WO1994001543A1 - Polypeptide humain de faible masse moleculaire semblable a la pro-urokinase, et sa production - Google Patents

Polypeptide humain de faible masse moleculaire semblable a la pro-urokinase, et sa production Download PDF

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Publication number
WO1994001543A1
WO1994001543A1 PCT/JP1993/000897 JP9300897W WO9401543A1 WO 1994001543 A1 WO1994001543 A1 WO 1994001543A1 JP 9300897 W JP9300897 W JP 9300897W WO 9401543 A1 WO9401543 A1 WO 9401543A1
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WIPO (PCT)
Prior art keywords
leu
polypeptide
human
pro
glu
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PCT/JP1993/000897
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English (en)
Japanese (ja)
Inventor
Youichi Kobayashi
Naoyuki Takagi
Mahito Ohhira
Hiroaki Nakamura
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Nippon Soda Co., Ltd.
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Publication of WO1994001543A1 publication Critical patent/WO1994001543A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6456—Plasminogen activators
    • C12N9/6462—Plasminogen activators u-Plasminogen activator (3.4.21.73), i.e. urokinase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21073—Serine endopeptidases (3.4.21) u-Plasminogen activator (3.4.21.73), i.e. urokinase

Definitions

  • the present invention relates to a human low-molecular-weight proproteinase-like polypeptide, and the polypeptide has a function as a thrombolytic agent.
  • Human auricular kinase is an enzyme present in trace amounts in human urine, has the effect of activating inactive plasminogen to plasmin, and the generated plasmin can dissolve thrombus. For this reason, human oral kinase is widely used clinically as a therapeutic agent for thrombosis.
  • human mouth kinase has an extremely low affinity for thrombus and is an active form, so it activates not only plasminogen present at the thrombus site but also plasminogen in circulating blood and produces large amounts in circulating blood. To generate plasmin. Excessive production of plasmin in the circulating blood leads to platelet function conversion, degrades fibrinogen, blood coagulation factors V and VIII, and has the danger of causing systemic hemorrhagic side effects and death. is there.
  • thrombotherapeutic would attack the thrombotic component but would not disrupt the coagulation system with the consumption of circulating platelets or blood coagulation factors, and would have a desirable body half-life.
  • human pro-prokinase and tissue brassica-genichi-ichi-ichi (hereinafter abbreviated as tPA) have been developed. These activate plasminogen in a thrombus-specific manner. Therefore, there is little danger of causing systemic bleeding tendency even with large doses.
  • Human protokinase has an increased thrombophilicity compared to human mouth kinase, but is still inadequate.
  • tPA has a large amount of inhibitor (plasminogen inhibitor Inhibitor I) in the blood, has a short half-life in blood, has no therapeutic effect unless administered in large amounts, and has an artery due to thrombolysis. There is a defect that reocclusion often occurs after reopening. Many attempts have been made to extend the half-life of the thrombolytic agent in the body, but as the half-life is prolonged, the thrombus affinity tends to decrease. There is. (Thrombosis Haemostasis Vol. 66, p. 88, 1991)
  • the present invention relates to a human small molecule prokinase-like polypeptide having a significantly higher thrombus affinity than human protokinase and an improved half-life in the body, and an economical use of the polypeptide. It is intended to provide a manufacturing method.
  • V31 [(Met.) May be a methionine that may be present in some cases]
  • Voligopeptide collectively for V30 and V31
  • a DNA segment, the DNA segment and control for its expression A plasmid containing a region and a DNA sequence necessary for replication in Escherichia coli, and a human low-molecular-weight properokinase-like polysaccharide comprising culturing the Escherichia coli and collecting from the cultured cells. This is a method for producing peptides.
  • the present invention provides a selective method by which, when added to the N-terminus of the human protokinetic kinase, a covalent bond with thrombus is rapidly and efficiently generated by the enzymatic action of human blood coagulation factor XIII.
  • This oligopeptide is a Gin- * 1 peptide proposed in Japanese Patent Application Laid-Open No. HEI 4-175584 as an oligopeptide having a structure capable of rapidly forming a covalent bond with a thrombus by the enzyme action of blood coagulation factor XIII.
  • the following oligopeptides selected from among the oligopeptides having the amino acid sequence of * —Va 1 — *-Pro—
  • the structure of the present oligopeptide at the N-terminus has a synergistically improved thrombus affinity due to the structure of the human small molecule prokinase.
