KR100538465B1 - Composition comprising kringle domain 1-2 peptide having anti-angiogenic activity and cancer growth inhibitory activity and its use thereof - Google Patents

Abstract

Kringle domain protein TK1-2 which inhibits angiogenesis and cancer growth derived from human tissue type plasminogen activator (t-PA), vectors and strains expressing the same, and the protein It relates to a pharmaceutical composition. RT-PCR confirmed cDNA, E. coli expression plasmid, yeast pichia expression plasmid, protein expression and isolation and purification. It was confirmed that the growth of cancer in the experimental animal model. The present invention can be usefully used for diseases such as rheumatoid arthritis, retinopathy, psoriasis as well as cancer.

Description

Composition comprising kringle domain 1-2 peptide having anti-angiogenic activity and cancer growth inhibitory activity and its use approximately}

The present invention relates to a protein having angiogenesis inhibitory activity and cancer growth inhibitory activity. More specifically, the present invention relates to a recombinant protein TK1-2 comprising Kringle domains 1 and 2 of human tissue tissue plasminogen activator (t-PA), a preparation method thereof, and a pharmaceutical composition containing the same.

Angiogenesis processes are essential for tumor growth and metastasis (Folkman, J., J. Natl. Cancer Inst. , 82 , pp 4-6, 1990; Millauer, B. et al. , Nature , 367 , pp 576-579, 1994). Many researchers have already tried to treat tumors by inhibiting the angiogenesis process, and as a result, various angiogenesis including angiostatin, endostatin, PK 5, prothrombin kringle 2, etc. Inhibitors have been reported (O'Reilly, MS et al , Cell , 79 , pp 315-328 1994; O'Reilly, MS et al. , Cell , 88 , pp 277-285, 1997; Cao, Y., J Biol. Chem. , 272 , pp 22924-22928, 1997; Lee TH, J. Biol. Chem ., 273 , pp 28805-28812, 1998). Some of these inhibitors, including angiostatin, are characterized by a common kringle structure. These anti-angiogenic agents are characterized by inadequate drug resistance, no toxicity, gastrointestinal symptoms and bone marrow suppression and no hair loss, which can increase the effectiveness of radiation therapy, and blood There is no brain barrier (BBB), which can compensate for the disadvantages of chemotherapeutic agents.

Angiogenesis inhibitors can also be used to inhibit cancer growth and metastasis, as well as to treat retinopathy, rheumatoid arthritis, psoriasis, etc. (Folkman, J., Nature Med. , 1 , pp27-31, 1995).

Tissue type plasminogen activator (t-PA) is a multi-domain serine protease and is involved in fibrinolysis and tumor metastasis (Rijken, DC, J. Biol. Chem ., 257 , pp 2920-2925, 1982; Mignatti, P., Cell , 47 , pp 487-498, 1986). t-PA is largely divided into five groups: finger domain (F), epithelial cell growth factor domain (G), two kringle domain (kringle II domain) and serine protease (P) from the N-terminal side. It consists of domains. The Kringle domain of about 70 to 80 amino acids is characterized by a loop structure linked by triple disulfide bonds, and the triple disulfide bonds are strictly preserved between the Kringle structures. t-PA kringles have relatively low amino acid sequence identity with plasminogen kringles (26-39%).

In the prior art, tissue-type plasminogen activator (t-PA) is disclosed as a fibrin soluble agent having coagulation lytic activity (Korean Patent Publication No. 1997-5251), and Korean Patent 1997-1237 discloses a novel plasminogen activity. Factors are disclosed as thrombolytics for the treatment of vascular diseases such as myocardial infarction, seizures, heart attacks, pulmonary embolism, deep vein thrombosis, peripheral artery occlusion and the like. There are many prior arts as thrombolytic agents that dissolve fibrin and inhibit blood coagulation, but it is not known until now whether some sequences have endothelial growth inhibition, angiogenesis inhibition, and cancer growth inhibition.

Therefore, the present inventors have conducted in vitro and in vivo tests on the Kringle domain of tissue type plasminogen activator (t-PA) for the development of a new anti-cancer drug with no drug resistance. The purpose of this study was to investigate whether angiogenesis inhibitory effect was observed in vivo and to investigate the anticancer effect in experimental animal models to find the possibility of new anticancer drugs. Established a method for efficiently producing recombinant proteins by establishing technologies for expression, refolding, and purification in E. coli and yeast as recombinant proteins with only two Kringle domains in t-PA. Production and purification in large quantities to secure a sufficient sample, and then confirmed the effect on the angiogenesis in vivo through the proliferation inhibitory effect on endothelial cells and CAM assay, and finally human lung cancer cells ( The present invention was completed by investigating the anticancer effect in an experimental animal model implanted with human lung cancer cells, and confirming the effect on cancer growth and angiogenesis through tissue staining.

An object of the present invention is to express and purify the Kringle portion of the plasminogen as a recombinant protein, thereby revealing an angiogenesis inhibitor by the recombinant protein of the present invention revealing vascular endothelial cell proliferation inhibitory effect, angiogenesis inhibitory effect and cancer growth inhibitory effect. It is to provide a novel use as a growth inhibitor of cancer.

In order to achieve the above object, the present invention provides a recombinant protein TK1 having an amino acid sequence as set forth in SEQ ID NO: 1, which is derived from human tissue type plasminogen activator (t-PA) and has an inhibitory activity on angiogenesis and cancer. Gives -2.

The present invention provides a recombinant plasmid expressing the protein, TK1-2 (inserted gene) / pET-15b (vector), which is a recombinant E. coli expression plasmid.

In addition, the present invention is a recombinant plasmid expressing the protein, His-TK1-2 (insert gene) / pPICZα-C (vector) and N-TK1-2 (insert gene) / pPICZα-C (recombinant yeast expression plasmid) Vector).

The present invention also provides a pharmaceutical composition for inhibiting endothelial cell growth containing the Kringle domain TK1-2 protein.

The present invention also provides a pharmaceutical composition for inhibiting angiogenesis containing the Kringle domain TK1-2 protein.

The present invention also provides a pharmaceutical composition for inhibiting growth of cancer containing the Kringle domain TK1-2 protein.

Hereinafter, the present invention will be described in detail.

The present invention provides a protein composition having the amino acid sequence set forth in SEQ ID NO: 1 derived from human tissue type plasminogen activator.

The TK1-2 protein is a part corresponding to 1 and 2 of the kringle domain of plasminogen, and the amino acid sequence from the 90th amino acid sequence serine to the 263th threonine of human plasminogen protein (SEQ ID NO: 1). It includes, it may be encoded by the nucleotide sequence represented by SEQ ID NO: 2.

The present invention provides a plasmid consisting of E. coli or yeast expression vector and a base sequence and a modified base sequence encoding the protein in order to express the protein in E. coli and yeast for the expression of the protein.

The modified base sequence may be used by selecting one or more of a base sequence encoding 5 to 10 histidine amino acids, an amino acid coding sequence cut by thrombin, and a c-myc epitope base sequence.

RT-PCR was performed on the whole RNA extracted from human blood, tissue, or cells to prepare cDNA, and PCR was performed using primers SEQ ID NO: 3 and 4 or SEQ ID NO: 5 and 6 to include SEQ ID NO: 2. cDNA can be amplified. By treating the PCR product with restriction enzymes, the plasmid for protein expression can be prepared by cloning the E. coli expression vector or yeast expression vector.

The present invention provides a recombinant E. coli expression plasmid TK1-2 / pET-15b comprising the nucleotide sequence of SEQ ID NO: 2 as a recombinant plasmid expressing the protein, recombinant E. coli transformed with TK1-2 / pET-15b, Preferably, BL21 (DE3) / TK1-2 / pET-15b strain is provided.

