JPH06321989A - New polypeptide, new dna and pharmaceutical composition - Google Patents

Abstract

PURPOSE:To obtain a new polypeptide having a specific amino acid sequence, exhibiting an inhibitory action on a proteinous protease which has having high Xa factor and elastase inhibiting action, and useful e.g. for the treatment of shock, pancreatitis, disseminated intravascular coagulation syndrome, etc. CONSTITUTION:This objective new polypeptide expressed by the formula and having an inhibitory action on proteinous protease is produced by using a chromosomal DNA library, screening the library with a DNA coding a partial amino acid sequence of a protease inhibitor, selecting a positive clone from the library, recovering the DNA, subjecting the DNA to site-specific mutagenesis and amplification by polymerase chain reaction to obtain a DNA coding the amino acid sequence of the formula (Xaa-1 is amino acid other than Arg; Xaa-2 is amino acid other than Gln; Xaa-3 is amino acid other than Tyr), linking the DNA to a proper expression vector and introducing the vector into a host cell to effect the transformation and expression.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a novel polypeptide, a novel DNA, a vector containing the novel DNA, a transformant producing the novel polypeptide, a pharmaceutical composition containing the novel polypeptide as an active ingredient, and an enzyme. Regarding inhibition method.

[0002]

As is well known, various proteases such as trypsin and chymotrypsin are present in the living body. While these proteases play important roles in the body, such as digestion, biological defense, and blood coagulation / fibrinolysis, it has been clarified in previous studies that they directly or indirectly cause diseases. Has been For example, shock, pancreatitis, disseminated intravascular coagulation (DI
C) and the like are typical diseases thought to be caused by abnormal activation of protease. At present, various protease inhibitors are used and developed for the purpose of treating diseases involving such proteases.

Among protease inhibitors, proteinaceous protease inhibitors generally have a wide spectrum of inhibition.
The type and sequence of amino acids at the active center are deeply related. For example, in general, the amino acid at position P ₁ , which is the major site of the active center of a protease inhibitor, is Lys or Ar.
g is a trypsin-like enzyme, Phe or Tyr is a chymotrypsin-like enzyme, and Ala, Ser or Val is an elastase (Laskowski M., Jr., Biochem. Phar
(Macol. 29, 2089-2094, 1980)
. Therefore, it is presumed that the inhibitory spectrum of proteinaceous protease inhibitors can be altered by substituting the amino acids that make up their active centers, and already some natural protease inhibitors have such methods. It is applied.

For example, Brinkmann et al. Reported that the inhibitory activity of aprotinin against chymotrypsin was enhanced by substituting the positions P ₁ and P ′ ₂ of the active center of aprotinin with hydrophobic amino acids such as Phe, Tyr or Leu. (Thomas Brinkmann et al., Eur J. Bioc
hem., 202, 95-99, 1991). Also, F
Ritz et al. prepared a substance in which the amino acid at the active center of bikunin (HI-30) was replaced with another amino acid, and its elastase inhibitory activity and trypsin inhibitory activity were measured (JP-A-3-255099, 1991, Europe). Published publication, EP 401508, 1990).

On the other hand, the present inventors have already surprisingly found that the active center shown by Wachter et al. And Sailer (Wachter E.
at al., Hoppe-Seyler's z. Physiol. Chem., 360
Volume, 1297-1303, 1979, and Jean-P.
hilippe Sailer, TIBS, Volume 15, Pages 435-439, 1
990), and succeeded in changing the enzyme inhibition spectrum by substituting an amino acid at a different site. That is, the present inventors succeeded in producing a characteristic protease inhibitor by mutating a polypeptide having the amino acid sequence of the following formula 7 (Japanese Patent Application No. 4-29).
7344). The polypeptide having the amino acid sequence of formula 7 was first discovered by the present inventors as a polypeptide having an inhibitory activity against activated blood coagulation factor X (hereinafter abbreviated as FXa) (Japanese Patent Application No. 03-
No. 325220). The amino acid sequence of this polypeptide corresponds to part of the amino acid sequence of urinary trypsin inhibitor (UTI) or bikunin (HI-30). However, the polypeptide clearly differs from UTI and HI-30 in that it exhibits a remarkable FXa inhibitory activity.

[0006]

Thus, it has become possible to change the characteristics of protease inhibitors by the substitution of amino acids. However, in order to develop a more effective pharmaceutical composition using a proteinaceous protease inhibitor, it is important to obtain an inhibitor having a stronger inhibitory activity. Among the proteases, protease and elastase of blood coagulation / fibrinolysis system such as thrombin and FXa are especially associated with multiple organ failure, disseminated intravascular coagulation syndrome (DIC), and adult respiratory failure syndrome (ARDS).
It is known to cause serious symptoms. Therefore, it is important to obtain a protease inhibitor having a high inhibitory activity against blood coagulation / fibrinolytic proteases and elastase. Therefore, the purpose of the present invention is to
It is intended to provide a novel polypeptide which is a proteinaceous protease inhibitor and has enhanced inhibitory activity against blood coagulation / fibrinolytic proteases and elastase. Another object of the present invention is a DNA having a nucleotide sequence encoding the novel polypeptide, a vector containing the DNA,
Transformant transformed with the DNA and DN
It is to provide a transformant transformed with a vector containing A. Further, it is an object of the present invention to provide a pharmaceutical composition containing the polypeptide as an active ingredient and a method for inhibiting an enzyme.

[0007]

Means for Solving the Problems As a result of further studies to solve the above problems, the present inventors have found that the amino acid sequence of formula 7 has an elastase inhibitory activity in addition to an FXa inhibitory activity. The inventors have succeeded in obtaining more enhanced and more useful substances, and completed the present invention.

Formula 7 Cys Asn Leu Pro Ile Val Arg Gly Pro Cys Arg Ala Phe Ile Gln Leu Trp Ala Phe Asp Ala Val Lys Gly Lys Cys Val Leu Phe Pro Tyr Gly Gly Cys Gln Gly Asn Gly Asn Lys Phe Tyr Ser Glu Lys Glu Cys Arg Glu Tyr Cys

That is, the first aspect of the present invention provides a novel polypeptide characterized by having the amino acid sequence represented by the formula 1.

The second aspect of the present invention provides a novel DNA having a base sequence encoding the novel polypeptide of the first aspect of the present invention.

The third aspect of the present invention provides a vector comprising the novel DNA of the second aspect of the present invention.

The fourth aspect of the present invention provides a transformant characterized by being transformed with the novel DNA of the second aspect of the present invention.

The fifth aspect of the present invention provides a transformant transformed with the vector of the third aspect of the present invention.

The sixth aspect of the present invention provides a pharmaceutical composition comprising the novel polypeptide of the first aspect of the present invention as an active ingredient.

The seventh aspect of the present invention provides a method for inhibiting FXa, which comprises using the novel polypeptide of the first aspect of the present invention.

The eighth aspect of the present invention provides a method for inhibiting elastase, which comprises using the novel polypeptide of the first aspect of the present invention.

The present invention will be described in detail below. The novel polypeptide of the first aspect of the present invention has an amino acid sequence represented by the following formula 1 (SEQ ID NO: 1 in the sequence listing). Formula 1 Cys Asn Leu Pro Ile Val Xaa-1 Gly Pro Cys Arg Ala Phe Ile Xaa-2 Leu Trp Ala Phe Asp Ala Val Lys Gly Lys Cys Val Leu Phe Pro Tyr Gly Gly Cys Gln Gly Asn Gly Asn Lys Phe Xaa-3 Ser Glu Lys Glu Cys Arg Glu Tyr Cys (where Xaa-1 is an amino acid other than Arg, Xaa-2 is Gln
Other amino acids, Xaa-3 is an amino acid other than Tyr)

Furthermore, a preferred example of the novel polypeptide of the present invention is a polypeptide having the amino acid sequence of the following formula 2 (SEQ ID NO: 2 in the sequence listing). Formula 2 Cys Asn Leu Pro Ile Val Ser Gly Pro Cys Arg Ala Phe Ile Lys Leu Trp Ala Phe Asp Ala Val Lys Gly Lys Cys Val Leu Phe Pro Tyr Gly Gly Cys Gln Gly Asn Gly Asn Lys Phe Asp Ser Glu Lys Glu Cys Arg Glu Tyr Cys

Further, the novel polypeptide of the present invention has an amino acid sequence in which any one or more amino acids are added to the N-terminal and / or C-terminal of the amino acid sequence represented by the above formula 1 or 2. It may be present (SEQ ID NO: 3 in the sequence listing). Here, the term "N-terminal and / or C-terminal" means that the addition of the arbitrary one or more amino acids may occur only at one of the N-terminal and the C-terminal.
It means that it may be at both the terminal and C-terminal. The type and number of amino acids added are not particularly limited as long as the characteristics of the novel polypeptide of the present invention are not completely lost. The polypeptide of the present invention more preferably has the amino acid sequence of the following formula 3 (SEQ ID NO: 4 in the sequence listing). The polypeptide having the following sequence has high elastase inhibitory activity and FXa inhibitory activity, and can be more easily produced in Escherichia coli.