  • a polypeptide from the leucine at position 144 to the leucine at position 411 of the entire amino acid sequence (abbreviated as CUK (—, k)).
  • the present human small molecule prokinase-like polypeptide refers to a combination of the present oligopeptide and the present human small molecule protease, ie, V30-CUK (-, k) and V31-CUK (1, k).
  • the DNA segment encoding the present low-molecular-weight prokinase-like polypeptide is composed of a DNA obtained by linking DNA encoding the present oligopeptide and DNA encoding the present low-molecular-weight prokinase.
  • Such DNA encodes each amino acid It is possible to use naturally occurring DNA as it is, but it is possible to use codon combinations that do not change the amino acid sequence by chemical synthesis or introduction of artificial mutation. well known.
  • the DNA segment encoding the present human low molecular weight pro-prokinase-like polypeptide is recombined into an expression type plasmid, and the gene is expressed by transforming into an appropriate host.
  • the confectionery, the human low molecular weight properokinase-like polypeptide is accumulated inside or outside the host cell.
  • the target substance is insolubilized and accumulated in host cells
  • the insoluble fraction is collected by crushing the cells with a homogenizer or the like, and then dissolved in an aqueous solution such as guanidine hydrochloride or urea. Air oxidation is performed below to restore the original three-dimensional structure of the target substance.
  • purification is carried out using a technique such as ammonium sulfate salting-out, hydrophobic interaction chromatography, affinity chromatography, etc., but other commonly used biochemical purification techniques can also be used.
  • the human protease-like polypeptide is economically produced at low cost.
  • the present invention will be described specifically with reference to examples.
  • Synthesis of this oligopeptide encoding double-stranded DNA was prepared by synthesizing each single-stranded DNA oligomer by the amidite method, and purified by a 0 PC cartridge (manufactured by Applied Biosystems). The strand DNA is dissolved in 20 mM Tris-HCl, lmM ethylene diamine tetraacetate buffer (pH 8.0), heated at 65 ° C for 15 minutes, and then gradually cooled to room temperature to anneal. Single-stranded DNA was obtained.
  • Plasmid pACUK (-, q) / 0 (described in JP-A-4-75584) and a double digested fragment with BamHI (about 0.8 Kb), and plasmid pFPUKCq, q) / 5 (described in JP-A-4-75584) Described above) and the double-digested fragment (about 5.1 Kb) with BamHI and the synthetic DNA oligomer V30 of step 1 were ligated with T4 DNA ligase to obtain plasmid pV30CUK (-, q) / 5.
  • Ball of plasmid pV30CUK (_, q) / 5 and a double digested fragment with BamHI (about 5.2 Kb) and Ball and BamHI of plasmid pAPUK (k, k) / 5 (described in JP-A-4-75584).
  • Ball and BamHI of plasmid pAPUK (k, k) / 5 (described in JP-A-4-75584).
  • T4DN A ligase was ligated with T4DN A ligase to obtain plasmid PV30CUK (-, k) / 5.
  • Escherichia coli L coli KY1436 containing the plasmid pV30CUK (-, k) / 5 was sent to the Institute of Microbial Industry and Technology of the National Institute of Advanced Industrial Science and Technology by the Research Institute of Microorganisms No. BP-43327 [F ERM BP-43227 [ (Transferred from No. P-131017 of Weiken Article))].
  • a double digested fragment (about 0.8 Kb) of plasmid pCUK (-, q) / 0 (described in JP-A-4-75584) with Sacl and BamHI, plasmid pFPUK (q, q) / 5 (JP-A-4-75584) Described above) was ligated with Ncol and BamHI (about 5.1 Kb) and the synthetic DNA oligomer V31 of step 1 using T4 DNA ligase to obtain plasmid pV31CUK (-, q) / 5.
  • Step 4 Expression of this human small molecule properokinase-like polypeptide gene by Escherichia coli
  • Plasmid pV30CUK (-, k) / 5 obtained in step 2 was transformed into Escherichia coli ⁇ 103 according to a conventional method.
  • the obtained recombinant was aerobically cultured in 50 ml of L-broth at 37 ° C, and when the absorbance at 600 nm reached about 0.5, 0.5 ml of lOOmM isopropylthiogalactobyranoside (IPTG) was added. Culture was continued for 4 hours to produce each human low molecular weight prokinetic kinase-like polypeptide V30CUK (-, k).
  • Step 5 Extraction and purification of gene products from E. coli.
  • the wet cells obtained in step 4 were suspended in 10 ml of 10 mM Tris-HC1 buffer (pH 8.0) per 10 g, and the cells were disrupted by ultrasonication. Subsequently, the precipitate was recovered by centrifugation at 12,000 rpni at 4 ° C. The obtained precipitate is suspended in 500 ml of 100 mM Tris-HCl buffer ( ⁇ (8.0), and further dissolved by adding an equal amount of guanidine hydrochloride, containing lmM EDTA and 0.2 mM reduced daltathione.
