buffer 5 400 mM 500 mM 50 mM 20mM 0.8 mM 1 mM 2 mM 1 mM 8.2 Buffer 6 800mM 500 mM 50 mM 20mM 0.8 mM 1 mM 2 mM 1 mM 8.2 Buffer 7 - 1M 50 mM 20mM 0.8 mM 1 mM 2 mM 1 mM 8.2 Buffer 8 400 mM 1M 50 mM 20mM 0.8 mM 1 mM 2 mM 1 mM 8.2 Buffer 9 800mM 1M 50 mM 20mM 0.8 mM 1 mM 2 mM 1 mM 8.2 Buffer 2-1 - - 50 mM 20mM 0.8 mM 1 mM 2 mM 1 mM 8.2 Buffer 2-2 100 mM - 50 mM 20mM 0.8 mM 1 mM 2 mM 1 mM 8.2 Buffer 2-3 200 mM - 50 mM 20mM 0.8 mM 1 mM 2 mM 1 mM 8.2 Buffer 2-4 300mM - 50 mM 20mM 0.8 mM 1 mM 2 mM 1 mM 8.2 Buffer 2-5 400 mM - 50 mM 20mM 0.8 mM 1 mM 2 mM 1 mM 8.2 Buffer 2-6 600mM - 50 mM 20mM 0.8 mM 1 mM 2 mM 1 mM 8.2 Buffer 2-7 800mM - 50 mM 20mM 0.8 mM 1 mM 2 mM 1 mM 8.2

The results of the experiment using each of the above buffers are shown in FIG. Experimental results showed that the protein yield was high when arginine buffer solution was used, especially when buffer 2-6 was used. Therefore, the inventors of the present invention have found that the use of a buffer solution of 2-6 in comparison with the buffer solution composed of other component combinations can yield the most active A20 protein at a high yield.

The size exclusion chromatography was carried out using 4M guanidine hydrochloric acid buffer solution, and the result of separation by time was shown in Fig.

The denatured and refolded proteins were electrophoresed and electrophoresed with Escherichia coli extracts and protein stained for more than 80% (FIG. 7).

In the following experiments, the fusion protein was prepared using buffer buffer 2-6, which is an optimal buffer solution.

[Example 2] NF-κB activation experiment

1) Experimental method

To investigate the effect of the TAT-A20 fusion protein of the present invention on NF-κB activation, IgB-Luc, which is a plasmid with an NF-κB reporter gene, was cloned into a control factor gene, -actin / Cells were transplanted and cultured for 2 days. (TAT-A20) was added to each cell group for one hour. TNF, which is known as a substance inducing NF-κB activity by an inflammatory cytokine, was added to the cells and the cells were further cultured for 6 hours Lt; / RTI > After lysing the cells, luciferase activity induced by NF-κB activity was measured by luminometry using the assay system (Promega). Separately, the activity of galactosidase in the cell lysate was measured to correct the Luciferase measurement value.

In order to examine whether the inhibitory effect of TAT-A20 is continuously exhibited, the HEK cells were stimulated with tumor necrosis factor (TNF) after adding 3 g / ml of the example of the present invention (TAT-A20) And the expression of luciferase was measured. TNF, and A20 protein treated groups.

2) Experimental results

As a result of the experiment, the TAT-A20 fusion protein of the present invention showed an inhibitory effect according to the treatment concentration, whereas the control group A20 showed a lower inhibitory effect than the example in the same concentration range (FIG. 8).

Measurement of NF-κB reporter gene expression over time after TNF stimulation revealed that the effect also occurred after 24 hours (FIG. 9).

[Example 3] Confirmation mechanism of NF-kB activity of fusion protein

1) Experimental method

To investigate the mechanism by which TAT-A20 inhibited NF-κB activation, A20 (control) and TAT-A20 were added to HEK293 cells and stimulated with TNF, an inducer of NF-κB activity.

NF-κB is normally inactivated by binding to IκBα (nuclear factor of kappa light polypeptide enhancer, alpha) protein in the cytoplasm, and then stimulated by TNFα or lipid polysaccharide , The NF-κB signaling pathway is activated and IκBα is degraded, and then NF-κB released from IκBα is transferred into the nucleus. The p65 protein is a subunit that constitutes NF-κB.

In order to examine the migration of NF-κB to the nucleus, HEK293 cells were stimulated with TNF, the cells were lysed, the nuclear extract was separated, and the p65 protein content was measured by Western blot. The nucleoprotein nucleoporin was measured as a control protein.

2) Experimental results

The migration of NF-κB p65 to the nucleus significantly inhibited the embodiment of the fusion protein of the present invention compared to the control (A20) (FIG. 10). This means that the fusion protein of the present invention inhibits the activation of NF-κB by inhibiting the higher step of NF-κB nuclear transfer such as IκBα degradation.

