JP2824392B2 - Culture supernatant containing a peptide having an action of promoting the activation of protein C by thrombin - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a culture supernatant of a microorganism or a cell as an intermediate in the genetic engineering production of a novel peptide having a function of promoting the activation of protein C of thrombin. More specifically, the present invention has a thrombolytic action, an anticoagulant action and a platelet aggregation inhibitory action. Regarding the culture supernatant . The present invention also provides a blood anticoagulant reaction,
The present invention relates to a culture supernatant as a crude preparation of the peptide, which can be used as a highly reproducible effector or a positive standard in a large-scale assay such as an activated protein C production reaction or a blood anticoagulation reaction.

[0002] In the present specification, amino acids and peptides are defined as IUPAC-IUB Biochemical Nomenclature Committee (C
BN). In addition, when there is an optical isomer for an amino acid or the like, the L-form is indicated unless otherwise specified. Further, unless otherwise specified, the left and right ends of the amino acid sequence of the peptide are the N-terminal and C-terminal, respectively.

Gln: Glutamine residue Asp: Aspartic acid residue Pro: Proline residue Tyr: Tyrosine residue Val: Valine residue Lys: Lysine residue Glu: Glutamic acid residue Ala: Alanine residue Asn: Asparagine residue Leu : Leucine residue Phe: Phenylalanine residue Gly: Glycine residue His: Histidine residue Ser: Serine residue Thr: Threonine residue Ile: Isoleucine residue Trp: Tryptophan residue Arg: Arginine residue Met: Methionine residue Cys : Cysteine residue

[0004] Polydeoxyribonucleotides and oligonucleotides are represented by deoxyribonucleotide sequences represented by the following abbreviations. A: 2'-deoxyadenylic acid residue C: 2'-deoxycytidylic acid residue G: 2'-deoxyguanylic acid residue T: thymidylic acid residue Unless otherwise specified, the left and right ends of the deoxyribonucleotide sequence are The 5 'end and the 3' end, respectively.

[0005]

2. Description of the Related Art Streptokinase and urokinase are currently used as thrombolytic agents. Heparin and warfarin are used as anticoagulants. Further, as a platelet aggregation inhibitor, aspirin, sulfinpyrazone, dipyridamole and the like are used. At present, these thrombolytic agents, anticoagulants and platelet aggregation inhibitors are used separately or in combination, for example, for myocardial infarction, thrombosis, embolism, peripheral vascular occlusion, atherosclerosis, vascular Blood coagulation syndrome (DI
C), for the treatment and prevention of diseases such as angina pectoris, transient ischemic attack, preeclampsia and the like. However, these thrombolytics, anticoagulants and platelet aggregation inhibitors only act on a very small part of the blood coagulation / fibrinolysis system, which consists of a very complex mechanism. Thus, there has been a demand for a drug that acts widely on the coagulation / fibrinolysis system of blood and has an excellent blood coagulation inhibitory action.

[0006] By the way, protein C is known as a vitamin K-dependent protein which plays an important role in the blood coagulation mechanism. In recent years, substances that promote the activation of protein C and suppress platelet activation and fibrin formation due to the action of thrombin are present in rabbit lung, bovine lung, human lung and human placenta, etc. It has been reported that it has an excellent blood coagulation inhibitory effect as compared with the aforementioned drugs.

[0007] With respect to substances present in rabbit lung, for example, see tea. Esmon (CT Esmon)
Proceding of National Academy of Science USA (Proc. Nat)
l. Acad. Sci. USA), 78, 2249 (1981); N. Esmon (NL Es)
mon) et al., The Journal of Biological
Chemistry (J. Biol. Chem.), 257
Vol. 859 (1982); tea. CT Esmon et al., The Journal of Biological Chemistry (J. Biol. Che).
m. ), 257, 7944 (1982);
NL Esmon et al., The Journal of Biological Chemistry (J. Bi)
ol. Chem. ), 258, 12238 (198
2 years).

For the substance present in bovine lung, reference can be made to, for example, Kusumoto et al., Biochemistry, 56, 890 (1984). Regarding substances present in the human placenta, for example, JP-A-60-199819; Kurosawa et al., Journal of the Japanese Society of Hematology, 47, 632 (1984).
Year); Etch Etch Salem (HH Sale)
m) et al., Journal of Biological Chemistry (J. Biol. Chem.), 259, 122
46 (1984); S. Kurosawa (S. Kuro)
sawa) et al., Thrombosis Research (Thromb)
ossis Research), 37, 353 (1
985). Regarding substances present in human lung, for example, Kusumoto et al., Biochemistry, 57, 11
Page 02 (1985).