The present invention also provides His-TK1-2 / pPICZα-C and N-TK1-2 / pPICZα-C, which are recombinant yeast expression plasmids comprising the nucleotide sequence of SEQ ID NO: 2 as a recombinant plasmid expressing the protein, This provides a yeast strain transformed with His-TK1-2 / pPICZα-C or a yeast strain transformed with N-TK1-2 / pPICZα-C.

The yeast strain is Pichia pastoris , preferably X-33 or GS115 can be used.

The cell line X-33 / N-TK1-2 / pPICZα-C, which produces the TK1-2 protein of the present invention, was deposited with KRIBB as Accession No. KCTC 10415BP on January 27, 2003.

In addition, the present invention provides a method for purifying TK1-2 protein expressed in E. coli, comprising the following steps.

1) preparing E. coli transformed with TK1-2 / pET-15b, an E. coli expression plasmid comprising SEQ ID NO: 2;

2) inducing and expressing protein expression in E. coli;

3) collecting and crushing the cultured Escherichia coli;

4) separating the soluble portion and the non-soluble portion;

5) washing the enclosure

6) passing the non-soluble portion through heat-tack affinity column chromatography;

7) refolding; And

8) obtaining a recombinant protein TK1-2; provides a method for producing a recombinant TK1-2 protein comprising a.

In a preferred embodiment of the present invention, a TK1-2 / pET-15b expression plasmid comprising a heat-tack sequence is transformed into Escherichia coli BL21 (DE3), and the transformant is 37 ° C using LB medium containing antibiotics. Incubate at until the absorbance (A ₆₀₀ ) reaches 0.6. In order to induce recombinant protein production, isopropyl thio-β-galactopyranoside (IPTG) was added to the medium to a final concentration of 1.0 mM, and then incubated again at 37 ° C. for 3 to 4 hours, and then 15 at 8,000 × g. Collect E. coli by centrifugation for a minute. The next step is to refold and purify the TK1-2 protein. The E. coli pellets were dissolved in lysis buffer (20 mM Tris-Cl, pH 7.9, 50 mM NaCl, 1 mM MgCl ₂ , 0.1 mM CaCl ₂ , 20). Suspension in μg / ml RNase A, 50 μg / ml DNase I), followed by sonication for 15-30 minutes to destroy E. coli, and centrifugation at 1,000 × g for 20 minutes for cell residue and inclusion body layer. After the separation was centrifuged at 4 ℃, 20,000 × g for 30 minutes to collect only the inclusion body pellet. IB wash buffer (IB wash buffer, 20 mM Tris-Cl, pH 7.9, 0.5 M NaCl, 2 M urea) was added to the harvested inclusion bodies, suspended by sonication and centrifuged at 20,000 × g for 30 minutes to obtain inclusion bodies. The inclusion body was suspended in IB soluble buffer (IB soluble buffer, 20 mM Tris-HCl, pH 7.9, 0.5 M NaCl, 5 mM imidazole, 8 M Urea), reacted overnight at 4 ° C. for 12 hours, and dissolved in IB dilution buffer ( Dilute urea to 6 M with IB dilution buffer, 20 mM Tris-HCl, pH 7.9, 0.5 M NaCl, 5 mM imidazole, 2 M Urea). The protein is purified by centrifugation at 18,000 × g for 30 minutes at 4 ° C. followed by His-tag affinity chromatography.

The band 10 ㎖ Pro ^TM resin (ProBond ^TM resin) is filled (packing) to the glass column. After washing with primary distilled water, which is three times the volume of resin, and doubling the charge buffer (putting 0.1 M of NiSO ₄ into the binding buffer), the volume of binding buffer is three times. (0.5 M NaCl, 20 mM Tris-HCl, pH 7.9, 5 mM Imidazole) and 3 volumes of urea binding buffer (0.5 M NaCl, 20 mM Tris-HCl, pH 7.9, 5 mM Imidazole, 6 M) Urea) to level out. After passing the sample, it was washed with 10 times volume of urea binding buffer of resin, and 2 times volume of wash buffer (wash buffer, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9, 20 mM imidazole, 6 M urea). Wash again with). Elution buffer (0.5 M NaCl, 20 mM Tris-HCl, pH 7.9, 1 M Imidazole, 6 M Urea) was fractionated by 10 ml.

Add 1.5 L of 3 M urea refolding buffer (0.1 M Tris-HCl, pH 8.0, 0.15 M NaCl, 3 M Urea) and add reduced glutathione (GSH) to a final concentration of 1 mM. Dilute urea concentration to 2 M with refolding dilution buffer (0.1 M Tris-HCl, pH 8.0, 0.15 M NaCl) and shake slowly for 24 hours at room temperature with GSH added to maintain final concentration of 1 mM. Then, add oxidized glutathione (GSSG) to 0.1 mM, shake slowly at room temperature for 4 to 6 hours, and adjust the pH to 5.5 with 4 M acetic acid. Recombinant TK1-2 purified by concentrating to 20 ml using a Millipore Labscale TFF system and dialyzed three times with 100-fold volume of 20 mM tris-acetate (pH 5.5) solution and third distilled water, respectively. Proteins can be obtained.

With some modification to the protein purification process, the TK1-2 protein of the present invention was expressed by expressing the protein in a cell line transformed with His-TK1-2 / pPICZα-C or N-TK1-2 / pPICZα-C. Can be obtained.

The purified TK1-2 protein of the present invention showed an excellent inhibitory effect in the endothelial cell growth assay, CAM analysis was confirmed an angiogenesis inhibitory function, inhibited the growth of cancer in the experimental animal model of lung cancer It was.

The present invention also provides a composition having an endothelial cell growth inhibitory activity containing the recombinant protein TK1-2.

The present invention also provides a composition having angiogenesis inhibitory activity containing the recombinant protein TK1-2.

The present invention also provides a composition having growth inhibitory activity of cancer containing the recombinant protein TK1-2.

The present invention also provides a pharmaceutical composition for the prevention and treatment of diseases caused by endothelial cell growth containing the recombinant protein TK1-2.

The present invention also provides a pharmaceutical composition for the prevention and treatment of diseases caused by angiogenesis containing the recombinant protein TK1-2.

The present invention also provides a pharmaceutical composition for the prevention and treatment of cancer diseases containing the recombinant protein TK1-2.

Diseases caused by angiogenesis include rheumatoid arthritis, osteoarthritis, sepsis arthritis, psoriasis, corneal ulcer, age-related macular degeneration, diabetic retinopathy, proliferative vitreoretinopathy, immature retinopathy, ophthalmic inflammation, cone cornea, Sjogren's syndrome, myopia ophthalmic tumor, corneal graft rejection, aberrant wound union, bone disease, proteinuria, abdominal aortic aneurysm, degenerative cartilage loss due to traumatic joint injury, demyelination of the nervous system, cirrhosis, renal glomeruli, immature tear , Inflammatory bowel disease, periodontal disease, arteriosclerosis, restenosis, inflammatory diseases of the central nervous system, Alzheimer's disease, skin aging, cancer infiltration and metastasis, the pharmaceutical composition of the present invention can be used for the treatment of diseases caused by angiogenesis have

The cancer diseases include lung cancer, non-small cell lung cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, gastric cancer, anal muscle cancer, colon cancer, breast cancer, and fallopian tube carcinoma , Endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penis cancer, prostate cancer , Chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, brain stem glioma, pituitary adenoma The pharmaceutical composition of the present invention can be used for the treatment of cancer.