Formula 3 Ala Ala Cys Asn Leu Pro Ile Val Ser Gly Pro Cys Arg Ala Phe Ile Lys Leu Trp Ala Phe Asp Ala Val Lys Gly Lys Cys Val Leu Phe Pro Tyr Gly Gly Cys Gln Gly Asn Gly Asn Lys Phe Asp Ser Glu Lys Glu Cys Arg Glu Tyr Cys Gly Val Pro Gly Asp Gly Asp Glu Glu Leu Leu Arg Phe Ser Asn

The polypeptide of the present invention may have any one or more amino acids added to its N-terminal and / or C-terminal in addition to the amino acid sequence represented by the formula 3 as long as the characteristics of the polypeptide are not completely lost. It may have the amino acid sequence shown. Also, in the amino acid sequences of the above formulas 1 to 3, the amino acids represented by other than Xaa-1, Xaa -2, and Xaa -3 are substituted with other amino acids,
It is included in the present invention as long as the characteristics of the polypeptide are not lost.

The novel polypeptide of the present invention includes both those having a sugar chain and those having no sugar chain.

Further, recent techniques have made it possible to subject polypeptides to various chemical modifications such as alkylation, oxidation, reduction and hydrolysis. In addition, it can form salts with pharmacologically acceptable acids and bases, and can be used as a drug delivery system (DD).
From the point of S), conjugating polyethylene glycol or the like to the polypeptide is also a generally performed technique. Therefore, the novel polypeptides of the present invention also include polypeptides having these modifications.

The novel polypeptide according to the first aspect of the present invention is
Preferably, one of its characteristics is to have protease inhibitory activity. The protease inhibitory activity is at least one inhibitory activity against trypsin, FXa, or elastase.

The novel polypeptide of the present invention may be obtained by any method. For example, the novel polypeptide of the present invention may be chemically synthesized using a peptide synthesizer (for example, Applied Biosystems 431 type). Further, by using the DNA encoding the novel polypeptide of the present invention, known recombinant DN
A technology (eg T. Maniatis et al., Molecular Clonin
g, a laboratory manual, 1982, Cold Spring Harbor
(See Laboratory). The production method using recombinant DNA technology is a more preferable production method as the production method of the polypeptide of the present invention.

A preferred example of the production by the recombinant technique will be described below. The method includes the following steps a) to d). a) A DNA having a nucleotide sequence encoding the novel polypeptide of the first aspect of the present invention is obtained. b) A vector containing the DNA obtained in a) is obtained. c) A host cell is transformed with the vector obtained in b) to obtain a transformant. d) The transformant obtained in c) is cultured to produce the polypeptide of the first aspect of the present invention, and the polypeptide is recovered from the culture mixture.

The DNA having the nucleotide sequence encoding the novel polypeptide of the first aspect of the present invention in the above a) is preferably the DNA of the second aspect of the present invention. The vector of b) above is preferably the vector of the third aspect of the present invention. Among them, in addition to the base sequence encoding the polypeptide of the first aspect of the present invention, a base sequence required for expression, and if necessary, a vector having a base sequence encoding a signal peptide is provided. preferable. Construction of the vector may be performed according to a known method, and details thereof will be described in the third aspect of the present invention. The transformant of the above c) is preferably the transformant of the fifth aspect of the present invention. The production of the transformant may be carried out according to a known method, and details thereof are described in the fifth aspect of the present invention. Described in.

In the step d), the transformant obtained in c) is used. The method for culturing the transformant, and the method for recovering and purifying the novel polypeptide of the present invention from the culture mixture can be performed by known methods. That is, "Biochemical Engineering" (by Shuichi Aiba, 1976, The University of Tokyo Press)
Or “Tissue culture” (edited by Junnosuke Nakai et al., 1976,
Asakura Shoten) and the like.
When the produced polypeptide is not secreted out of the transformant, it is preferably isolated from the transformant, and when secreted, from the culture supernatant. Purification and recovery of the novel polypeptide from the mixture containing the novel polypeptide according to the first aspect of the present invention can be performed by any means commonly used for purification of polypeptides, such as "Biochemistry Laboratory Course 1 Protein Chemistry "(edited by the Japanese Biochemical Society, 1976)
It can be carried out by referring to the methods described in many literatures and books such as Tokyo Kagaku Dojin, etc. When the polypeptide of the present invention is an inclusion body, it is solubilized before or after purification,
Denature, refolding may be performed (eg, Thomas E. Creighton, J. Mol. Biol., Vol. 87,
563-577, 1974).

An example of a method for purifying a polypeptide is as follows:
Salting out method, ultrafiltration method, isoelectric focusing method, gel filtration method, ion exchange chromatography, affinity chromatography such as hydrophobic chromatography and antibody chromatography, chromatofocusing method, adsorption chromatography and reverse method. Since there are phase chromatography and the like, a method suitable for obtaining the novel polypeptide of the first aspect of the present invention can be appropriately selected from these and, if necessary, can be performed in an appropriate order using an HPLC system or the like. Good. The preferred method for obtaining the polypeptide of the present invention using the recombinant technique has been described above.
It is also possible to transform an appropriate host with A and obtain the novel polypeptide of the present invention from the culture mixture of the transformant.

Next, the novel DNA of the second aspect of the present invention will be described. The novel DNA of the present invention is a DNA having a nucleotide sequence encoding the novel polypeptide of the first aspect of the present invention. That is, the DNA of the present invention has a nucleotide sequence encoding a polypeptide having the amino acid sequence of the above formula 1, preferably the amino acid sequence of the above formula 2, and more preferably the amino acid sequence of the above formula 3. DN
It is A. The novel DNA of the present invention is the novel polypeptide of the first aspect of the present invention, which is obtained by introducing the DNA into an appropriate host cell by an appropriate method to transform the host cell. DNA having any base sequence may be used as long as it is a DNA capable of producing As is well known, considering that there are a plurality of codons corresponding to one amino acid, the nucleotide sequence encoding the novel polypeptide of the first aspect of the present invention is not limited to only one type, and thus the nucleotide sequence of the present invention is not limited. The base sequence of the novel DNA is not limited to one kind. However, the base sequence encoding the amino acid sequence of the above formulas 1 to 3 is:
The following formula 4 (SEQ ID NO: 5 in the sequence listing), formula 5 (SEQ ID NO: 6 in the sequence listing) and formula 6 (SEQ ID NO: 7 in the sequence listing), respectively
It is preferable that it is the base sequence described in.

Formula 4 TGT AAT CTA CCA ATA GTC Xnn-1 GGC CCC TGC CGA GCC TTC ATC Xnn-2 CTC TGG GCA TTT GAT GCT GTC AAG GGG AAG TGC GTC CTC TTC CCC TAC GGG GGC TGC CAG GGC AAC GGG AAC AAG TTC Xnn-3 TCA GAG AAG GAG TGC AGA GAG TAC TGC (However, Xnn -1 is a nucleotide sequence other than CGG, Xnn-2 is CAG
Other than TAC, Xnn-3 is a base sequence other than TAC. )

Formula 5 TGT AAT CTA CCA ATA GTC AGC GGC CCC TGC CGA GCC TTC ATC AAG CTC TGG GCA TTT GAT GCT GTC AAG GGG AAG TGC GTC CTC TTC CCC TAC GGG GGC TGC CAG GGC AAC GGG AAC AAG TTC GAC TCA GAG AAG GAG TGC AGA GAG TAC TGC

Formula 6 GCC GCC TGT AAT CTA CCA ATA GTC AGC GGC CCC TGC CGA GCC TTC ATC AAG CTC TGG GCA TTT GAT GCT GTC AAG GGG AAG TGC GTC CTC TTC CCC TAC GGG GGC TGC CAG GGC AAC GGG AAC AAG TTC GAC TCA GAG AAG GAG TGC AGA GAG TAC TGC GGT GTC CCT GGT GAT GGT GAT GAG GAG CTG CTG CGC TTC TCC AAC

The DNA of the present invention has D having a base sequence in which any one or more bases are added to its 5'end and / or 3'end in addition to the base sequences of the above formulas 4 to 6.
It may be NA. Here, the term "5'-end and / or 3'-end" means that any one or more bases may be added only to the 5'-end or the 3'-end, and
It indicates that it may be at both ends. As long as the finally obtained DNA can encode the polypeptide of the first aspect of the present invention, any kind and number of bases can be added. For example, a sequence encoding a start codon, a promoter, a ribosome binding site, a signal peptide may be added at the 5'end, and a stop codon may be added at the 3'end. Furthermore, a sequence recognized by an appropriate restriction enzyme may be added to the 5'end and / or the 3'end, and the novel polypeptide of the first aspect of the present invention may be a fusion protein with another polypeptide. A nucleotide sequence encoding another polypeptide may be added to the 5 ′ end and / or the 3 ′ end for the purpose of producing

The novel DNA of the present invention may be obtained by any method. For example, it may be chemically synthesized, or may be prepared by recombinant DNA technology. Novel DN of the present invention
The chemical synthesis of A is performed as follows, for example. That is, a desired base sequence is designed by dividing it into appropriate fragments, and oligomers corresponding thereto are chemically synthesized using a fully automatic DNA chemical synthesizer (for example, 394 type, manufactured by Applied Biosystems). If necessary, the 5'end of the DNA oligomer obtained using T4 polynucleotide kinase is phosphorylated at the 5'end and then annealed. Then, if necessary, each of the annealed fragments is ligated with T4 DNA ligase and then cloned into an appropriate vector.