[Example 4] Confirmation of intracellular inflow of fusion protein

1) Experimental method

(A20) and the example of the present invention (TAT-A20) were reacted with fluorescein isothiocyanate (FITC) as a fluorescent substance to detect whether the produced recombinant protein was introduced into cells. Recombinant proteins were prepared. The cells were incubated for 1 hour with RAW264.7 macrophages at a concentration of 1 g / ml each of the fluorescence-labeled control (A20) and the present invention example (TAT-A20), and the cells were washed several times with phosphate buffered saline .

2) Experimental results

The cells (TAT-A20) of the present invention were introduced into the cells at the measured concentrations as a result of observation with an optical microscope (LM) and a fluorescence microscope (FITC) (Fig. 11).

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> Industry Academic Cooperation Foundation of Catholic University <120> Methods of TAT-A20 fusion protein with good cell penetration and          use thereof <130> PN1405-153 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 33 <212> PRT <213> TAT polypeptide sequence <400> 1 Thr Tyr Arg Gly Leu Tyr Ala Arg Gly Leu Tyr Ser Leu Tyr Ser Ala   1 5 10 15 Arg Gly Ala Arg Gly Gly Leu Asn Ala Arg Gly Ala Arg Gly Ala Arg              20 25 30 Gly     <210> 2 <211> 33 <212> DNA <213> TAT polynucleotide sequence <400> 2 tacggtcgta aaaaacgtcg tcagcgtcgt cgt 33 <210> 3 <211> 790 <212> PRT <213> amino acid sequence A20 <400> 3 Met Ala Glu Gln Val Leu Pro Gln Ala Leu Tyr Leu Ser Asn Met Arg   1 5 10 15 Lys Ala Val Lys Ile Arg Glu Arg Thr Pro Glu Asp Ile Phe Lys Pro              20 25 30 Thr Asn Gly Ile Ile His His Phe Lys Thr Met His Arg Tyr Thr Leu          35 40 45 Glu Met Phe Arg Thr Cys Gln Phe Cys Pro Gln Phe Arg Glu Ile Ile      50 55 60 His Lys Ala Leu Ile Asp Arg Asn Ile Gln Ala Thr Leu Glu Ser Gln  65 70 75 80 Lys Lys Leu Asn Trp Cys Arg Glu Val Arg Lys Leu Val Ala Leu Lys                  85 90 95 Thr Asn Gly Asp Gly Asn Cys Leu Met His Ala Thr Ser Gln Tyr Met             100 105 110 Trp Gly Val Gln Asp Thr Asp Leu Val Leu Arg Lys Ala Leu Phe Ser         115 120 125 Thr Leu Lys Glu Thr Asp Thr Arg Asn Phe Lys Phe Arg Trp Gln Leu     130 135 140 Glu Ser Leu Lys Ser Gln Glu Phe Val Glu Thr Gly Leu Cys Tyr Asp 145 150 155 160 Thr Arg Asn Trp Asn Asp Glu Trp Asp Asn Leu Ile Lys Met Ala Ser                 165 170 175 Thr Asp Thr Pro Met Ala Arg Ser Gly Leu Gln Tyr Asn Ser Leu Glu             180 185 190 Glu Ile His Ile Phe Val Leu Cys Asn Ile Leu Arg Arg Pro Ile Ile         195 200 205 Val Ile Ser Asp Lys Met Leu Arg Ser Leu Glu Ser Gly Ser Asn Phe     210 215 220 Ala Pro Leu Lys Val Gly Gly Ile Tyr Leu Pro Leu His Trp Pro Ala 225 230 235 240 Gln Gly Cys Tyr Arg Tyr Pro Ile Val Leu Gly Tyr Asp Ser His His                 245 250 255 Phe Val Pro Leu Val Thr Leu Lys Asp Ser Gly Pro Glu Ile Arg Ala             260 265 270 Val Pro Leu Val Asn Arg Asp Arg Gly Arg Phe Glu Asp Leu Lys Val         275 280 285 His Phe Leu Thr Asp Pro Glu Asn Glu Met Lys Glu Lys Leu Leu Lys     290 295 300 Glu Tyr Leu Met Val Ile Glu Ile Pro Val Gln Gly Trp Asp His Gly 305 310 315 320 Thr He Leu Ile Asn Ala Ala Lys Leu Asp Glu Ala Asn Leu Pro                 325 330 335 Lys Glu Ile Asn Leu Val Asp Asp Tyr Phe Glu Leu Val Gln His Glu             340 345 350 Tyr Lys Lys Trp Gln Glu Asn Ser Glu Gln Gly Arg Arg Glu Gly His         355 360 365 Ala Gln Asn Pro Met Glu Pro Ser Val Pro Gln Leu Ser Leu Met Asp     370 375 380 Val Lys Cys Glu Thr Pro Asn Cys Pro Phe Phe Met Ser Val Asn Thr 385 390 395 400 Gln Pro Leu Cys His Glu Cys Ser Glu Arg Arg Gln Lys Asn Gln Asn                 405 410 415 Lys Leu Pro Lys Leu Asn Ser Lys Pro Gly Pro Glu Gly Leu Pro Gly             420 425 430 Met Ala Leu Gly Ala Ser Arg Gly Glu Ala Tyr Glu Pro Leu Ala Trp         435 440 445 Asn Pro Glu Glu Ser Thr Gly Gly Pro His Ser Ala Pro Pro Thr Ala     450 455 460 Pro Ser Pro Phe Leu Phe Ser Glu Thr Thr Ala Met Lys Cys Arg Ser 465 470 475 480 Pro Gly Cys Pro Phe Thr Leu Asn Val Gln His Asn Gly Phe Cys Glu                 485 490 495 Arg Cys His Asn Ala Arg Gln Leu His Ala Ser His Ala Pro Asp His             500 505 510 Thr Arg His Leu Asp