The composition containing the TK1-2 protein of the present invention as an active ingredient can be used as a single agent or in combination with other active ingredients that can enhance the efficacy.

In this case, a pharmaceutically acceptable diluent may be used, and the diluent includes, but is not limited to, saline solution, buffered saline solution, dextrose, maltodextrin, water, glycerol, ethanol and combinations thereof.

The TK1-2 protein composition of the present invention may be administered in a pharmaceutical composition with one or more pharmaceutically acceptable excipients.

The specific therapeutically effective amount for a particular patient is determined by the specific composition, including the type and extent of the reaction to be achieved and whether other agents are used in some cases, the age, weight, general health, sex and diet of the patient, and the time of administration. It is desirable to apply differently depending on various factors and similar factors well known in the medical field, including the route of administration and the rate of release of the composition, the duration of treatment, and the drug (eg, chemotherapeutic agent) used or co-used with the specific composition.

The unit dosage of the TK1-2 protein composition of the present invention is preferably 5 mg to 2 g, most preferably 10 mg to 1 g.

In addition, depending on the age and weight of the patient as well as the type of disease and the extent of the disease caused by angiogenesis, the dosage and the method of administration vary, but can generally be administered at 0.05 to 200mg / ㎏ body weight 1 to 3 times a day It is preferable to administer.

The pharmaceutical compositions of the present invention may be administered in a conventional manner via the oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, nasal, inhalation, intraocular or intradermal routes.

Parenteral administration means administration modes including intravenous, intramuscular, intraperitoneal, intrasternal, transdermal and intraarterial injection and infusion. Parenteral administration of the pharmaceutical compositions of the present invention may be carried out in unit dosage form in combination with a pharmaceutically acceptable carrier, i.e., nontoxic to the receptor and compatible with other agent components at the concentrations and dosages employed, under the desired purity. It is preferable to prepare.

In addition, the formulation of the pharmaceutical composition of the present invention can be applied to any formulation, and the prepared formulation can be used for oral, injection, and application. The formulation may be prepared in an injectable form (solution, suspension or emulsion), and may be prepared orally for tablets, capsules, soft capsules, infusions, granules, pills and the like.

The composition is prepared by filling the capsule with TK1-2 protein without excipients or by uniformly sufficient contact with the particulate solid or liquid carrier or both. The product is then shaped into the desired formulation if necessary.

Examples of such carrier excipients include starch, water, saline, Ringer's solution and dextrose solution. Suitable formulations known in the art are preferably used as disclosed in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the examples.

Example 1 TK1-2 Protein Expression, Purification and Efficacy Screening in Escherichia Coli

1-1. TK1-2 / pET-15b Plasmid Construction

After obtaining total RNA of umbilical vein endothelial cells (see HUVEC, 1-6-2) using a Qiagen RNA isolation kit, cDNA was prepared using oligo-d (T) primer and cDNA synthesis kit (First strand cDNA synthesis kit, CDNA was prepared using Roche Molecular Biochemicals. Subsequently, cDNA encoding amino acids from the 90th amino acid alanine of human t-PA to 263 threonine was produced by PCR using Pfu transcriptase (Stratagene). At this time, primer 1 (5'-ATCGGATCCCATGGCCACGTGCTACGAGGACCAG-3 ') and primer 2 (5'-ATCGGATCCTCAGGTGGAGCAGGAGGGCACATC-3') described in SEQ ID NO: 3 were used. The amplified cDNA was treated with BamHI, ligated to dephosphorylated pET-15b (Novagen) to produce a recombinant plasmid TK1-2 / pET-15b, and the plasmid structure is shown in FIG. 1. The recombinant plasmid has 26 additional amino acids at its N-terminal, including a his-tag sequence and a thrombin cleavage site. The prepared plasmid was identified using a DNA sequencing version (T7 sequencing version 2.0 DNA sequencing kit, Amersham Pharmacia Biotech). For protein expression, TK1-2 / pET-15b was transformed into BL21 (DE3) Escherichia coli by conventional methods in the art.

1-2. TK1-2 Protein Expression and Purification

E. coli transformants were incubated at 37 ° C. using 3 liter LB medium (containing 100 μg / ml ampicillin) and incubated until absorbance (A ₆₀₀ ) reached 0.6. In order to induce recombinant protein production, IPTG (isopropyl thio-β-galactopyranoside, Amresco) was added to the medium to a final concentration of 1.0 mM, and then incubated at 37 ° C. for about 3 hours, and then 20 at 8,000 × g. Cells were collected by centrifugation for a minute.

The next step was to refold and purify the TK1-2 protein. The cell pellet is suspended in 40 ml of lysis buffer (lysis buffer, 20 mM Tris-Cl, pH 7.9, 50 mM NaCl, 1 mM MgCl ₂ , 0.1 mM CaCl ₂ , 20 µg / ml RNase A, 50 µg / ml DNase I). Sonication was performed for 15-30 minutes. After centrifugation at 1,000 x g for 20 minutes to separate the cell residue and the inclusion body layer was centrifuged at 4 ℃, 20,000 × g for 30 minutes to obtain only the inclusion body pellets.

When the recombinant protein expression of E. coli (TK1-2 / pET-15b) expressing TK1-2 was induced, almost all proteins appeared in the form of inclusion bodies (IB) (see FIG. 2). The inclusion body pellet was added to 40 ml of IB wash buffer (IB wash buffer, 20 mM Tris-Cl, pH 7.9, 0.5 M NaCl, 2 M urea), suspended by sonication and centrifuged at 20,000 × g for 30 minutes to wash the inclusion body. It was. The washed inclusion body was suspended in 18 ml of IB soluble buffer (IB soluble buffer, 20 mM Tris-HCl, pH 7.9, 0.5 M NaCl, 5 mM imidazole, 8 M Urea) and reacted overnight at 4 ° C. for 12 hours. The urea concentration was diluted to 6 M with dilution buffer (IB dilution buffer, 20 mM Tris-HCl, pH 7.9, 0.5 M NaCl, 5 mM imidazole, 2 M Urea). The proteins were purified by centrifugation at 18,000 × g for 30 minutes at 4 ° C. followed by His-tag affinity chromatography (see Examples 1-3 below). 1.5 L of 3 M urea refolding buffer (0.1 M Tris-HCl, pH 8.0, 0.15 M NaCl, 3 M Urea) was added and reduced glutathione (glutathione, GSH, Amresco) was added to a final concentration of 1 mM. . The dilution buffer solution (0.1 M Tris-HCl, pH 8.0, 0.15 M NaCl) was diluted to 2 M urea and the GSH was added to maintain a final concentration of 1 mM and shaken slowly at room temperature for 24 hours. Then, oxidized glutathione (GSSG, Sigma) was added to 0.1 mM, shaken slowly at room temperature for 4-6 hours, and adjusted to pH 5.5 with 4 M acetic acid. The solution was concentrated to 20 ml using a Millipore Labscale TFF system (Milliipore) and dialyzed three times with 100-fold volume of 20 mM tris-acetate (pH 5.5) solution and tertiary distilled water, respectively. Proteins were identified by SDS-PAGE (see Figures 2 and 3).