On the other hand, the novel DNA of the present invention is used as a recombinant DNA.
For example, suitable cDNA libraries, chromosomal DNA libraries or the above formula 7 can be prepared using techniques.
Site-directed mutagenesis method (for example, Kramer. W et al., Nucleic Acid Res., Vol. 12, 944) using DNA encoding the amino acid sequence of
1-9456, 1984 or Kunkel, TA, etc.
Methods in Enzymology, Volume 154, 367-382
Page, 1987) and the polymerase chain reaction (PCR) method (for example,
PCR Protocols, aguide to methods and applications,
Michakl AI, et al., 1990, Academic Press) may be used to modify the nucleotide sequence and amplify the DNA by a known method. The DNA library used here is a commercially available c
An appropriate one may be selected from a DNA library, a chromosomal DNA library and the like. In addition, known methods (for example, Molecular Cloning, a laboratory manual, T. Mani
atis et al., Cold Spring Harbor Laboratory, 1982)
According to the above, a cDNA library or a chromosomal library may be prepared from an appropriate tissue or cell and used. Further, the DNA encoding the amino acid sequence of the above formula 7 may be chemically synthesized, but may be hybridized (eg, Wallace RB, Nucleic
Acid Res., Vol. 9, 879-894, 1981), etc., and may be obtained from an appropriate DNA library by appropriately using known recombinant DNA technology. Of course, if necessary, a portion of the desired DNA was prepared by recombinant DNA technology by chemical synthesis D
Chemical synthesis and recombinant DNA technology may be combined, such as ligation with NA fragments. According to the disclosure of the novel DNA of the present invention, a DNA having a sequence complementary thereto
And RNA can also be obtained.

A third aspect of the present invention is a vector containing the novel DNA of the second aspect of the present invention. The vector may be used for any purpose. Usually, when a host is transformed with a vector containing a DNA encoding a desired polypeptide to express the desired polypeptide, the vector is required in addition to the DNA encoding the desired polypeptide and expression A vector containing a base sequence such as a promoter and an arbitrary ribosome binding site is used. In addition, the desired polypeptide,
In order to secrete it from the host body, a vector having a base sequence encoding a signal peptide in addition to the base sequence required for the above expression is usually used as a vector. Therefore, the vector of the present invention also preferably has, in addition to the novel DNA of the second aspect of the present invention, a nucleotide sequence such as a promoter and a ribosome binding site, and if necessary, a nucleotide sequence encoding a signal peptide. Vectors also containing are preferred. In addition, the above promoter,
The ribosome binding site, the nucleotide sequence encoding the signal peptide, and the like may be any sequences as long as they function in the host used.

In order to obtain the preferred vector of the present invention, it is necessary for expression and secretion of the novel polypeptide of the first aspect of the present invention, such as a promoter, a ribosome binding site, and a nucleotide sequence encoding a signal peptide. The novel D of the second aspect of the present invention can be added to a plasmid vector, a phage vector, a virus vector or the like which already has a nucleotide sequence.
The novel DNA of the second aspect of the present invention is obtained by introducing NA or chemically synthesizing the base sequence required for the expression.
At the same time, it may be introduced into an appropriate position such as a plasmid vector, a phage vector, or a viral vector. Since various vectors are commercially available at present,
It can be appropriately selected and used from these. In order to introduce DNA into the vector, known methods (for example, Molecular Cloning, a laboratory manual, T. M.
aniatis, etc., Cold Spring Harbor Laboratory, 19
1982).

The fourth aspect of the present invention is a transformant transformed with the novel DNA of the second aspect of the present invention.
The transformant of the fourth aspect of the present invention preferably produces the novel polypeptide of the first aspect of the present invention. The transformant may be a transformant that accumulates the produced novel polypeptide in the body or a transformant that secretes it outside the body.

The transformant of the fourth aspect of the present invention can be transformed into an appropriate host cell by a known method such as a calcium chloride method, a method using a calcium phosphate-DNA complex, a microinjection method, and a perforation method by electric pulse. It can be prepared by introducing the novel DNA of the second aspect of the invention. The novel DNA of the second aspect of the present invention used here is a novel DNA of the first aspect of the present invention such as a promoter and a ribosome binding site in addition to the nucleotide sequence encoding the novel polypeptide of the first aspect of the present invention. Those having a base sequence required for peptide expression are preferable. In particular, when the obtained transformant is intended to produce and secrete the polypeptide of the present invention, the DNA used here encodes a signal peptide in addition to the nucleotide sequence required for expression. Those having a base sequence are preferable. The promoter, the ribosome binding site, the base sequence encoding the signal peptide and the like may be any sequences as long as they function in the host used.

The host cells into which the novel DNA of the second aspect of the present invention is introduced are HeLa cells, COS cells, as long as they are cells suitable for expressing the novel polypeptide of the first aspect of the present invention.
It may be a eukaryotic cell typified by CHO cells or yeast, or may be a prokaryotic cell typified by Escherichia coli or Bacillus subtilis. Appropriate cells may be selected from these cells and used as host cells.

The fifth aspect of the present invention is a transformant transformed with the vector of the third aspect of the present invention. The transformant of the present invention preferably produces the novel polypeptide of the present invention. The transformant may be one that accumulates the produced novel polypeptide in the body or one that secretes it outside the body. The transformant of the fifth aspect of the present invention is a calcium chloride method, a rubidium chloride method,
Hanahan's Method (by Hanahan, D, Techniques
for Transformation of E, coli. In: DNA cloning, v
ol 1, Glover, DM (ed.), pp. 109-136, IRL Pr
ess, 1985), etc., and a suitable host is transformed with the vector of the third aspect of the present invention. The host cell used in the fifth aspect of the present invention into which the vector of the third aspect of the present invention is introduced is HeL as long as it is a cell suitable for expressing the novel polypeptide of the first aspect of the present invention.
It may be a eukaryotic cell represented by a cell, COS cell, CHO cell, yeast or the like, or a prokaryotic cell represented by Escherichia coli, Bacillus subtilis or the like. However,
The host cell is preferably E. coli.

As is well known, a host cell and a vector are
Since they function with each other, it is preferable to combine the vector and the host cell so that the novel polypeptide of the first aspect of the present invention can be expressed, and obtain the transformant of the present invention. In particular, the combination of the origin of the vector, the sequence required for expression of the vector, and the host cell is important. For example, when a mammalian cell is used as the host cell and the vector of the third aspect of the present invention is a viral vector, the vector is
In addition to the DNA of the second aspect of the present invention, a sequence required for expression of a promoter, a ribosome binding site, etc., and an RN
It may be a vector having an A splicing region and a poly A addition signal.

A specific example of the combination of the expression vector and the host cell is simian virus 40 (SV4
0) an expression vector containing the early promoter and C
There are combinations of OS-7 cells, combinations of an expression vector derived from plasmid pBR322 and containing a tryptophan promoter and a nucleotide sequence encoding a tryptophan SD sequence, and Escherichia coli HB101 strain.

The inventors of the present invention transformed the preferred transformant of the present invention with the Escherichia coli JE5505 strain transformed with the vector of the fifth aspect of the present invention, which will be described later in detail in Examples, to Yatabe, Tsukuba-gun, Ibaraki prefecture. It has been deposited at the Patent Microorganism Depositary Center, Institute of Microbial Technology, Ministry of International Trade and Industry, which is located at 1-3, Machihigashi. The names and deposit numbers for identifying microorganisms are shown below. Date of Deposit Deposit Number Labeling of Microorganisms April 28, 2005 Microtechnology Research Institute No. 4285 Escherichia coli JE5505 (pM765) (FERM BP-4285) Depositor Hideaki Morishita

Next, the pharmaceutical composition according to the sixth aspect of the present invention will be described. The pharmaceutical composition provided by the present invention contains the novel polypeptide of the first aspect of the present invention as an active ingredient. The sixth pharmaceutical composition of the present invention comprises only the polypeptide of the first aspect of the present invention (for example, one which has been treated in a pharmaceutically necessary step such as freeze-drying or sterile filtration). Also, it is possible to sufficiently provide the effect to the medical field, but in addition to the above-mentioned polypeptide, a pharmaceutically acceptable auxiliary component may be contained in a pharmaceutically acceptable amount. .