Pro Gly Lys Cys Gln Ala Cys Leu Gln Asp Val         515 520 525 Thr Arg Thr Phe Asn Gly Ile Cys Ser Thr Cys Phe Lys Arg Thr Thr     530 535 540 Ala Glu Ala Ser Ser Seru Ser Thr Ser Leu Pro Ser Ser Cys His 545 550 555 560 Gln Arg Ser Ser Ser Ser Prop Ser Arg Leu Val Arg Ser Ser Ser Pro                 565 570 575 His Ser Cys His Arg Ala Gly Asn Asp Ala Pro Ala Gly Cys Leu Ser             580 585 590 Gln Ala Ala Arg Thr Pro Gly Asp Arg Thr Gly Thr Ser Lys Cys Arg         595 600 605 Lys Ala Gly Cys Val Tyr Phe Gly Thr Pro Glu Asn Lys Gly Phe Cys     610 615 620 Thr Leu Cys Phe Ile Glu Tyr Arg Glu Asn Lys His Phe Ala Ala Ala 625 630 635 640 Ser Gly Lys Val Ser Pro Thr Ala Ser Arg Phe Gln Asn Thr Ile Pro                 645 650 655 Cys Leu Gly Arg Glu Cys Gly Thr Leu Gly Ser Thr Met Phe Glu Gly             660 665 670 Tyr Cys Gln Lys Cys Phe Ile Glu Ala Gln Asn Gln Arg Phe His Glu         675 680 685 Ala Lys Arg Thr Glu Glu Gln Leu Arg Ser Ser Gln Arg Arg Asp Val     690 695 700 Pro Arg Thr Thr Gln Ser Thr Ser Arg Pro Lys Cys Ala Arg Ala Ser 705 710 715 720 Cys Lys Asn Ile Leu Ala Cys Arg Ser Glu Glu Leu Cys Met Glu Cys                 725 730 735 Gln His Pro Asn Gln Arg Met Gly Pro Gly Ala His Arg Gly Glu Pro             740 745 750 Ala Pro Glu Asp Pro Pro Lys Gln Arg Cys Arg Ala Pro Ala Cys Asp         755 760 765 His Phe Gly Asn Ala Lys Cys Asn Gly Tyr Cys Asn Glu Cys Phe Gln     770 775 780 Phe Lys Gln Met Tyr Gly 785 790 <210> 4 <211> 2373 <212> DNA <213> A20 cDNA sequence <400> 4 atggctgaac aagtccttcc tcaggctttg tatttgagca atatgcggaa agctgtgaag 60 atacgggaga gaactccaga agacattttt aaacctacta atgggatcat tcatcatttt 120 aaaaccatgc accgatacac actggaaatg ttcagaactt gccagttttg tcctcagttt 180 cgggagatca tccacaaagc cctcatcgac agaaacatcc aggccaccct ggaaagccag 240 aagaaactca actggtgtcg agaagtccgg aagcttgtgg cgctgaaaac gaacggtgac 300 ggcaattgcc tcatgcatgc cacttctcag tacatgtggg gcgttcagga cacagacttg 360 gtactgagga aggcgctgtt cagcacgctc aaggaaacag acacacgcaa ctttaaattc 420 cgctggcaac tggagtctct caaatctcag gaatttgttg aaacggggct ttgctatgat 480 actcggaact ggaatgatga atgggacaat cttatcaaaa tggcttccac agacacaccc 540 atggcccgaa gtggacttca gtacaactca ctggaagaaa tacacatatt tgtcctttgc 600 aacatcctca gaaggccaat cattgtcatt tcagacaaaa tgctaagaag tttggaatca 660 ggttccaatt tcgccccttt gaaagtgggt ggaatttact tgcctctcca ctggcctgcc 720 caggaatgct acagataccc cattgttctc ggctatgaca gccatcattt tgtacccttg 780 gtgaccctga aggacagtgg gcctgaaatc cgagctgttc cacttgttaa cagagaccgg 840 ggaagatttg aagacttaaa agttcacttt ttgacagatc ctgaaaatga gatgaaggag 900 aagctcttaa aagagtactt aatggtgata gaaatccccg tccaaggctg ggaccatggc 960 acaactcatc tcatcaatgc cgcaaagttg gatgaagcta acttaccaaa agaaatcaat 1020 ctggtagatg attactttga acttgttcag catgagtaca agaaatggca ggaaaacagc 1080 gagcagggga ggagagaggg gcacgcccag aatcccatgg aaccttccgt gccccagctt 1140 tctctcatgg atgtaaaatg tgaaacgccc aactgcccct tcttcatgtc tgtgaacacc 1200 cagcctttat gccatgagtg ctcagagagg cggcaaaaga atcaaaacaa actcccaaag 1260 ctgaactcca agccgggccc tgaggggctc cctggcatgg cgctcggggc ctctcgggga 1320 gt; ccaccgacaga cacccagccc ttttctgttc agtgagacca ctgccatgaa gtgcaggagc 1440 cccggctgcc ccttcacact gaatgtgcag cacaacggat tttgtgaacg ttgccacaac 1500 gcccggcaac ttcacgccag ccacgcccca gaccacacaa ggcacttgga tcccgggaag 1560 tgccaagcct gcctccagga tgttaccagg acatttaatg ggatctgcag tacttgcttc 1620 aaaaggacta cagcagaggc ctcctccagc ctcagcacca gcctccctcc ttcctgtcac 1680 cctcgttcca agtcagatcc ctcgcggctc gtccggagcc cctccccgca ttcttgccac 1740 agagctggaa acgacgcccc tgctggctgc ctgtctcaag ctgcacggac tcctggggac 1800 aggacgggga cgagcaagtg cagaaaagcc ggctgcgtgt attttgggac tccagaaaac 1860 aagggctttt gcacactgtg tttcatcgag tacagagaaa acaaacattt tgctgctgcc 1920 tcagggaaag tcagtcccac agcgtccagg ttccagaaca ccattccgtg cctggggagg 1980 gaatgcggca cccttggaag caccatgttt gaaggatact gccagaagtg tttcattgaa 2040 gctcagaatc agagatttca tgaggccaaa aggacagaag agcaactgag atcgagccag 2100 cgcagagatg tgcctcgaac cacacaaagc acctcaaggc ccaagtgcgc ccgggcctcc 2160 tgcaagaaca tcctggcctg ccgcagcgag gagctctgca tggagtgtca gcatcccaac 2220 cagaggatgg gccctggggc ccaccggggt gagcctgccc ccgaagaccc ccccaagcag 2280 cgttgccggg cccccgcctg tgatcatttt ggcaatgcca agtgcaacgg ctactgcaac 2340 gaatgctttc agttcaagca gatgtatggc taa 2373