1-3: Chromatography Using His-tag Affinity Column

A 10 ㎖ pro band ^TM resin (ProBond ^TM resin, Invitrogen Corporation, Carlsbad, CA) was packed (packing) to the glass column. After washing with primary distilled water, which is three times the volume of resin, and doubling the charge buffer (putting 0.1 M of NiSO ₄ into the binding buffer), the volume of binding buffer is three times. (0.5 M NaCl, 20 mM Tris-HCl, pH 7.9, 5 mM Imidazole) and 3 volumes of urea binding buffer (0.5 M NaCl, 20 mM Tris-HCl, pH 7.9, 5 mM Imidazole, 6 M) Urea) was flushed and leveled. After passing the sample, it was washed with 10 times volume of urea binding buffer of resin, and 2 times volume of wash buffer (wash buffer, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9, 20 mM imidazole, 6 M urea). ) And washed again. Elution buffer (elution buffer, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9, 1 M Imidazole, 6 M Urea) was eluted and fractionated 10 ml.

Approximately 5 g of the cell pellet was obtained from the culture of the 5 L flask, and 3 to 4 g of the inclusion body were obtained from the cell pellet. After purification and refolding through a His-tag affinity column, the final 7-12 mg was obtained. / l TK1-2 protein was obtained.

The expressed and purified protein samples were subjected to SDS-PAGE and amino acid sequence analysis and protein mass spectrometry of Examples 1-4.

FIG. 2 is a photograph of Coomassie blue staining of the samples obtained for each step of protein purification on 14% SDS-polyacrylamide gel under electrophoresis (using tris-glycine buffer solution) under reducing conditions. Lane 1 is the MW standard protein Mark12 (Invitrogen), lane 2 is the E. coli protein, lane 3 is the soluble fraction of E. coli protein, lane 4 is the non-soluble fraction of E. coli protein, and lane 5 is dissolved in 6M urea solution. Lane 6 is a fraction obtained by passing a sample of lane 5 through a His-tag Ni + affinity column, lane 7 is a fraction eluted with a 20 mM imidazole solution, and lane 8 is 1M from a His-tag affinity column. Sample eluted with imidazole solution and lane 9 is the final sample obtained after dialysis. Purified TK1-2 appeared as a single band on SDS-PAGE under reducing conditions, and the size was 22 to 23 kilodaltons.

Lane 1 of FIG. 3 is electrophoresed in purified TK1-2 under non-reducing conditions, and lane 2 is electrophoresized under reducing conditions. The obtained protein showed faster migration on SDS-PAGE under non-reducing conditions, compared to the expected molecular weight of 22,075 Daltons. In the reducing condition in which β-mercaptoethanol is present, it was confirmed that the mobility of the molecule was reduced during electrophoresis.

1-4: Protein Analysis

The amino acid sequences were analyzed using the Procise 491 protein sequencing system (Applied Biosystems) of the Korea Basic Science Institute (Seoul, Korea). MALDI mass spectrometry (Matrix-assisted laser desorption ionization mass spectrometry) was also performed by the Voyager Biospectrometry workstation (PerSeptive Biosystems, Framinghum, MA, USA).

As a result of the amino acid analysis, N-terminal methionine was lost, which was 21,947 Da when MALDI mass spectrometry (see FIG. 4) was consistent with the calculated molecular weight of 21,944 Da where methionine was lost. Samples were prepared to have an endotoxin level of 0.3 EU / mg or less, and were prepared for animal testing.

1-5: Investigation of affinity of lysine sepharose of recombinant protein TK1-2

 The Kringle domain of human tissue type plasminogen (t-PA) is known to have an affinity for lysine residues, and the recombinant protein TK1-2 expressed and purified according to the present invention is properly treated during purification. To determine if refolding occurred, we examined the ability of binding to lysine sepharose. 10 g of lysine sepharose was swelled in secondary distilled water for 1 hour at room temperature, then packed into a column and equilibrated with buffer A (50 mM phosphate: pH 7.5), which is three times the resin volume. Then, the prepared sample was passed. Buffer A was washed three times and then washed three times with Buffer B (50 mM phosphate: pH 7.5, 0.5 M NaCl) and with elution buffer (0.2 M ε-aminocaproic acid (ε-ACA) in distilled water). After elution and fractionation of 5 ml each, it was electrophoresed under reducing conditions using tris-glycine buffer on 14% SDS-polyacrylamide gel.

5 and 6 are coomassie blue staining by electrophoresis of the protein sample obtained after passing through the lysine-sepharose column, lane 1 of Figure 5 is a refolded TK1-2 protein, lane 2 is 50mM Tris- Fractions obtained by washing the column with Cl solution, lanes 3, 4 and 5 are the fractions obtained after washing the column with 0.5 M sodium chloride solution, lanes 6, 7 and 8 are the elution fractions. Lane A in FIG. 6 is purified TK1-2 after passing through a lysine-sepharose column and lane B is purified fraction after passing through a heat-tack affinity column.

As shown in FIGS. 5 and 6, a protein fraction of about 22 KDa bound to lysine-sepharose and eluted with a solution of 0.2 M ε-ACA was identified. Therefore, it was determined that the Kringle structure of the protein of the present invention, which had been purified, was properly refolded to form a lysine-binding pocket.

1-6: Inhibitory Effects of TK1-2 on Endothelial Cells

(1-6-1) TK1-2 Inhibitory Effect on Bovine Capillary Endothelial Cell Proliferation

On bovine capillary endothelial (BCE) cell growth stimulated by bFGF (basic fibroblast growth factor), the inhibitory effect of the purified TK1-2 protein of the present invention was observed. BCE cell proliferation assays were performed by O'Reilly, MS et al .; Cell , 79 , pp315-328, 1994; Lee, HS et al . ; Arch. Biochem. Biophys. , 375 , pp359-363, 2000 Was performed according to

12,500 BCE cells per well were placed on gelatin coated 24-well culture plates and incubated for 24 hours in DMEM medium (10% FBS, with 1% antibiotics). Existing medium was then replaced with 0.25 ml DMEM medium (5% FBS, with 1% antibiotic) and TK1-2 protein samples or angiostatin at concentrations from 0 nM to 320 nM. After 30 min incubation, 0.25 ml DMEM (5% FBS, 1% antibiotic) and 2 ng / ml bFGF (R & D system) were added to each well. After 72 hours, the cells were spread out by trypsinization and the number of cells was counted by trypan blue exclusion method.

As shown in Figure 7, protein TK1-2 showed a growth inhibitory effect on bFGF-stimulated BCE cells, and showed a protein-dependent pattern. The concentration of TK1-2 showing a 50% inhibitory effect (ED ₅₀ ) was about 40 nM. In the case of angiostatin, the concentration showing 50% inhibitory effect was about 80nM, whereas TK1-2 showed a significant inhibitory effect.

(1-6-2) Inhibitory Effect of TK1-2 on Proliferation of Umbilical Vein Endothelial Cells

Human Umbilical Vein Endotherial Cells (HUVECs) are known from the umbilical cord of humans obtained by caesarean section (Jaffe et al . ; J. Clin. Invest. , 52 , pp 2745-2756, 1973). Isolated by application, M199 medium (20% FBS, 30 μg / ml endothelial cell growth supplement (Sigma), 90 μg / ml heparin, 25 mM Hepes, 2.2 g / L sodium bicarbonate, 2 mM L- Glutamine and 1% antibiotic). Cells were passaged to use cells of the 3, 4 and 5 generations for the experiment. Using cultured endothelial cells, a [ ³ H] -thymidine incorportion assay was performed as follows.

20,000 umbilical cord endothelial cells per well were placed in gelatin coated 24-well culture plates and incubated for 24 hours in M199 medium (10% FBS, 90 μg / ml heparin, with 1% antibiotics). Existing medium was then added 0.25 mL M199 medium (5% FBS, 90 μg / mL heparin, with 1% antibiotic) and His-TK1-2 protein samples at concentrations from 0 nM to 320 nM. After 30 min incubation, 0.25 ml M199 (5% FBS, 90 μg / ml heparin, 1% antibiotic) and 6 ng / ml bFGF (R & D system) were added to each well. After 18 hours, [ ³ H] -thymidine was added to 1 μCi (0.037 MBq) and further incubated for 6 hours. After incubation, the cells were fixed with methanol, washed three times with cold 10% TCA, dissolved in 0.25 N NaOH, 1% SDS, and radioactivity was measured using a liquid scintillation counter.