The auxiliary ingredients that can be contained in the pharmaceutical composition of the present invention include bases, stabilizers, preservatives, preservatives, emulsifiers,
Suspending agents, solubilizers, solubilizers, lubricants, corrigents, coloring agents, fragrances, soothing agents, excipients, binders, thickeners, buffers, etc. Include calcium carbonate, lactose, saccharose, sorbit, mannitol, starch, amylopectin, cellulose derivatives, gelatin, cocoa butter,
Distilled water for injection, sodium chloride solution, Ringer's solution,
Examples include glucose solution and human serum albumin (HSA). Among them, gelatin and albumin are useful as protein stabilizers. In selecting the auxiliary components used in the pharmaceutical composition of the present invention, the list of pharmaceutical additives (published by the Pharmaceutical Affairs Law Committee of the Tokyo Pharmaceutical Industry Association and the Pharmaceutical Affairs Law Research Committee of Osaka Pharmaceutical Association) can be referred to. It may be appropriately determined depending on the drug form of the pharmaceutical composition.

The dosage form of the pharmaceutical composition of the present invention is not particularly limited as long as it has a form suitable for the method of use. Generally, the dosage form of the pharmaceutical composition is an injection, tablet, capsule, pill, granule, suppository, solution, suspension, emulsion, powder, ointment, cream, gel, poultice, lotion. Etc., and the pharmaceutical composition of the present invention can take any form thereof.
The dose of the pharmaceutical composition of the present invention is appropriately selected depending on the content of the active ingredient, the condition of the patient to be treated, age, sex, body weight and the like. A preferable example of the dose is 0.1 mg to 1000 mg / kg, more preferably 0.2 mg to 50 mg / kg, as the amount of the active ingredient. More preferably, it is 0.2 mg to 20 mg / kg. In addition, the pharmaceutical composition of the present invention is orally administered, intramuscularly, intraperitoneally, intradermally, subcutaneously, intravenously, intraarterially, rectally, or vaginally depending on the patient's condition. Further, it can be used in various administration methods such as inhalation into the respiratory tract as an aerosol, dissolved in the oral cavity, absorbed transdermally, absorbed transmucosally, etc., but used by a method administered intravenously. Preferably.

The pharmaceutical composition of the present invention prevents and / or prevents diseases associated with at least one of FXa and elastase.
Alternatively, it is preferably used for therapy. As diseases involving FXa, diseases associated with abnormal blood coagulation / fibrinolysis system in vivo, such as DIC, acute liver failure, ischemic heart disease, and multiple organ failure are known. In addition, as diseases involving elastase, for example, adult respiratory distress syndrome (ARDS) and emphysema are known. The pharmaceutical composition of the present invention can, of course, also be used when two or more diseases involving FXa and elastase are combined. The pharmaceutical composition of the present invention has, due to its protease inhibitory activity, in addition to the above diseases, surgical invasion, shock, pancreatitis, nephritis, cirrhosis, re-occlusion during revascularization, edema due to vascular permeability enhancement, and adult respiratory distress. Syndrome, rheumatoid arthritis,
It may be used for the prevention and / or treatment of diseases such as arthritis and allergies. Further, the pharmaceutical composition of the present invention can be used as a blood coagulation inhibitor, not limited to a specific disease. In addition to being directly administered to the human body, on the surface of medical equipment such as artificial blood vessels, artificial organs, and catheters,
It can also be used for the purpose of binding and adsorbing with a cross-linking agent or the like to prevent blood coagulation.

Next, the seventh and eighth aspects of the present invention will be described. A seventh aspect of the present invention is a method of inhibiting the activity of FXa using the novel polypeptide of the first aspect of the present invention.

The eighth aspect of the present invention is a method for inhibiting the activity of elastase using the novel polypeptide of the first aspect of the present invention.

The method comprises reacting FXa or elastase with the novel polypeptide of the first aspect of the present invention under appropriate conditions, for example, at an appropriate temperature, an appropriate pH, and an appropriate time. As a method of inhibiting the activity, the reaction may be performed in vitro such as in a test tube or in an animal body (in vivo or ex vivo).
In addition to the polypeptide of the first aspect of the present invention, other substances or agents may be added for the reaction if necessary. The novel polypeptide used in the enzyme inhibition method may be in the form of a composition containing auxiliary components and the like.

[0053]

EXAMPLES The present invention will be described in more detail with reference to examples below, but these are given as examples and the present invention is not limited thereto.
The abbreviations used in the following description are based on conventional abbreviations in the field. The various operations in the examples were carried out with reference to the following magazines and books.

1. Laboratory Manual Genetic Engineering, Masamitsu Muramatsu, 1989, Maruzen Co., Ltd. 2. Gene manipulation experiment method, edited by Yasutaka Takagi, 1980, Kodansha 3. Gene Operation Manual, edited by Yasutaka Takagi, 1982
Year, Kodansha 4. Molecular Cloning, a laboratory manual, T. Man
iatis et al., 1982, Cold Spring Harbor Laborat
ory 5. Methods in Enzymology, Volume 65, L. Grossman et al., 1980, Academic Press 6. Methods in Enzymology, Volume 68, edited by R. Wu, 19
1979, Academic Press 7. PCR Protocols, a guide to methods and applica
tions, Michael del A. I et al., 1990, Academic P
ress 8. Molecular Cloning, a laboratory manual, Second
editionT. Maniatis et al., 1989, Cold Spring
Harbor Laboratory

Example 1. Preparation of DNA of the Present Invention The DNA of the present invention having the nucleotide sequence of the above formula 4 was obtained by the following method. Construction of plasmid pM594 Using plasmid pM552 (see Japanese Patent Application No. 3-325220), site-directed mutagenesis (Site-dire
cted mutagenesis) (Kunkel TA et al., Methods in Enz
ymology, 154, 367, 1987), pM594 was prepared by the following method. The plasmid pM552 contains a polypeptide represented by the amino acid sequence of formula 8 below, and a base sequence encoding TN70,
That is, it is a plasmid having a DNA represented by the nucleotide sequence of the following formula 9 and further having a tryptophan promoter, a nucleotide sequence encoding an alkaline phosphatase signal peptide, and a kanamycin resistance gene (see FIG. 1).

Formula 8 Thr Val Ala Ala Cys Asn Leu Pro Ile Val Arg Gly Pro Cys Arg Ala Phe Ile Gln Leu Trp Ala Phe Asp Ala Val Lys Gly Lys Cys Val Leu Phe Pro Tyr Gly Gly Cys Gln Gly Asn Gly Asn Lys Phe Tyr Ser Glu Lys Glu Cys Arg Glu Tyr Cys Gly Val Pro Gly Asp Gly Asp Glu Glu Leu Leu Arg Phe Ser Asn

Formula 9 ACC GTC GCC GCC TGC AAT CTC CCC ATA GTC CGG GGC CCC TGC CGA GCC TTC ATC CAG CTC TGG GCA TTT GAT GCT GTC AAG GGG AAG TGC GTC CTC TTC CCC TAC GGG GGC TGC CAG GGC AAC GGG AAC AAG TTC TAC TCA GAG AAG GAG TGC AGA GAG TAC TGC GGT GTC CCT GGT GAT GGT GAT GAG GAG CTG CTG CGC TTC TCC AAC

First, the HindIII primer (FIG. 2) and the AN68 primer (FIG. 3) were combined with a chemical synthesizer (381).
A, Applied by Systems) was used for chemical synthesis and purification. First PCR using HindIII primer as a sense primer, AN68 primer as an antisense primer, and pM552 as a template was performed using Gene Amp Kit ^R (Takara Shuzo Co., Ltd.). The reaction was repeated at 94 ° C. for 1 minute, 55 ° C. for 2 minutes, and 72 ° C. for 3 minutes as one cycle, and 30 cycles were repeated. Primary PC
When a part of the amplified product after R was subjected to 1.5% agarose gel electrophoresis, a target DNA fragment of about 130 bp in size was confirmed. Amplification products treated with phenol /
After purification by ethanol precipitation, it was dissolved in TE buffer. Second PCR using the amplified product of this first PCR as a sense primer and plasmid pM552 as a template
Was performed in the same manner as the first PCR. As the antisense primer, pBR Bam obtained by chemical synthesis was used.
The HI primer (Figure 4) was used. When a part of the amplification product after the PCR was subjected to 1.5% agarose gel electrophoresis, a band of the target size of about 350 bp was confirmed. The obtained amplification product was purified by phenol treatment / ethanol precipitation and then digested with restriction enzymes HindIII and BamHI to obtain a DNA fragment having a size of about 300 bp.

Plasmid pM463 (Kanamori)
T. Et al., Gene 66, 259-300, 19
1988) (Fig. 5) with the restriction enzymes HindIII and Bam.
Double digested with HI. This and the above size of about 300bp
The DNA fragment of was ligated using T4 DNA ligase to obtain plasmid pM594.