  • the crude product is dissolved in 50 mM Tris-HCl buffer (pH 8.0) containing 1 M guanidine hydrochloride and 1 M ammonium sulfate, and adsorbed on a Phenyl Sepharose (Pharmacia) column equilibrated with the same buffer. After sufficient washing with the liquid, the column was eluted with a 50 mM Tris-HCl buffer (PH8.0) containing 1 M guanidine hydrochloride and 0.6 M ammonium sulfate, and an eluted fraction containing the target compound was collected.
  • Tris-HCl buffer pH 8.0
  • PH8.0 50 mM Tris-HCl buffer
  • This fraction was adsorbed to a column of zinc chelate Sepharose (Pharmacia), washed thoroughly with a 50 ⁇ phosphate buffer (pH 7.0) containing 0.5 M sodium chloride, and then washed with 50 mM sodium acetate containing 0.5 M sodium chloride.
  • the fraction containing the target compound was eluted with a lithium buffer solution ( ⁇ 5.6), concentrated and desalted using an ultrafiltration membrane PM-10 (manufactured by Namicon Co., Ltd.).
  • a purified product of the polypeptide V30CUK (-, k) was obtained.
  • the plasmid PV31CUK (-, k) / 5 obtained in Step 3 was treated in the same manner as in Steps 4 and 5, to obtain a purified product of the human small molecule properokinase-like polypeptide V31CUK (-, k). From purified product (purification yield 4 3. 6%) of specific activity ultraviolet absorption intensity of the degradation activity and refined products against the Chikarabisha synthetic substrate S- 24 4 4 (A 280) , L 73X 10 5 IZA 28 . And was calculated.
  • Tris-HCl buffer 50 mM Tris-HCl, 38 mM salt, 0.01% Triton X-100, pH 7.5
  • Thrombin solution Sigma human thrombin 100 units / ml Tris-HCl buffer
  • Plasmin solution Human mold Plasmin 15 cu / ml distilled water
  • Triscin inhibitor solution Sigma's trypsin inhibitor 5 mg / ml distilled water
  • Test sample solution Test sample 5000 international units Zml Tris-HCl buffer (Method)
  • Plasma with citrate George King's normal human plasma
  • Heparin-added plasma Human plasma (freeze-dried product) manufactured by Kaketsuken dissolved in 25 mM calcium chloride aqueous solution containing 1 unit of Zm1 heparin.
  • citrate-supplemented plasma 25 1 into the bottom of a 2 ml Etpendorf tube and add An aqueous solution 3 ⁇ 1 containing 50 units / m1 of bottle and 0.5M calcium chloride was added, and the mixture was kept at 37 ° C for 30 minutes to form a thrombus.
  • heparin-added plasma 100 1 and each test sample 51 were sequentially added to an eppendorf tube in which a thrombus had formed, and incubated at 37 ° C for 1 hour.
  • the amount of protein contained in the thrombus was quantified by the tyrosine method, and the amount of the dissolved thrombus was estimated.
  • Test Example 3 Stability test in rabbit circulating blood
  • test substance was injected through the force june of the common jugular vein. During the experiment, heparin was injected at a rate of 500 units / Kg through the same vein. The dose of the test substance was 100, 001 U / kg, respectively, and the number of administrations was 5 in each case.
  • a plasma fraction was obtained by centrifugation. After adding 2 ml of 0.01% acetic acid to the plasma 1001, the Euglobulin fraction obtained by centrifugation was collected. The amount of perokinase contained in the Euglobulin fraction was measured by the decomposition activity of S-244 (manufactured by Mold).
  • Table 1 below shows the test results of Test Examples 1 to 3.
  • the human protokinase (or human low-molecular-weight prokinase) -like polypeptide used for comparison is the N-terminal of each of the human low-mouth kinase derivatives shown in Table 2 below. Having a structure added to Table 2
  • V3 (Met.) Gln. Glu. Gin. Val. Ser. Pro. Gin. Thr. Leu. Leu. Lys.
  • Human protokinetic kinase derivative (Amino acid No. indicates amino acid N 0. of natural human protokinetic kinase)
  • V30CUK (—, k) and V31CUK (—, k) have high thrombophilicity and thrombolytic properties, have long blood half-lives, and have been proposed in Japanese Patent Application Laid-Open No. 4-755558. It was found to be the best thrombolytic agent among kinase-like polypeptides.
  • the human low molecular weight properokinase-like polypeptides ⁇ V30CUK (-, k) and V31CUK (1-, k) ⁇ have high thrombus specificity and thrombolytic properties, and have low thrombus penetration and half-life in vivo due to their low molecular weight. It has a long period and is the best as a thrombolytic agent. For this reason, the human low molecular weight properokinase-like polypeptide ⁇ V30CUK (—, k) and V31CUK (—, k) ⁇ is used as a thrombolytic agent that is significantly better than human protokinase.
  • the DNA and amino acid sequences of V30CUK (-, k) and V31CUK (-, k) are shown in the following table.
  • SEQ ID NO: 1 V30CUK (-, k)
  • Sequence type nucleic acid
  • Sequence type other nucleic acids (synthetic DNA) and cDNA to mRNA
  • Organism name human
  • na naq AI3 usy nig sA SJV dJi
  • Sequence type other nucleic acids (synthetic DNA) and cDNA to mRNA
  • Organism name human
  • 066 33D10I0091 IVOIVDVOVO 013X090V1V J, VV903i 0V DVV9VV101 3V31V0010J, 066 086 016 096 096 0 ⁇ 6