As shown in Figure 8, protein TK1-2 showed a growth inhibitory effect on bFGF-stimulated HUVE cells, and showed a protein-dependent pattern.

1-7: Observation of angiogenesis inhibitory effect of TK1-2 in vivo by CAM assay

In order to confirm the antiangiogenic effect in vivo, Chorioallanotic Membrane (CAM) analysis was performed (Nguyen, M. et al .; Microvascular Res ., 47 , pp31-40, 1994). Briefly, the CAM analysis method is an experiment in which a drug is placed on a CAM of a 4.5-day fertilized egg, a dried coverslip and a microscope is observed two days after the drug angiogenesis occurs. As a normal control group for comparison, only coverslips without drug dropping were placed on the CAM and observed after 2 days to observe normal vascular morphology.

Fertilized eggs (Pulmuone, Korea) were incubated at 37 ° C. with 80-90% humidity. On day 2 the albumin part was removed and on day 3 the upper shell was removed to create a window. On day 4.5 of cultivation, to Termanox coverslip (Thermanox coverslip, Nunc, Roskilde, Denmark; cut to 1/4 size of coverslip), 5 μg, 20 μg and 40 μg of protein TK1-2 were added dropwise, respectively. Each fertilized egg was placed on the CAM and cultured for 2 days, and then 20% fat emulsion was injected into the CAM to observe the angiogenesis inhibition zone.

In FIG. 9, the area indicated by the arrow is the site where angiogenesis is suppressed, and in the result of FIG. 10, as the amount of TK1-2 increases, the ratio of CAM having a site without blood vessels increases. Suppression was observed. No toxicity by TK1-2 was observed in fertilized eggs.

1-8: Investigation of anticancer effect using experimental animal model

(1-8-1) Cell Culture

PC14, a non-small cell lung cancer cell distributed at the Catholic Institute of Medicine and Development, is a T75 flask with DMEM medium containing 10% FBS (Sigma) (Cheil Biotech Service, Daegu, Korea). Incubated at 5% CO ₂ , 37 ℃.

(1-8-2) Experiment Animal

Laboratory animals (Hamamatsu, Japan) received BALB / c nude mice weighing about 18g at 4 weeks of age, and sterilized solid feeds without any antibiotics (Samyang Feed Co., Ltd.) and tap water and supplied them in a laboratory environment. Was used in the experiment.

(1-8-3) Transplantation of Tumor Cells

A total of 30 rats were suspended in 200 μl of PBS with 3 × 10 ⁶ tumor cells of lung cancer cell line PC14 and injected with 26 gauge needles subcutaneously. After 7 days after implantation, tumor nodule was formed at 80 ~ 110mm3 on the graft site, and TK1-2 protein was administered from this time.

(1-8-4) Administration and Observation of Recombinant Proteins

One week after tumor cell transplantation, the experimental animals were divided into three groups and the recombinant protein was administered. Of the three groups, 300 μl of PBS was added to the control group, 150 μl of 1.5 mg of TK1-2 / ml to the 10 mg / kg group, and 3 mg of TK1-2 to 3 mg / ml to the 50 mg / kg group. Μl was injected daily into the abdominal cavity. Tumor size was measured by the long diameter and short diameter in mm units using a vernier caliper (long diameter ² x short diameter x 0.52 (mm ³ )), and the weight was measured every other day using an electronic balance. The measurement result was calculated as the average of 10 animals, and the error was taken as the standard deviation (SD) value. Significance was verified by comparison with the control group by t-test.

Seven days of administration did not show a significant difference between the three groups, but after 10 days, the difference between the control group and the other treatment groups began to show. After 30 days of administration, the difference in size increased by about five times or more. 11, 12 and 13 are photographs taken on day 40 of treatment. 11 is a control group not treated with TK1-2, FIG. 12 is a group administered with 10 mg / kg of TK1-2, and FIG. 13 is a group treated with 50 mg / kg of TK1-2. On the other hand, it was observed that the tumor size of the 50 mg / kg administration group remained almost ungrown (see FIG. 14). 15 shows the result table for survival rate. The control group survived to 50 days of treatment. All the 10 mg / kg dosed groups were sacrificed on the day that the control group died, and the survival rate was 60%. The growth of the cancer was so strong that the control group began to die from the 30th day of treatment and then died every 4 to 5 days. The overall survival of the control group was determined to be about 58 days. The 50 mg / kg TK1-2 administration group had a 100% survival rate (see FIG. 15), and showed an excellent anticancer activity because cancer growth was inhibited by more than 97%. When administered at 50 mg / kg, 5% of the 50 mice stopped the TK1-2 protein but continued to develop cancer as in the remaining 5 mice treated with TK1-2 until 20 days after stopping. Did not see at all.

1-9: Amino Acid Sequence Comparison of Human TK1-2 and Mouse TK1-2

TK1-2 recombinant protein was prepared based on human t-PA sequences, and in the experiments of the present invention, mice were used as experimental animals, and the differences between these sequences were compared. In comparison, about 76.6% homogeneity was confirmed on the human and mouse TK1-2 amino acid sequences (see FIG. 16).

Therefore, since the amino acid sequence of mouse and human TK1-2 is 75.9%, it was examined whether antibodies were formed when prolonged administration of recombinant TK1-2 in BALB / c nude mice as in the following example.

1-10: Investigation of antibody production by Western blot analysis using mouse blood

Blood was collected on the 40th day of treatment in each group (control group, 10 mg / kg TK1-2 administration group, 50 mg / kg TK1-2 administration group). The collected blood was centrifuged at 14,000 rpm for 5 minutes, divided into plasma and blood cells, and only plasma was transferred to a new tube. Recombinant protein TK1-2 was SDS-PAGE and electrically blotted onto a 0.2 μm nitrocellulose membrane (Schleicher & Schuell, Germany). The protein-transferred membrane was immersed in blocking buffer (5% skim milk in TBST; 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% Tween 20) for 1 hour, and then stirred. Serum obtained from the / kg TK1-2 administration group, 50mg / kg TK1-2 administration group was diluted in blocking buffer at a ratio of 1: 1000 and primary binding for 1 hour. In addition, TK1-2 antibody (Body-Tech Co., Chuncheon, Korea) was used to confirm the immune response. The nitrocellulose membrane was washed three times with TBST three times for 5 minutes and bound for 2 hours using an anti-mouse IgG-HRP secondary antibody diluted 1: 2000 with blocking buffer. After completion of the reaction, washing with TBST was repeated three times for 5 minutes and developed with WEST-ZOL ^™ solution (Intron Biotechnology, Seongnam, Korea).

Referring to the results of FIG. 17, lane A is a Western blot using TK1-2 antibody, lane B is the serum of control group, lane C is the serum of 10 mg / kg TK1-2 administration group, lane D is a result of reacting the serum of a 50 mg / kg administration group. It was found that the antibody was not recognized even in the treatment group administered at the dose of 50 mg / kg. Therefore, the anti-cancer effect of recombinant TK1-2 is not reduced by the production of antibody in the experimental animal model.