Construction of plasmid pM710 The DNA shown in the nucleotide sequence of FIG. 6 was designed by dividing it into 5 fragments. Of these five fragments, S2, S3,
S4 was phosphorylated at the 5'end with T4 polynucleotide kinase in the presence of ATP. After annealing each piece,
Ligation was performed using T4 DNA ligase (Takara Shuzo Co., Ltd.). 8% of sample after ligation
About 150bp by polyacrylamide gel electrophoresis
Was isolated and prepared. The plasmid pM594 constructed in 1. was double-digested with the restriction enzymes HindIII and ApaI to obtain a DNA fragment of about 3.2 kbp. This approximately 3.2 kbp DNA fragment and the approximately 150 kbp DNA fragment previously obtained by chemical synthesis were ligated with T4 DNA ligase to prepare plasmid pM710.

Construction of plasmid pM748 Using the plasmid pM710 as a template, PCR was carried out twice. The PCR conditions were followed. The chemically synthesized Y46D primer (Fig. 7) was used as the sense primer for the first PCR, and the pBRBamHI primer was used as the antisense primer. When the obtained amplification product was subjected to 1.5% agarose gel electrophoresis, a target band of about 120 bp in size was observed. This amplification product was purified by phenol treatment / ethanol precipitation and then dissolved in TE buffer. This first
Using the amplification product of the next PCR as an antisense primer,
A second PCR was carried out using the indIII primer as a sense primer and the plasmid pM710 as a template.
When the obtained amplification product was subjected to 1.5% agarose gel electrophoresis, a band of the desired size of about 380 bp was observed. The amplified product of this secondary PCR was purified by phenol treatment / ethanol precipitation and then incorporated into plasmid pM463 in the same manner as described above to obtain plasmid pM748.
Was produced.

Construction of plasmid pM727 PCR was carried out twice using the plasmid pM748 as a template. The PCR conditions were followed. The HindIII primer was used as the sense primer for the first PCR.
A Q19K primer (FIG. 8) was chemically synthesized and used as an antisense primer. When the obtained amplification product was subjected to 1.5% agarose gel electrophoresis, a band of the desired size of about 210 bp was observed. This amplification product was purified by phenol treatment / ethanol precipitation and then dissolved in TE buffer. This first PCR
The amplification product of pBRBamHI was used as a sense primer.
Secondary PCR was performed using the primer as an antisense primer and the plasmid pM748 as a template. When the obtained amplification product was subjected to 1.5% agarose gel electrophoresis, a band of the desired size of about 380 bp was observed. Amplification product of this second PCR is treated with phenol /
After purification by ethanol precipitation, it was incorporated into plasmid pM463 in the same manner as in
7 was produced.

Construction of plasmid pM765 PCR was performed twice using the plasmid pM727 as a template. The PCR conditions were followed. HindIII primer was used as the sense primer for the first PCR, and R1 obtained by chemical synthesis was used as the antisense primer.
The 1S primer (Figure 9) was used. When the obtained amplification product was subjected to 1.5% agarose gel electrophoresis, a target band of about 180 bp in size was observed. This amplification product was purified by phenol treatment / ethanol precipitation and then dissolved in TE buffer. This primary PC
Using the R amplification product as a sense primer, pBRBamH
A second PCR was carried out using the I primer as an antisense primer and the plasmid pM727 as a template. When the amplification product after the second PCR was subjected to 1.5% agarose gel electrophoresis, the target size was about 380 bp.
Band was confirmed. The amplification product of this secondary PCR was purified by phenol treatment / ethanol precipitation and
I got NA. The obtained DNA of the present invention was incorporated into the plasmid pM463 by the same method as described above to prepare the expression plasmid pM765. The plasmid pM765 was double-digested with the restriction enzymes HindIII and BamHI, and the desired fragment of about 340 bp was extracted and purified. DN
Sequencing was performed using A sequencer (DNA Sequencer 370A, Applied Biosystems). Hind containing the DNA encoding the novel polypeptide of the present invention in plasmid pM765
The confirmed nucleotide sequence from the III digestion site to the BamHI digestion site and the corresponding amino acid sequence are shown in Fig. 10 (see SEQ ID NO: 2 in the sequence listing).

Example 2. Production of Polypeptide of the Present Invention The polypeptide of the present invention was produced by the following method.

Preparation and Culture of Transformant Method of Hanahan (Hanahan, D, Techniques for Trans
formation of E, coli. In: DNA cloning, vol 1, Glov
er, DM (ed.), pp. 109-136, IRL Press, 1985), Escherichia coli JE5505 was transformed with the plasmid pM765 obtained in Example 1 to obtain E. coli strain JE5.
505 (pM765) was prepared. The resulting transformant, JE5505 (pM765), was treated with ampicillin 50 μm.
g / ml containing L-broth at 37 ° C. for about 8 hours,
After culturing before the pre-incubation, inoculate in about 100 times the same amount of medium,
The cells were cultured overnight (preculture) at ° C. Then, 50 times as much of M9CA medium containing 50 μg / ml of ampicillin was inoculated as the main culture, and cultured at 37 ° C. for about 1 hour. 3β- in the medium
Indole acrylic acid (manufactured by Wako Pure Chemical Industries, Ltd.) was added to a final concentration of 10 μg / ml, and the mixture was further cultured for 16 hours. The obtained culture mixture was mixed with Benchmar.
k GX (membrane (Membren), pore size 0.
2 μm) and then concentrated at 4 ° C. to about 10,000 ×
The cells were centrifuged for 20 minutes to recover the bacterial cells.

The microbial cell obtained by the solubilization and reduction treatment was disrupted with a solution (0.5% Trito).
It was suspended in nX-100, 10 mM EDTA) and crushed under pressure at 800 bar using a minilab (RANNIE). Then, it was centrifuged at 4 ° C. for 20 minutes at about 10,000 × g to collect the pellet. Add the crushing solution to the recovered pellets, and again at 4 ° C for 20 minutes, about 100
Centrifuge at 00 × g. This operation was performed twice more, and the pellets were collected. Solubilization buffer (5M guanidine hydrochloride, 0.005% Tween8) was added to the recovered pellet.
0,50 mM Tris hydrochloric acid pH 8.0,5 mM ED
TA, 2 mM reduced glutathione, 0.02 mM oxidized glutathione) was added, 2-mercaptoethanol was further added to a final concentration of 50 mM, and the mixture was stirred overnight at 4 ° C. After this solution was concentrated using an ultrafiltration membrane (YM-5, Grace Japan), the pore size was 0.44.
Filtered using a μm filter. Sephacr
A column (5 cmφ × 95 cm) packed with yl S-100 HR (Pharmacia) was equilibrated with the above-mentioned solubilization buffer, and the concentrated filtrate was added thereto. Gel filtration was performed at a flow rate of 3.5 ml / min while monitoring the protein content using the above-mentioned solubilization buffer as a developing solution and the absorbance at a wavelength of 280 nm as an index. 30 ml of each fraction was collected, and a part of each fraction was collected and subjected to SDS-PAGE. SDS-PAGE is a PAGEL ^R (SPU-15
S, Ato Co., Ltd.) was used according to the instruction manual. Staining with Coomassie Brilliant Blue, a fraction containing a large amount of the desired molecular weight polypeptide was selected. A solubilization buffer was used to adjust the protein content of this fraction to approximately 0.5 mg / ml, and this was used as a sample for refolding treatment.

The sample for refolding obtained by refolding was dialyzed twice with a solution obtained by removing guanidine hydrochloride from the solubilization buffer used in 2. Then, dialysis was performed three times using distilled water in an amount of about 15 times the amount of the sample. After dialysis, use hydrochloric acid to adjust the sample to pH 2
Adjusted to. This sample was purified as follows.

Reverse Phase Chromatography A sample after refolding was added to a PLRP-S column (25 mmφ × 150 mm, manufactured by Polymer Laboratories) preliminarily equilibrated with a 0.1% TFA solution, and a 0.1% TFA solution was added. Was used for adsorption. Elution is
Using a 0.1% TFA / acetonitrile solution, a linear gradient of acetonitrile (0-70% acetonitrile /
0.1% TFA solution / 30 minutes, 70-100% acetonitrile / 0.1% TFA solution / 3 minutes), flow rate 5 m
Elution was performed at 1 / min while monitoring the absorbance at a wavelength of 280 nm, and 5 ml each was collected. The trypsin inhibitory activity of each fraction was measured according to the method of Example 3 (1), and the fraction in which activity was observed was freeze-dried and then dissolved in 70% formic acid to a concentration of about 100 μM. Then, add cyanogen bromide so that the molar concentration becomes 2000 times,
It was left standing in the dark at 25 ° C for 24 hours. Distilled water was added to this to dilute it by a factor of 2 to obtain a sample for ion exchange chromatography.