Claims (11)

Culturing a transformant transformed with an expression vector containing a gene encoding a TAT-A20 fusion protein in which a cell permeable TAT protein and an A20 protein are fused;
Obtaining a culture of the transformant and disrupting the cells to recover and wash the inclusion bodies;
Solubilizing the inclusion body; And
The solubilized TAT-A20 fusion protein was incubated at 37 ° C under conditions of pH 8.2 with 600 mM L-arginine, 45-55 mM Tris, 18-22 mM NaCl, 0.7-0.9 mM KCl, 0.8-1.2 mM EDTA, 1.8-2.2 mM glutathione Dialyzed and refolded in a solution containing a reducing agent (reducing type) and 0.8 to 1.2 mM glutathione (an oxycyclic), and refolding the resultant. The method according to claim 1,
In the step of culturing the transformant,
i) preparing a DNA sequence encoding a cell permeable TAT protein and an A20 protein,
Ii) introducing the DNA sequence operatively fused to an expression vector to produce a recombinant expression vector,
Iii) transforming the host cell with a recombinant expression vector, and
Iv) culturing the transformant to express a TAT-A20 fusion protein. The method according to claim 1,
Wherein the lysogen is used for lysing the cells. The method according to claim 1,
Wherein the step of solubilizing is carried out by adding a solution of guanidine chloride as a modifier. delete delete delete delete delete delete delete

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