1-11: Investigation of effects on angiogenesis and cancer through immunohistochemistry staining

Each group was sacrificed for about 50 days, and then sacrificed. The organs (liver, spleen, lungs, kidneys, heart), including tumor tissues, were extracted for signs of toxicity and PCNA (Proliferating Cell Nuclear Antigen), bFGF, VEGF. (Vascular Endothelial Growth Factor), Angiogenin (angiogenin), von Willebrand Factor (vWF), Matrix Metallo Proteinase 9 (MMP 9), and MMP 10 expression were examined.

FIG. 18 shows the harvested organs of the control group, FIG. 19 shows the harvested organs of the 10 mg / kg administration group, and FIG. 20 shows tumor tissues extracted from the control group. When the tissue was chopped and analyzed, no traces of tissue lesions or metastases could be observed.

Each tissue was extracted for 4 hours in 10% formaldehyde (formaldehyde) solution and immersed in 100% ethanol. The tissue was then fixed with paraffin. After continuous cutting to 7 μm thickness, tissue sections were stained with hematoxylin (hematoxylin, Sigma) and eosin (eosin, Sigma). The metastasized cancer cells could not be observed in the stained sections of each tissue. In the case of the control group, symptoms showing jaundice, a poor health condition, were observed in liver tissues of the experimental animals (see FIG. 21).

Tumor tissues of the control group and the 10 mg / kg TK1-2 group were treated with VEGF, bFGF, vWF, angiogenin, MMP9, MMP10, and PCNA (DAKO, Glostrup, Denmark) with immunoperoxidase. Staining with monoclonal or polyclonal antibodies was performed (see Figures 22 and 23).

In Figure 22, A is a picture of hematoxylin / eosin stained tumor tissue sections of the control group and TK1-2 administration group, B is a picture of the reaction with PCNA monoclonal antibody, C is a picture of the reaction of VEGF monoclonal antibody D Is a photograph reacted with bFGF antibody. The black line in the picture means 25㎛, and the picture is magnified 400 times.

In FIG. 23, A is a tumor tissue section of the control group and the TK1-2 administration group with an angiogenin monoclonal antibody, B is a reaction with vWF polyclonal antibody, C is a photograph of the reaction with MMP9 polyclonal antibody, D is a photograph reacted with MMP10 monoclonal antibody.

When staining cancer tissues, mitotic cells were easily observed in the control group, whereas apoptotic cells composed of fragmented DNA were frequently observed in the treatment group. It became. However, when stained with a single antibody against PCNA, all of the cell proliferation is active even though there may be a difference in the number of cells in the M phase because there is no difference between the control and treatment groups. It can be seen (see Fig. 22). No difference in the proliferation index between the treated and treated groups is consistent with the results reported in the treatment of angiostatin (O'Reilly et al .; Cell , 79 , pp315-328, 1994). . Although the number of cells with Pyknotic nuclei (fragmented DNA) is more observed in the administration group, more quantitative experimental analysis is required.

When expression was examined for angiogenic factors, angiogenin and MMP9 and MMP10, which are important factors for cancer metastasis, MMP9 was found to be less expressed in PC14 cell lines, whereas bFGF, VEGF, MMP10 and angiogenin were relatively high in the control group and interestingly, the expression was significantly decreased in the treatment group (see FIGS. 22 and 23). The expression of tissues was altered by the administration of recombinant TK1-2, which was determined by the angiogenic factors bFGF and VEGF when plasminogen kringles 1-3, angiostatin, was administered. This pattern is consistent with the decrease in expression (Joe, YA et al., Int. J. Cancer , 82 , pp694-699, 1999). Since vWF is generated by endothelial cells constituting the vascular wall, blood vessels can be stained at the time of staining. Unlike other transplant cancer models, blood vessels are difficult to find in the control group as well as in the control group, and thus quantitative evaluation is impossible (see FIG. 23). ).

Example 2 TK1-2 Protein Expression, Purification and Efficacy Screening in Yeast, Pichia

2-1: Preparation of TK1-2 Expressing Pichia Vector

Two recombinant plasmids expressing TK1-2 were prepared, including a plasmid with a His-tag expression, a His-TK1-2 with a non-tag plasmid, and N-TK1-2. . DNA fragments of 525 bp encoding the 89 th amino acid arginine to the 263 th amino acid threonine of human tissue type plasminogen were obtained by PCR using Pfu transcriptase (Stratagene). When preparing His-TK1-2 plasmid, using primer 3 of SEQ ID NO: 5 (5'-CAGTATCGATCAGGGCCACGTGCTACGAG-3 '(ClaI)) and primer 4 of SEQ ID NO: 6 (5'-CTGATCTAGAGTGGAGCAGGAGGGCAC-3' (XbaI)) In preparing the N-TK1-2 plasmid, primer 3 of SEQ ID NO: 5 (5'-CAGTATCGATCAGGGCCACGTGCTACGAG-3 '(ClaI)) and primer 5 of SEQ ID NO: 7 (5'-CTGATCTAGATCAGGTGGAGCAGGAGGG-3' (XbaI)) Was used. The PCR product thus obtained was treated with restriction enzymes ClaI and XbaI.

The vector was prepared with pPICZα-C vector (Invitrogen) dephosphorylated after treatment with the same restriction enzymes ClaI and XbaI, and the vector and PCR products were ligated using T4 DNA ligase (Takara). After attaching, E. coli TOP10F '(Invitrogen) was transformed. DNA sequencing was performed with the plasmid of the transformant to confirm the base sequence of the inserted DNA. As a result, the recombinant plasmids His-TK1-2 / pPICZα-C (see FIG. 24) and N-TK1-2 / pPICZα-C (See FIG. 25), all of which contain an additional amino acid (Glu-Ala-Glu-Ser-Ile) at the N-terminus.

2-2: P.pastoris  Transformation to

One kind of yeast, Pichia pastoris GS115 or X-33, Invitrogen) strains were transformed with His-TK1-2 / pPICZα-C and N-TK1-2 / pPICZα-C of Example 2-1 treated with restriction enzyme SacI. Approximately 3 μg of linear plasmid DNA was transformed by electroporation (Gene Pulser, Bio-Rad, 0.2 cm cuvette, conditions: 1.5 kV, 25 kV, 400 Ω). Immediately after the electric shock, 1 ml of cold 1M sorbitol solution was added to the cuvette, and then incubated at 30 ° C. for 1 hour without shaking. Cultured yeast cells were plated on YPD (yeast extract peptone dextrose medium, 1% yeast extract, 2% peptone, 2% dextrose) agar medium to which zeocin was added at a concentration of 1000 μg / ml, and at 30 ° C. 16 hours of incubation yielded the transformants.

2-3: Mut Phenotype Screening

To screening that uses methanol as a carbon source, in Example 2 the obtained transformant in MM (1.34% YNB, 4 × 10 -5 biotin, 0.5% methanol, 1.5% agar) or MMH medium (0.4 to MM medium was streaked in that the addition of histidine%), and MD (1.34% YNB, 4 × 10 -5 biotin, 2% dextrose, 1.5% agar) or MDH medium (not containing added 0.4% histidine in the medium MD). Mut ⁺ control (GS115 / His + / β-galactosidase, Invitrogen) and Mut ^s control (GS115 / His + / albumin, Invitrogen) were also tested for comparison. After 48 hours of incubation at 30 ° C., Mut ⁺ colonies grew significantly more than Mut ^s colonies.