Cation Exchange Chromatography SP-Toyopearl (30 mmφ × 150 mm, Tosoh Corporation) was equilibrated with a 10% formic acid solution, and then cation exchange chromatography was performed by the following method using an FPLC system (existing). It was That is, the sample obtained in (2) was added to SP-Toyopearl and adsorbed using a 10% formic acid solution. Use NaCl / 10% formic acid
Linear gradient of Cl (0-1.2M NaCl / 10%
Formic acid solution / 100 minutes) at a flow rate of 8 ml / minute,
Elution was performed while monitoring the absorbance at a wavelength of 280 nm.
32 ml of each fraction was collected, and the trypsin inhibitory activity of each fraction was measured by the method of Example 3 (1). The obtained active fraction was used as a sample for reverse phase chromatography.

Reversed-phase chromatography PLRP-S column (25 mmφ × 150 mm, already mentioned)
Was equilibrated with 0.1% TFA solution, and then the active fraction obtained in (1) was added and adsorbed with 0.1% TFA solution. A 0.1% TFA / acetonitrile solution was used for elution, and a linear concentration gradient of acetanilyl (0-70% / acetonitrile / 0.1% TFA / 15 minutes, 70-100%).
Acetonitrile / 0.1% TFA solution / 3 min) at a flow rate of 10 ml / min. Elution was performed while monitoring the absorbance at a wavelength of 280 nm, and each peak was fractionated, and the trypsin inhibitory activity of each fraction was measured by the method of Example 3.
The obtained active fraction was freeze-dried to obtain a purified sample.

[0071] was subjected to purified preparation obtained by SDS-PAGE in SDS-PAGE using PAGEL ^R (supra). As a result of silver staining, a single band was recognized.

The purified polypeptide obtained by the analysis of the amino acid sequence was dissolved in 50% acetic acid to prepare a model 477A protein sequencing system-120A PTH.
The amino acid sequence was analyzed using an analyzer (Applied Biosystems). 27 PTH Amino Acids
The retention time was measured by detecting it by ultraviolet absorption at 0 nm, and the amino acid was identified based on the retention time of a standard PTH amino acid (Applied Biosystems) previously separated by the same method. As a result, it was confirmed that the purified polypeptide obtained in the above was the desired polypeptide of the present invention (see SEQ ID NO: 4 in the sequence listing).

Example 3. Measurement of Enzyme Inhibitory Activity Using the purified preparation of the polypeptide of the present invention obtained in Example 2, trypsin, FXa and elastase inhibitory activity were measured according to the following methods. Regarding FXa and elastase inhibitory activity, the inhibitory activity is determined by using the polypeptide AN68.
Compared with. Polypeptide AN68 is a polypeptide purified from the culture supernatant of transformant JE5505 (pM594) transformed with plasmid pM594 prepared in Example 1. The amino acid sequence is shown below.

Ala Ala Cys Asn Leu Pro Ile Val Arg Gly Pro Cys Arg Ala Phe Ile Gln Leu Trp Ala Phe Asp Ala Val Lys Gly Lys Cys Val Leu Phe Pro Tyr Gly Gly Cys Gln Gly Asn Gly Asn Lys Phe Tyr Ser Glu Lys Glu Cys Arg Glu Tyr Cys Gly Val Pro Gly Asp Gly Asp Glu Glu Leu Leu Arg Phe Ser Asn

(1) Trypsin Inhibitory Activity A purified preparation of the polypeptide of the present invention was dissolved in 100 μl of distilled water, and then 0.1% BSA / 0.2M triethanolamine-hydrochloric acid buffer solution (pH 7.8) was used. Serially diluted to obtain a solution for activity measurement. Using a synthetic substrate S-2444 (manufactured by Daiichi Pure Chemicals Co., Ltd.), the method of Kassel (Kassell B. et al., Methods in Enzymol., Vol. 19, 8)
44-852, 1970), and the trypsin inhibitory activity of the activity measurement solution was measured. That is, bovine trypsin (Type XIII, manufactured by Sigma) was used at a concentration of 13
0.001MH to be 600BAEEU / ml
After being dissolved in Cl, it was further diluted with 0.1% BSA / 0.2M triethanolamine-hydrochloric acid buffer (pH 7.8) to prepare a trypsin solution having a concentration of 1.2BAEEU / ml. Separately, the concentration of the synthetic substrate S-2444 is 2 m.
It was dissolved in distilled water so as to have M, to give an S-244 solution. 100 μl of the above-mentioned activity measuring solution and 100 μl of bovine trypsin solution were mixed and left at 37 ° C. for 10 minutes. To this, 50 μl of the above S-2444 solution was added, and 37
After reacting at 50 ° C for 15 minutes, 50 µl of 50% acetic acid was added to stop the reaction, and a wavelength of 405 n
The absorbance at m was measured. In addition, in order to subtract the influence of the various solutions used here on the absorbance, 50% acetic acid was added.
100 μl of the above-mentioned bovine trypsin solution to which μl had been added in advance
100 μl of the activity measurement solution and 50 μl of the S-2444 solution were used as a blank. As a result, it was confirmed that the novel polypeptide of the present invention inhibits trypsin in a concentration-dependent manner.

(2) Human Trypsin Inhibitory Activity A purified preparation of the polypeptide of the present invention was dissolved in 100 μl of distilled water. UTI (Mochida Pharmaceutical Co., Ltd., Haruo Onishi, etc.
(See Japanese Pharmacology Journal, Vol. 85, pp. 1-6, 1985)
The concentration of the polypeptide in this solution was calculated from the bovine trypsin inhibitory activity measured by the method (1) using the above as a standard. Then, the polypeptide solution was diluted to various concentrations with 0.03% BSA / 0.2M triethanolamine-hydrochloric acid buffer solution (pH 7.8) to prepare a solution for activity measurement. Separately, human trypsin (manufactured by Calbiochem) dissolved in 1 mM HCl at a temperature of 1000 SU / ml was diluted with 0.03% BSA / 0.2 M triethanolamine-hydrochloric acid buffer solution (pH 7.8),
A human trypsin solution of 1.25 SU / ml was prepared, and the human trypsin inhibitory activity was measured in the same manner as in (1) using the synthetic substrate S-2444 (existing) as a substrate. As a result, it was confirmed that the novel polypeptide of the present invention inhibits human trypsin in a concentration-dependent manner (Fig. 13).

(3) Inhibitory activity against FXa The purified preparations of the polypeptide of the present invention and the polypeptide AN68 were each dissolved in 100 μl of distilled water. UTI
As a standard, the concentration of the polypeptide in each solution was calculated from the bovine trypsin inhibitory activity measured by the method (1). This solution is 0.1% BSA / 150 mM
NaCl / 5mM CaCl ₂ / 50mM Tris-HCl buffer with (pH 8.3), diluted to various concentrations, was activity measurement solution. The FXa inhibitory activity of the activity measurement solution was measured using a synthetic substrate S-2222 (Daiichi Pure Chemicals Co., Ltd.) by a method such as Ohno (Ohno H. et al., T.
hromb. Res., 19, 579-588, 1980). First, human FXa (manufactured by American Diagnostics Co., Ltd.) was dissolved in distilled water so that the concentration was 10 PEU / ml, and then 0.1% BSA / 150 m
Diluted with M NaCl / 5mMCaCl ₂ / 50mM Tris-HCl buffer (pH8.3), 0.1PEU /
A ml FXa solution was prepared. Separately, S-2222 was dissolved in distilled water so that the concentration was 4 mM, and then 0.1% BSA / 150 mM NaCl / 5 mM CaCl was added.
_2/50 mM Tris-HCl buffer (pH 8.3) was diluted with was prepared concentration 2mM of S-2222 solution. 25 μl of each activity measurement solution, 0.1% BSA / 150 mM
NaCl / 5mM CaCl ₂ / 50mM Tris-HCl buffer (pH 8.3) 100 [mu] l and FXa solution 25
μl was mixed and left at 37 ° C. for 10 minutes. Then S
100 μl of −2222 solution was added and reacted at 37 ° C. for 30 minutes. After the reaction was stopped by adding 50 μl of 50% acetic acid, the absorbance at a wavelength of 405 nm was measured using a spectrophotometer. In order to subtract the influence of the various solutions used here on the absorbance, 50 μl of 50% acetic acid was added in advance to 25 μl of the above FXa solution, and
5 μl, 0.1% BSA / 150 mM NaCl / 5 mM
CaCl _2/50 mM Tris-HCl buffer (pH 8.
3) 100 μl and 100 μl of S-2222 solution were used as a blank.

The results are shown in FIG. In the figure, the abscissa represents the concentration of the polypeptide in the reaction solution as a bovine trypsin inhibitory activity (U / ml). In the figure, the residual human FXa
In the above assay system, the activity was 0.1% BSA / 150 mM NaCl / 5 mM C instead of the activity assay solution.
NaCl _2/50 mM Tris-HCl buffer (pH 8.3) 2
The absorbance is expressed as a percentage, with the absorbance when 5 μl was added and reacted as 100%. The FXa inhibitory activity of the novel polypeptide of the present invention judged from the figure was about 8 times the FXa inhibitory activity of the polypeptide AN68.