The above prior art documents describe the general properties of the above substances. However, the structure of the substance, such as the amino acid sequence, has not been elucidated, and the substance has not yet been identified. Therefore, whether the substance reported in the above prior art document is a single substance,
Moreover, it is completely unknown whether the same substance can be repeatedly obtained according to the descriptions in these prior art documents.

[0010]

SUMMARY OF THE INVENTION In the above-mentioned technical background, the present inventors have not only activated protein C, which is a factor in the coagulation / fibrinolysis system of blood, but also suppressed blood coagulation. As a result of intensive studies to find a substance that enhances the dissolution action, surprisingly, as described later, a peptide having a specific amino acid sequence is converted into a protein C by thrombin.
It has been found that not only can activation be promoted to control blood coagulation, but also fibrinolysis can be promoted and that it is useful as an agent for controlling blood coagulation. In addition, the present inventors have isolated DNA encoding the protein, and have further found that the peptide can be easily produced in a large amount and easily by a recombinant DNA technique in a pure form containing no other human-derived protein. The present invention has been completed based on these findings.

[0011] That is, an object of the present invention, intermediates, as the culture supernatant of the microorganism or cell in the novel <br/> peptide genetic engineering preparation having an effect of promoting the activation of protein C by thrombin To provide a liquid .
The present invention also provides a blood anti-coagulation reaction, for example in mass assays, such as activated protein C production reaction or blood thinning reaction, it can be used as effector or positive standard height repeatability, the culture as a crude preparation of the peptide and to provide a supernatant <br/>. Furthermore, another object of the invention is that it is commonly used as a transformed microorganism or cell, preferably as a cell for producing genetically engineered products,
Using any one selected from COS cells, CHO cells, C ₁₂₇ cells, was cultured, obtained by expressing either recombinant DNA encoding the peptide culture
To provide a supernatant .

[0012]

That is, according to the present invention,
A culture supernatant of a microorganism or a cell is used. (A) At least as an amino acid sequence, (i) the following formula (I): Val Glu Pro Val Asp Pro Cys Phe Arg Ala Asn Cys Glu Tyr Gln Cys Gln Pro Leu ShnGn Ser Tyr Leu Cys Val Cys Ala Glu Gly Phe Ala Pro Ile Pro His Glu Pro His Arg Cys Gln Met Phe Cys Asn Gln Thr Ala Cys Pro Ala Asp Cys Asp Pro Asn Thr Gln Ala Ser Cys Glu Cys Pro Glu Gly Tyr Ile Leu Asp Asp Gly Phe Ile Cys Thr Asp Ile Asp Glu Cys Glu Asn Gly G lyPheCysSerGlyValCysHisAsnLeuProGlyThrPheGluCysIleCysGlyProAspSerAlaLeuValArgHisIleGly in the amino acid sequence of the amino acid sequence in the amino acid sequence. A base which encodes a soluble peptide having an amino acid sequence in which one or several amino acids have been deleted, substituted or added, and which is soluble and has an effect of promoting the activation of protein C by thrombin. Ligating a DNA containing the sequence to a replicable expression vector to obtain a replicable recombinant DNA containing the DNA and the replicable expression vector; and (b) a microorganism comprising the replicable recombinant DNA. Or transform the cells Allowed forming a transformant, (c) a transformant was selected from the parent cells of the microorganism or cells, (d) culturing the transformant, the DN in transformant
A culture supernatant of a microorganism or a cell containing the peptide, wherein A is expressed and a culture supernatant is collected from the culture is provided. In the specification of the present invention, the culture supernatant does not substantially contain these microorganisms or cells prepared by removing microorganisms or cells.

[0013] The culture supernatant of the present invention may contain a peptide substantially consisting of the amino acid sequence represented by the formula (I), or may contain an amino acid sequence represented by the formula (I); The peptide may further comprise an amino acid sequence of at least one other peptide bound to its N-terminal and / or C-terminal. Examples of the peptide containing the amino acid sequence represented by the formula (I) and the amino acid sequence of at least one other peptide include the following peptides (1) to (5).

(1) The following amino acid sequence is added to the N-terminus of the amino acid sequence represented by the formula (I): Cys Ser Val Glu Asn Gly Gly Cys Glu His Ala Cys Asn Ala Ile Pro Gly Ala Pro Arg Cys Glyn Cys Glyn Cry Glyn Cry Gly Ala Ala Leu Gln Ala Asp Gly Arg Ser Cys Thr Ala Ser Ala Thr Gln Ser Cys Asn Asp Leu Cys Glu His Phe Cys Val Pro Asn Pro Asp Gln Pro Gly Ser Tyr Ser Cys Met Cys Glu Thr Gly Tyr Arg Leu Ala Ala Asp Gln His Arg Cys Glu Asp Val Asp Asp Cys Ile Leu Glu ro Ser Pro Cys Pro Gln Arg Cys Val Asn Thr Gln Gly Gly Phe Glu Cys His Cys Tyr Pro Asn Tyr Asp Leu Val Asp Gly Glu Cys is bound peptide.