2-4: TK1-2 Expression Screening

Of the P. pastoris transformants 10㎖ BMG (100mM potassium phosphate (pH 6.0), 1.34% YNB , 4 × 10 -5 biotin, 1% glycerol) or BMGH medium (which will be added to 0.4% histidine in the culture medium BMG) Were incubated at 30 ° C., 250 rpm and incubated until the OD ₆₀₀ reached 3.0. By collecting the cultured cells by centrifugation of 2㎖ BMM (100mM potassium phosphate (pH 6.0), 1.34% YNB , 4 × 10 -5 biotin, 0.5% methanol) or BMMH medium (having the addition of 0.4% histidine in the BMM medium ) Is suspended. Methanol was added to a concentration of 0.5% every 24 hours. In order to confirm the secretion of TK1-2, SDS-PAGE and coomassie blue staining was performed using a culture medium (see FIGS. 26, 27a and 27b).

Lane 1 of Figure 26 His-TK1-2-4 transformant, Lane 2 His-TK1-2-5, Lane 3 His-TK1-2-6, Lane 4 His-TK1-2-7 to be.

Figure 27a is a screening of the protein secretion of the N-TK1-2 GS115 transformants, lane 1 is N-TK1-2-4, lane 2 is N-TK1-2-6, lane 3 is N-TK1 -2-7, lane 4 shows N-TK1-2-8, lane 5 shows the protein expression patterns of N-TK1-2-9.

Figure 27b is a screening of the protein secretion of N-TK1-2 X-33 transformants, lane 1 is N-TK1-2-1, lane 2 is N-TK1-2-2, lane 3 N-TK1-2-3, lane 4 is N-TK1-2-4, lane 5 is N-TK1-2-5, lane 6 is N-TK1-2-6, lane 7 is N-TK1-2- 7, lane 8 shows N-TK1-2-8, lane 9 shows the protein expression patterns of N-TK1-2-9.

2-5: TK1-2 Protein Expression and Purification

In order to express a large amount of TK1-2 protein, P.pastoris transformants were inoculated into a 2-liter flask containing 500 ml of BMG or BMGH medium and incubated at 30 ° C. at 250 rpm until OD ₆₀₀ = 3. The cultured cells were then collected by centrifugation (1500 × g, 5 minutes) and suspended in 100 ml of BMM or BMMH medium. Pure methanol was added every 24 hours to a final concentration of 0.5%. After 120 hours, cells were centrifuged (1500 × g, 5 minutes) and the cultures stored at −70 ° C. until use. The crude supernatant (200 ml) containing the TK1-2 protein was centrifuged at 14000 × g for 20 minutes to obtain a clear supernatant, and the original volume was obtained by ultrafiltration using a YM10 membrane (Amicon). After concentration to about 10% of the buffer was exchanged using 50mM phosphate buffer (pH 7.5). The sample was then dialyzed with 50 mM phosphate buffer (pH 7.5). Since the kringle domain of tPA has a property of binding to a lysine residue, Lysine-Sepharose 4B (Phamacia) was used as an affinity substrate. After passing the sample through a lysine-Sepharose column packed and equilibrated with 50 mM phosphate buffer (pH 7.5), the column was washed with 1.5 volumes of 50 mM phosphate buffer (pH 7.5). The column was then washed again with 1.5 volumes of PBS (pH 7.4) of resin. Recombinant His-TK1-2 and N-TK1-2 proteins were eluted with 0.2M ε-amino-N-caproic acid. The elution fractions were dialyzed with deionized water for 48 hours and then lyophilized. 28 and 29 are photographs stained by electrophoresis of recombinant His-TK1-2 protein and N-TK1-2 protein purified by lysine sepharose column, lane 1 is a protein before purification, and lane 2 is prothrough Fraction (flow through fraction), lane 3 is the fraction washed with 50 mM phosphate buffer, lane 4 is the fraction washed with PBS (pH 7.4), lanes 5 and 6 are 0.2M ε-amino-N-caproic acid Eluted fraction.

2-6: Western blot analysis

Protein samples were analyzed by performing 14% SDS-PAGE and electrically blotted onto nitro cellulose membranes. Protein samples were searched using polyclonal antibodies that respond to recombinant TK1-2 protein expressed from E. coli . Primary antibody / antigen complexes were searched using mouse IgG (Santa Cruz biotechnology) conjugated with Hoolradish peroxidase and photosensitized using Western blot chemical fluorescent reagent from NEN. As a result, referring to Figure 30, lane 1 is a recombinant His-TK1-2 protein, lane 2 is a recombinant N-TK1-2 protein, and the antibody of recombinant TK1-2 protein expressed in Escherichia coli is Reaction with the purified protein was observed.

2-7: Oxidation Degree Analysis

To confirm the presence of disulfide bonds, the purified recombinant protein was treated with a buffer containing β-mercaptoethanol. Control samples were prepared without treatment of β-mercaptoethanol. Two protein samples were analyzed via SDS-PAGE. Lane 1 of FIG. 31 is a photograph of electrophoresis of the reducing His-TK1-2 protein treated with a reducing agent, and lane 2 is the recombinant His-TK1-2 protein not reduced. 32 is a photograph of electrophoresis of a reducing agent-treated recombinant N-TK1-2 protein, and lane 2 is a non-reduced recombinant N-TK1-2 protein. It can be seen that the reduced protein is slow in electrophoretic migration.

2-8: Inhibitory Effect of His-TK1-2 on Umbilical Vein Endothelial Cells (HUVEC)

As in Example (1-6-2), [ ³ H] -thymidine incorporation assay was performed on His-TK1-2 to investigate the effect of inhibiting proliferation on HUVEC. As shown in FIG. 33, the protein His-TK1-2 showed a growth inhibitory effect on HUVE cells stimulated by bFGF, and showed a protein dependent dependence.

As described above, the recombinant TK1-2 protein expressed in E. coli and purified TK1-2 protein expressed in E. coli, which corresponds to the Krinkle domains 1 and 2 of the plasminogen according to the present invention, are endothelial cells. By inhibiting the growth of, inhibiting angiogenesis, and inhibiting the growth of cancer, it can be usefully used for the treatment of diseases caused by angiogenesis and the treatment of cancer diseases.

1 is a diagram showing a plasmid structure of the produced TK1-2 / pET-15b of the present invention,

Figure 2 is a SDS-PAGE photograph of the sample obtained by the step of TK1-2 protein purification,

3 is a SDS-PAGE photograph of purified TK1-2 protein under non-reducing and reducing conditions,

4 is a MALDI result of recombinant TK1-2 protein,

5 is a SDS-PAGE photograph of a protein sample obtained after passing through a lysine-sepharose column.

6 is an electrophoresis picture of TK1-2 protein passed through lysine-sepharose column and TK1-2 protein passed through a heat-tack affinity column,

7 is a diagram showing the inhibition of capillary endothelial (BCE) cell proliferation of bovine by TK1-2 protein, which is compared with the effect of angiostatin,

8 is a diagram showing observation of cell proliferation inhibition of umbilical vein endothelial cells (HUVEC) by TK1-2 protein,

9 is a photograph of angiogenesis inhibitory site (>) observed at 48 hours after treatment with 20 μg of TK1-2 in chicken embryos.