(4) Human elastase inhibitory activity Novel polypeptide and polypeptide AN68 of the present invention
Each of the purified preparations was dissolved in 100 μl of distilled water. The polypeptide concentration of each solution, as a standard the UTI (supra), after obtaining from the trypsin-inhibiting _{activity, 0.1% BSA / 27mM CaCl 2} / 133m
It was diluted with M Tris-hydrochloric acid buffer solution (pH 7.5) to various concentrations to prepare a solution for measuring human elastase inhibitory activity. Human elastase inhibitory activity of the activity measurement solution
Using STANA (Peptide Institute) as a substrate, Ogawa (O
gawa) et al. (Ogawa M. et al. Res. Commun. Chem. Pat.
Hol. Pharmacol., 55, 271-274, 1987).

[0080] First, human neutrophil elastase (Calbiochem) at 27 mM CaCl _2/133 mM Tris-HCl buffer (pH 7.5), was prepared in 100 [mu] g / ml, further, 0.1% BSA / 27mM CaCl
_{It was} diluted with 2/133 mM Tris-HCl buffer (pH 7.5) to prepare a 4 μg / ml elastase solution. On the other hand, the synthetic substrate STANA was added to N-methyl-2-pyrrolidone.
(N-methyl-2-pyrrolidone ) in dissolved in 100 mM, _{further, 0.1% BSA / 27mM CaCl 2} / 133m
20m diluted with M Tris-HCl buffer (pH 7.5)
A M STANA solution was prepared. 50 μl of the solution for measuring human elastase inhibitory activity, 50 μl of elastase solution
l, and _{0.1% BSA / 27mM CaCl 2/} 1
50 μl of 33 mM Tris-HCl buffer (pH 7.5) was mixed and left standing at 37 ° C. for 10 minutes. Then STANA
50 μl of solution was added to start the reaction. 2 at 37 ° C
After reacting for 0 minutes, 50 μl of 50% acetic acid was added to stop the reaction, and the absorbance at a wavelength of 405 nm was measured with a spectrophotometer. In the data processing, 50 μl of the elastase solution described above was used as a control for subtracting the influence of the various solutions used here on the absorbance.
50 μl of acetic acid was added, and 50 μl of a solution for activity measurement, 0.1
% BSA / 27mMCaCl ₂ / 133mM Tris-HCl buffer (pH7.5) 50μl, STANA solution 50μ
What added 1 was used.

The results are shown in FIG. In the figure, the residual human elastase activity was 0.1% BSA / 2 in the above assay system instead of the activity assay solution or the control.
7 mM CaCl _2/133 mM Tris-HCl buffer (p
H7.5) was added to 50 μl and reacted to give an absorbance of 1
It is shown as a percentage as 00%.

The elastase inhibitory activity of the polypeptide of the present invention judged from the figure was about 10 times that of the control polypeptide AN68.

From the above, it was confirmed that the polypeptide of the present invention has both extremely high FXa inhibitory activity and elastase inhibitory activity.

Example 4. Safety The following experiment was conducted to confirm the safety of the novel polypeptide of the present invention. First, obtained according to the method of Example 2,
Each purified preparation of the polypeptide of the present invention is dissolved in physiological saline, and Omega cell (100K) (Filtron)
LPS was removed by ultrafiltration using and the animals were subjected to the following animal experiments. Male and female Wi pre-bred for 1 week
Star rats were divided into groups of 10 rats. Each of the above polypeptide solutions was intravenously administered to rats so that the dose of the polypeptide would be 10 mg / kg / day. This was designated as a polypeptide administration group, and symptoms and changes in body weight were observed over a week. In addition, the same number of animals administered with physiological saline was used as a control group. As a result, no significant side effect was observed in any of the polypeptide administration groups, and the survival rate and body weight change were similar to those of the control group.

Example 5. Preparation of Formulation (1) A purified preparation of the polypeptide of the present invention was dissolved in distilled water, and LPS was removed using omegacell (see above).
It was dried under reduced pressure. 1% containing 0.1% (w / v) of pyrogen-free gelatin prepared using distilled water for injection
15M phosphate buffer (pH 7.4) was added to give a concentration of 2 mg.
/ Ml polypeptide solution was prepared. Sodium chloride to a final concentration of 75 mM and mannitol to a final concentration of 2% (w / v),
For each, in addition to the polypeptide solution, a sterilized pore size of 0.
22 μm membrane filter (disposable
After sterilization by filtration using a Sterile filter system (manufactured by Corning Inc.), 5 ml each was dispensed into a glass container.

(2) The purified preparation of the polypeptide of the present invention was dissolved in distilled water, LPS was removed by using Omega cell (see above), and then dried under reduced pressure. 0.01M containing 0.14m sodium chloride prepared using distilled water for injection
Phosphate buffer (pH 7.4) was added to prepare a 2 mg / ml polypeptide solution. Human serum albumin was added to the polypeptide solution so that the final concentration was 1% (w / v), and a sterilized membrane filter with a pore size of 0.22 μm (disposable sterilary filter system, already mentioned) was used. After sterilization by filtration, 5 ml of each was poured into the glass solution, and freeze-dried under sterile conditions.
Sealed.

[0087]

INDUSTRIAL APPLICABILITY The present invention provides a novel polypeptide and a method for producing the same. The present invention also provides a DNA encoding the above polypeptide, a vector containing the sequence of the DNA, and a transformant transformed with the DNA or the vector. Furthermore, the present invention provides a pharmaceutical composition containing the above-mentioned polypeptide as an active ingredient and an enzyme inhibition method characterized by using the above-mentioned polypeptide. Since the polypeptide of the present invention is a proteinaceous protease inhibitor having a high FXa and elastase inhibitory activity, it provides an effective means for treating diseases associated with FXa or elastase. Since the polypeptide of the present invention has blood coagulation-inhibiting activity, it can be used by binding or adsorbing it onto the surface of medical equipment such as artificial blood vessels, artificial organs and catheters using a crosslinking agent or the like.

[0088]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 51 Sequence type: Amino acid Sequence type: Peptide Sequence: Cys Asn Leu Pro Ile Val Xaa-1 Gly Pro Cys Arg Ala Phe Ile Xaa-2 Leu Trp Ala Phe Asp Ala Val Lys Gly Lys Cys Val Leu Phe Pro Tyr Gly Gly Cys Gln Gly Asn Gly Asn Lys Phe Xaa-3 Ser Glu Lys Glu Cys Arg Glu Tyr Cys

SEQ ID NO: 2 Sequence length: 51 Sequence type: Amino acid Sequence type: Peptide Sequence: Cys Asn Leu Pro Ile Val Ser Gly Pro Cys Arg Ala Phe Ile Lys Leu Trp Ala Phe Asp Ala Val Lys Gly Lys Cys Val Leu Phe Pro Tyr Gly Gly Cys Gln Gly Asn Gly Asn Lys Phe Asp Ser Glu Lys Glu Cys Arg Glu Tyr Cys

SEQ ID NO: 3 Sequence length: 68 Sequence type: Amino acid Sequence type: Peptide Sequence: Ala Ala Cys Asn Leu Pro Ile Val Xaa-1 Gly Pro Cys Arg Ala Phe Ile Xaa-2 Leu Trp Ala Phe Asp Ala Val Lys Gly Lys Cys Val Leu Phe Pro Tyr Gly Gly Cys Gln Gly Asn Gly Asn Lys Phe Xaa-3 Ser Glu Lys Glu Cys Arg Glu Tyr Cys Gly Val Pro Gly Asp Gly Asp Glu Glu Leu Leu Arg Phe Ser Asn

SEQ ID NO: 4 Sequence length: 68 Sequence type: Amino acid Sequence type: Peptide Sequence: Ala Ala Cys Asn Leu Pro Ile Val Ser Gly Pro Cys Arg Ala Phe 1 5 10 15 Ile Lys Leu Trp Ala Phe Asp Ala Val Lys Gly Lys Cys Val Leu 20 25 30 Phe Pro Tyr Gly Gly Cys Gln Gly Asn Gly Asn Lys Phe Asp Ser 35 40 45 Glu Lys Glu Cys Arg Glu Tyr Cys Gly Val Pro Gly Asp Gly Asp 50 55 60 Glu Glu Leu Leu Arg Phe Ser Asn 65 68

SEQ ID NO: 5 Sequence length: 153 Sequence type: Nucleic acid Sequence type: Other nucleic acid Sequence: TGT AAT CTA CCA ATA GTC Xnn-1 GGC CCC TGC CGA GCC TTC ATC Xnn-2 CTC TGG GCA TTT GAT GCT GTC AAG GGG AAG TGC GTC CTC TTC CCC TAC GGG GGC TGC CAG GGC AAC GGG AAC AAG TTC Xnn-3 TCA GAG AAG GAG TGC AGA GAG TAC TGC