(2) The following amino acid sequence is added to the N-terminus of the amino acid sequence represented by the formula (I): Thr Phe Leu Asn Ala Ser Gln Ile Cys Asp Gly Leu Arg Gly His Leu Met Thr Val Arg Ser Ser Val Ala Ala Asp Val Ile Ser Leu Leu Leu Asn Gly Asp Gly Gly Val Gly Arg Arg Arg Leu Trp Ile Gly Leu Gln Leu Pro Pro Gly Cys Gly Asp Pro Lys Arg Leu Gly Pro Leu Arg ly Phe Gln Trp Val Thr Gly Asp Asn Asn Thr Ser Tyr Ser Arg Trp Ala Arg Leu Asp Leu Asn Gly Ala Pro Leu Cys Gly Pro Leu Cys Val Ala Val Ser Ala Ala Glu Ala Thr Val Pro Ser Glu Pro Ile Trp Glu Glu Gln Gln Cys Glu Val Lys Ala Asp Gly Phe Leu Cys Glu Phe His Phe Pro Ala Thr Cys Arg Pro Leu Ala Val Glu Pro Gly Ala Ala Ala Ala Ala Val Ser Ile Thr Tyr Gly Thr Pro Phe Ala Ala Arg Gly Ala Asp Phe Gln Ala eu Pro Val Gly Ser Ser Ala Ala Val Ala Pro Leu Gly Leu Gln Leu Met Cys Thr Ala Pro Pro Gly Ala Val Gln Gly His Trp Ala Arg Glu Ala Pro Gly Ala Trp Asp Cys Ser Val Glu Asn Gly Gly Cys Glu His Ala Cys Asn Ala Ile Pro Gly Ala Pro Arg Cys Gln Cys Pro Ala Gly Ala Ala Leu Gln Ala Asp Gly Arg Ser Cys Thr Ala Ser Ala Thr Gln Ser Cys Asn Asp Leu Cys Glu His Phe Cys Val Pro Asn Pro Asp Gln Pro Gly Ser Tyr er Cys Met Cys Glu Thr Gly Tyr Arg Leu Ala Ala Asp Gln His Arg Cys Glu Asp Val Asp Asp Cys Ile Leu Glu Pro Ser Pro Cys Pro Gln Arg Cys Val Asn Thr Gln Gly Gly Phe Glu Cys His Cys Tyr Pro Asn Tyr Asp A peptide formed by binding Leu Val Asp Gly Glu Cys.

(3) The following amino acid sequences are provided at the N-terminal and C-terminal of the amino acid sequence represented by the formula (I): Cys Ser Val Glu Asn Gly Gly Cys Glu His Ala Cys Asn Ala Ile Pro Gly Ala Pro Arg Cys Gln Cys Pro Ala Gly Ala Ala Leu Gln Ala Asp Gly Arg Ser Cys Thr Ala Ser Ala Thr Gln Ser Cys Asn Asp Leu Cys Glu His Phe Cys Val Pro Asn Pro Asp Gln Pro Gly Ser Tyr Ser Cys Met Cys Glu Thr Gly Tyr Arg Leu Ala Ala Asp Gln His Arg Cys Glu Asp Val Asp Asp Cys I e Leu Glu Pro Ser Pro Cys Pro Gln Arg Cys Val Asn Thr Gln Gly Gly Phe Glu Cys His Cys Tyr Pro Asn Tyr Asp Leu Val Asp Gly Glu Cys and Asp Ser Gly Lys Val Asp Gly Gly Asp Ser Gly Ser Gly Glu Pro Pro Peptide comprising Pro Ser Pro Thr Pro Gly Ser Thr Leu Thr Pro Pro Ala Val Gly Leu Val His Ser Gly bonded thereto.