10 is a diagram showing the angiogenesis inhibitory effect of dose-dependent TK1-2 protein,

FIG. 11 is a photograph of control mice 40 days old without TK1-2 in mice transplanted with lung cancer cells,

12 is a photograph of a mouse treated with 10 mg / kg TK1-2 for 40 days after transplanting lung cancer cells,

FIG. 13 is a photograph of a mouse treated with 50 mg / kg TK1-2 for 40 days after transplanting lung cancer cells,

14 is a diagram showing the tumor size growth inhibition of human lung cancer cells PC14 by time by the recombinant TK1-2 protein of the present invention,

15 is a diagram showing the survival rate of the mouse control and TK1-2 treated group transplanted with lung cancer cells,

16 is a diagram comparing amino acid sequences of mouse-TK1-2 and human-TK1-2;

17 is a Western blot result photograph observing whether or not the production of antibodies to TK1-2 protein in TK1-2 administered mice,

18 are the organs extracted from the control group,

19 shows organs extracted from mice treated with 10 mg / kg TK1-2,

20 is tumor tissue extracted from the control group,

Figure 21 is a hematoxylin / eosin stained picture of the liver tissue section of the control group,

Figure 22 is a photograph of the tumor tissue sections of the control group and TK1-2 administration group hematoxylin / eosin staining, B is a photograph of the reaction with PCNA monoclonal antibody, C is a photograph of the reaction of VEGF monoclonal antibody D Is a photograph reacted with bFGF antibody. The black line in the picture means 25㎛, and the picture is magnified 400 times.

Figure 23 is a reaction of tumor tissue sections of the control group and TK1-2 administration group with angiogenin monoclonal antibody, B is a reaction with vWF polyclonal antibody, C is a photograph of the reaction with MMP9 polyclonal antibody, D is a photograph reacted with MMP10 monoclonal antibody,

Fig. 24 is a preparation diagram of His-TK1-2 / pPICZα-C, which is a pichia expression plasmid of the present invention,

Fig. 25 is a preparation diagram of N-TK1-2 / pPICZα-C which is a pichia expression plasmid of the present invention,

26 is a screen for the degree of protein secretion in His-TK1-2 transformant (GS115),

Figure 27a is a screening of the degree of protein secretion in the N-TK1-2 transformant (GS115),

Figure 27b is a screen for the degree of protein secretion in the N-TK1-2 transformant (X-33),

Figure 28 is a photograph stained by electrophoresis of recombinant His-TK1-2 protein purified by lysine Sepharose column,

29 is a photograph stained by electrophoresis of recombinant N-TK1-2 protein purified by lysine sepharose column,

30 is a Western blot picture of recombinant His-TK1-2 protein and N-TK1-2 protein,

Figure 31 is a photograph of the electrophoresis of the reducing His-TK1-2 protein treated with,

Figure 32 is a photograph of electrophoresis of the reducing agent-treated recombinant N-TK1-2 protein.

33 is a graph showing the proliferation inhibitory effect of umbilical vein endothelial cells (HUVEC) on recombinant His-TK1-2 protein.

<110> JOE, YOUNG-AE <120> Composition comprising TK1-2 peptide having anti-angiogenic activity and cancer growth inhibitory activity and its use <160> 9 <170> KopatentIn 1.71 <210> 1 <211> 174 <212> PRT <213> Homo sapiens <400> 1 Ala Thr Cys Tyr Glu Asp Gln Gly Ile Ser Tyr Arg Gly Thr Trp Ser 1 5 10 15 Thr Ala Glu Ser Gly Ala Glu Cys Thr Asn Trp Asn Ser Ser Ala Leu 20 25 30 Ala Gln Lys Pro Tyr Ser Gly Arg Arg Pro Asp Ala Ile Arg Leu Gly 35 40 45 Leu Gly Asn His Asn Tyr Cys Arg Asn Pro Asp Arg Asp Ser Lys Pro 50 55 60 Trp Cys Tyr Val Phe Lys Ala Gly Lys Tyr Ser Ser Glu Phe Cys Ser 65 70 75 80 Thr Pro Ala Cys Ser Glu Gly Asn Ser Asp Cys Tyr Phe Gly Asn Gly 85 90 95 Ser Ala Tyr Arg Gly Thr His Ser Leu Thr Glu Ser Gly Ala Ser Cys 100 105 110 Leu Pro Trp Asn Ser Met Ile Leu Ile Gly Lys Val Tyr Thr Ala Gln 115 120 125 Asn Pro Ser Ala Gln Ala Leu Gly Leu Gly Lys His Asn Tyr Cys Arg 130 135 140 Asn Pro Asp Gly Asp Ala Lys Pro Trp Cys His Val Leu Lys Asn Arg 145 150 155 160 Arg Leu Thr Trp Glu Tyr Cys Asp Val Pro Ser Cys Ser Thr 165 170 <210> 2 <211> 522 <212> DNA <213> Homo sapiens <400> 2 gccacgtgct acgaggacca gggcatcagc tacaggggca cgtggagcac agcggagagt 60 ggcgccgagt gcaccaactg gaacagcagc gcgttggccc agaagcccta cagcgggcgg 120 aggccagacg ccatcaggct gggcctgggg aaccacaact actgcagaaa cccagatcga 180 gactcaaagc cctggtgcta cgtctttaag gcggggaagt acagctcaga gttctgcagc 240 acccctgcct gctctgaggg aaacagtgac tgctactttg ggaatgggtc agcctaccgt 300 ggcacgcaca gcctcaccga gtcgggtgcc tcctgcctcc cgtggaattc catgatcctg 360 ataggcaagg tttacacagc acagaacccc agtgcccagg cactgggcct gggcaaacat 420 aattactgcc ggaatcctga tggggatgcc aagccctggt gccacgtgct gaagaaccgc 480 aggctgacgt gggagtactg tgatgtgccc tcctgctcca cc 522 <210> 3 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> primer 1 <400> 3 atcggatccc atggccacgt gctacgagga ccag 34 <210> 4 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer 2 <400> 4 atcggatcct caggtggagc aggagggcac atc 33 <210> 5 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer 3 <400> 5 cagtatcgat cagggccacg tgctacgag 29 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer 4 <400> 6 ctgatctaga gtggagcagg agggcac 27 <210> 7 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer 5 <400> 7 ctgatctaga tcaggtggag caggaggg 28

Claims (13)

Recombinant protein TK1-2 having the amino acid sequence as set forth in SEQ ID NO: 1 having an inhibitory activity on angiogenesis and cancer, derived from human tissue type plasminogen activator, was selected for rheumatoid arthritis, osteoarthritis, psoriasis, diabetic retinopathy, Pharmaceutical composition for the prevention and treatment of atherosclerosis or cancer disease. The pharmaceutical composition according to claim 1, wherein the recombinant protein TK1-2 is a recombinant E. coli expression plasmid comprising the nucleotide sequence of SEQ ID NO: 2. The pharmaceutical composition according to claim 1, wherein the recombinant protein TK1-2 is produced from His-TK1-2 / pPICZα-C and N-TK1-2 / pPICZα-C, which are recombinant yeast expression plasmids comprising the nucleotide sequence of SEQ ID NO: 2 . PET-15b / TK1-2 which is a recombinant E. coli expression plasmid expressing the TK1-2 protein of claim 1. E. coli strain transformed with pET-15b / TK1-2 of claim 4, expressing the TK1-2 protein. His-TK1-2 / pPICZα-C and N-TK1-2 / pPICZα-C, which are recombinant Pichia expression plasmids expressing the TK1-2 protein of claim 1. Yeast Pichia strain expressing TK1-2 protein transformed with N-TK1-2 / pPICZα-C of claim 6 (Accession No .: KCTC 10415BP). A method for producing a TK1-2 protein comprising the following steps; 1) preparing E. coli transformed with TK1-2 / pET-15b, an E. coli expression plasmid comprising SEQ ID NO: 2; 2) inducing and expressing protein expression in E. coli; 3) collecting and crushing the cultured Escherichia coli; 4) separating the soluble portion and the non-soluble portion; 5) washing the enclosure 6) passing the non-soluble portion through heat-tack affinity column chromatography; 7) refolding; And 8) obtaining the recombinant protein TK1-2. delete delete delete delete delete