SEQ ID NO: 6 Sequence length: 153 Sequence type: Nucleic acid Sequence type: Other nucleic acid Sequence: TGT AAT CTA CCA ATA GTC AGC GGC CCC TGC CGA GCC TTC ATC AAG CTC TGG GCA TTT GAT GCT GTC AAG GGG AAG TGC GTC CTC TTC CCC TAC GGG GGC TGC CAG GGC AAC GGG AAC AAG TTC GAC TCA GAG AAG GAG TGC AGA GAG TAC TGC

SEQ ID NO: 7 Sequence length: 204 Sequence type: Nucleic acid Sequence type: Other nucleic acid Sequence: GCC GCC TGT AAT CTA CCA ATA GTC AGC GGC CCC TGC CGA GCC TTC ATC AAG CTC TGG GCA TTT GAT GCT GTC AAG GGG AAG TGC GTC CTC TTC CCC TAC GGG GGC TGC CAG GGC AAC GGG AAC AAG TTC GAC TCA GAG AAG GAG TGC AGA GAG TAC TGC GGT GTC CCT GGT GAT GGT GAT GAG GAG CTG CTG CGC TTC TCC AAC

SEQ ID NO: 8 Sequence length: 343 Sequence type: Nucleic acid Sequence type: Other nucleic acid Origin organism name: Escherichia coli strain name: JE5505 (pM765) Sequence: AAGCTTAAAA AAGGGTATAA AATAAA ATG AAA CAA AGT ACT ATT GCA CTG 50 Met Lys Gln Ser Thr Ile Ala Leu -20 -15 GCA CTC TTA CCG TTA CTG TTT ACC CCT GTG ACA AAG GCC GCT GTG 95 Ala Leu Leu Pro Leu Leu Phe Thr Pro Val Thr Lys Ala Ala Val -10 -1 1 CTA CCG CAA GAA GAA GAA GGC TCG GGT ATG GCC GCC TGT AAT CTA 140 Leu Pro Gln Glu Glu Glu Gly Ser Gly Met Ala Ala Cys Asn Leu 5 10 15 CCA ATA GTC AGC GGC CCC TGC CGA GCC TTC ATC AAG CTC TGG GCA 185 Pro Ile Val Ser Gly Pro Cys Arg Ala Phe Ile Lys Leu Trp Ala 20 25 30 TTT GAT GCT GTC AAG GGG AAG TGC GTC CTC TTC CCC TAC GGG GGC 230 Phe Asp Ala Val Lys Gly Lys Cys Val Leu Phe Pro Tyr Gly Gly 35 40 45 TGC CAG GGC AAC GGG AAC AAG TTC GAC TCA GAG AAG GAG TGC AGA 275 Cys Gln Gly Asn Gly Asn Lys Phe Asp Ser Glu Lys Glu Cys Arg 50 55 60 GAG TAC TGC GGT GTC CCT GG T GAT GGT GAT GAG GAG CTG CTG CGC 320 Glu Tyr Cys Gly Val Pro Gly Asp Gly Asp Glu Glu Leu Leu Arg 65 70 75 TTC TCC AAC TGACAACTGG ATCC 343 Phe Ser Asn 80

[Brief description of drawings]

FIG. 1 shows the plasmid pM552.

FIG. 2 is a view showing a nucleotide sequence of HindIII primer.

FIG. 3 shows the nucleotide sequence of AN68 primer.

FIG. 4 is a diagram showing the nucleotide sequence of pBR BamHI primer.

FIG. 5 shows the plasmid pM463.

FIG. 6 is a view showing a base sequence of a linker 710.

FIG. 7 shows the nucleotide sequence of Y46D primer.

FIG. 8 shows the nucleotide sequence of Q19K primer.

FIG. 9 shows the nucleotide sequence of R11S primer.

FIG. 10 is a diagram showing the nucleotide sequence from the HindIII digestion site to the BamHI digestion site of plasmid pM765 and the corresponding amino acid sequence.

FIG. 11: Polypeptide R11S / Q19K of the present invention
8 is a graph showing the FXa inhibitory activity of / Y46D.

FIG. 12: Polypeptide R11S / Q19K of the present invention
2 is a graph showing the elastase inhibitory activity of / Y46D.

FIG. 13: Polypeptide of the invention R11S / Q19K
3 is a graph showing the human trypsin inhibitory activity of / Y46D.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Reference number within the agency FI Technical indication location A61K 37/64 ABN ABS ACD ACE ACJ ACS 8314-4C ACV C12N 1/21 7236-4B 15/11 C12P 21/02 C 8214-4B // (C12N 1/21 C12R 1:19) (C12P 21/02 C12R 1:19) C07K 99:00

Claims (14)

[Claims] 1. A novel polypeptide having an amino acid sequence represented by the following formula 1. Formula 1 Cys Asn Leu Pro Ile Val Xaa-1 Gly Pro Cys Arg Ala Phe Ile Xaa-2 Leu Trp Ala Phe Asp Ala Val Lys Gly Lys Cys Val Leu Phe Pro Tyr Gly Gly Cys Gln Gly Asn Gly Asn Lys Phe Xaa-3 Ser Glu Lys Glu Cys Arg Glu Tyr Cys (where Xaa-1 is an amino acid other than Arg, Xaa-2 is Gln
Other amino acids, Xaa-3 is an amino acid other than Tyr) 2. The novel polypeptide according to claim 1, wherein the amino acid sequence represented by the above formula 1 is the amino acid sequence represented by the following formula 2. Formula 2 Cys Asn Leu Pro Ile Val Ser Gly Pro Cys Arg Ala Phe Ile Lys Leu Trp Ala Phe Asp Ala Val Lys Gly Lys Cys Val Leu Phe Pro Tyr Gly Gly Cys Gln Gly Asn Gly Asn Lys Phe Asp Ser Glu Lys Glu Cys Arg Glu Tyr Cys 3. A novel polypeptide having an amino acid sequence represented by the following formula 3. Formula 3 Ala Ala Cys Asn Leu Pro Ile Val Ser Gly Pro Cys Arg Ala Phe Ile Lys Leu Trp Ala Phe Asp Ala Val Lys Gly Lys Cys Val Leu Phe Pro Tyr Gly Gly Cys Gln Gly Asn Gly Asn Lys Phe Asp Ser Glu Lys Glu Cys Arg Glu Tyr Cys Gly Val Pro Gly Asp Gly Asp Glu Glu Leu Leu Arg Phe Ser Asn 4. A novel DNA having a base sequence encoding the novel polypeptide according to any one of claims 1 to 3. 5. The novel DNA according to claim 4, wherein the base sequence has a base sequence represented by the following formula 4. Formula 4 TGT AAT CTA CCA ATA GTC Xnn-1 GGC CCC TGC CGA GCC TTC ATC Xnn-2 CTC TGG GCA TTT GAT GCT GTC AAG GGG AAG TGC GTC CTC TTC CCC TAC GGG GGC TGC CAG GGC AAC GGG AAC AAG TTC Xnn-3 TCA GAG AAG GAG TGC AGA GAG TAC TGC (However, Xnn-1 is a nucleotide sequence other than CGG, Xnn-2 is CAG
Other than TAC, Xnn-3 is a base sequence other than TAC. ) 6. The novel DNA according to claim 4 or 5, wherein the base sequence is a base sequence represented by the following formula 5. Formula 5 TGT AAT CTA CCA ATA GTC AGC GGC CCC TGC CGA GCC TTC ATC AAG CTC TGG GCA TTT GAT GCT GTC AAG GGG AAG TGC GTC CTC TTC CCC TAC GGG GGC TGC CAG GGC AAC GGG AAC AAG TTC GAC TCA GAG AAG GAG TGC AGA GAG TAC TGC 7. The novel DNA according to any one of claims 4 to 6, wherein the base sequence is a base sequence represented by the following formula 6. Formula 6 GCC GCC TGT AAT CTA CCA ATA GTC AGC GGC CCC TGC CGA GCC TTC ATC AAG CTC TGG GCA TTT GAT GCT GTC AAG GGG AAG TGC GTC CTC TTC CCC TAC GGG GGC TGC CAG GGC AAC GGG AAC AAG TTC GAC TCA GAG AAG TGC AGA GAG TAC TGC GGT GTC CCT GGT GAT GGT GAT GAG GAG CTG CTG CGC TTC TCC AAC 8. A vector comprising the novel DNA according to any one of claims 4 to 7. 9. A transformant transformed with the novel DNA according to any one of claims 4 to 7. 10. A transformant transformed with the vector according to claim 8. 11. A pharmaceutical composition comprising the novel polypeptide according to any one of claims 1 to 3 as an active ingredient. 12. The pharmaceutical composition comprises at least FXa,
The novel pharmaceutical composition according to claim 9, which is used for prevention and / or treatment of a disease involving any of elastase. 13. A method for inhibiting FXa, which comprises using the novel polypeptide according to any one of claims 1 to 3. 14. A method for inhibiting elastase, which comprises using the novel polypeptide according to any one of claims 1 to 3.