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Abstract

Polypeptide humain de faible masse moléculaire semblable à la pro-urokinase. On produit ce polypeptide par la liaison d'un oligopeptide, de la formule suivante, et formant une liaison covalente avec un thrombus par l'action enzymatique du facteur XIII humain de coagulation sanguine: (Met.)Gln.Glu.Gln.Val.Ser.Pro.Leu.Thr.Leu.Leu.Lys.(Met.)Gln.Glu.Gln.Val.Ser.Pro.Leu.Thr.Leu.Leu.Glu, à l'extrémité N-terminale de la partie polypeptidique constituée de la 144-leucine jusqu'à la 411-leucine, dans toute la séquence d'acides aminés de la pro-urokinase humaine naturelle. Ce polypeptide présente une spécificité des thrombus et une activité thrombolytique élevées, une excellente activité d'infiltration des thrombus, et une longue demi-vie in vivo, et constitue donc un excellent agent thrombolytique.
PCT/JP1993/000897 1992-07-03 1993-06-30 Polypeptide humain de faible masse moleculaire semblable a la pro-urokinase, et sa production WO1994001543A1 (fr)

Applications Claiming Priority (2)

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JP4/200222 1992-07-03
JP20022292 1992-07-03

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5951981A (en) * 1996-12-02 1999-09-14 Diatide, Inc. Thrombolytic agents with antithrombotic activity

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0475584A (ja) * 1989-06-13 1992-03-10 Nippon Soda Co Ltd ヒトプロウロキナーゼ様ポリペプチド及びその製造法

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0475584A (ja) * 1989-06-13 1992-03-10 Nippon Soda Co Ltd ヒトプロウロキナーゼ様ポリペプチド及びその製造法

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5951981A (en) * 1996-12-02 1999-09-14 Diatide, Inc. Thrombolytic agents with antithrombotic activity

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