(4) At the N-terminal and C-terminal of the amino acid sequence represented by the formula (I), the following amino acid sequences are respectively: Pro Gly Pro Ala Thr Phe Leu Asn Ala Ser Gln Ile Cys Asp Gly Leu Arg Gly His Leu Met Thr Val Arg Ser Ser Val Ala Ala Asp Val Ile Ser Leu Leu Leu Asn Gly Asp Gly Gly Val Gly Arg Arg Arg Leu Trp Ile Gly Leu Gln Leu Pro Pro Gly Cys Gly Asp Pro Lys Arg Leu Gly P o Leu Arg Gly Phe Gln Trp Val Thr Gly Asp Asn Asn Thr Ser Tyr Ser Arg Trp Ala Arg Leu Asp Leu Asn Gly Ala Pro Leu Cys Gly Pro Leu Cys Val Ala Val Ser Ala Ala Glu Ala Thr Val Pro Ser Glu Pro Ile Trp Glu Glu Gln Gln Cys Glu Val Lys Ala Asp Gly Phe Leu Cys Glu Phe His Phe Pro Ala Thr Cys Arg Pro Leu Ala Val Glu Pro Gly Ala Ala Ala Ala Ala Val Ser Ile Thr Tyr Gly Thr Pro Phe Ala Ala Arg Gly Ala Asp P e Gln Ala Leu Pro Val Gly Ser Ser Ala Ala Val Ala Pro Leu Gly Leu Gln Leu Met Cys Thr Ala Pro Pro Gly Ala Val Gln Gly His Trp Ala Arg Glu Ala Pro Gly Ala Trp Asp Cys Ser Val Glu Asn Gly Gly Cys Glu His Ala Cys Asn Ala Ile Pro Gly Ala Pro Arg Cys Gln Cys Pro Ala Gly Ala Ala Leu Gln Ala Asp Gly Arg Ser Cys Thr Ala Ser Ala Thr Gln Ser Cys Asn Asp Leu Cys Glu His Phe Cys Val Pro Asn Pro Asp Gln Pro G y Ser Tyr Ser Cys Met Cys Glu Thr Gly Tyr Arg Leu Ala Ala Asp Gln His Arg Cys Glu Asp Val Asp Asp Cys Ile Leu Glu Pro Ser Pro Cys Pro Gln Arg Cys Val Asn Thr Gln Gly Gly Phe Glu Cys His Cys Tyr Pro bound to the Asn Tyr Asp Leu Val Asp Gly Glu Cys and Asp Ser Gly Lys Val Asp Gly Gly Asp Ser Gly Ser Gly Glu Pro Pro Pro Ser Pro Thr Pro Gly Ser Thr Leu Thr Pro Pro Ala Val Gly Leu Val His Ser Gly Peptide.

(5) The following amino acid sequences are provided at the N-terminal and C-terminal of the amino acid sequence represented by the formula (I), respectively: Pro Gly Pro Ala Thr Phe Leu Asn Ala Ser Gln Ile Cys Asp Gly Leu Arg Gly His Leu Met Thr Val Arg Ser Ser Val Ala Ala Asp Val Ile Ser Leu Leu Leu Asn Gly Asp Gly Gly Val Gly Arg Arg Arg Leu Trp Ile Gly Leu Gln Leu Pro Pro Gly Cys Gly Asp Pro Lys Arg Leu Gly P o Leu Arg Gly Phe Gln Trp Val Thr Gly Asp Asn Asn Thr Ser Tyr Ser Arg Trp Ala Arg Leu Asp Leu Asn Gly Ala Pro Leu Cys Gly Pro Leu Cys Val Ala Val Ser Ala Ala Glu Ala Thr Val Pro Ser Glu Pro Ile Trp Glu Glu Gln Gln Cys Glu Val Lys Ala Asp Gly Phe Leu Cys Glu Phe His Phe Pro Ala Thr Cys Arg Pro Leu Ala Val Glu Pro Gly Ala Ala Ala Ala Ala Val Ser Ile Thr Tyr Gly Thr Pro Phe Ala Ala Arg Gly Ala Asp P e Gln Ala Leu Pro Val Gly Ser Ser Ala Ala Val Ala Pro Leu Gly Leu Gln Leu Met Cys Thr Ala Pro Pro Gly Ala Val Gln Gly His Trp Ala Arg Glu Ala Pro Gly Ala Trp Asp Cys Ser Val Glu Asn Gly Gly Cys Glu His Ala Cys Asn Ala Ile Pro Gly Ala Pro Arg Cys Gln Cys Pro Ala Gly Ala Ala Leu Gln Ala Asp Gly Arg Ser Cys Thr Ala Ser Ala Thr Gln Ser Cys Asn Asp Leu Cys Glu His Phe Cys Val Pro Asn Pro Asp Gln Pro G y Ser Tyr Ser Cys Met Cys Glu Thr Gly Tyr Arg Leu Ala Ala Asp Gln His Arg Cys Glu Asp Val Asp Asp Cys Ile Leu Glu Pro Ser Pro Cys Pro Gln Arg Cys Val Asn Thr Gln Gly Gly Phe Glu Cys His Cys Tyr Pro Asn Tyr Asp Leu Val Asp Gly Glu Cys and Asp Ser Gly Lys Val Asp Gly Gly Asp Ser Gly Ser Gly Glu Pro Pro Pro Ser Pro Thr Pro Gly Ser Thr Leu Thr Pro Pro Ala Val Gly Leu Val His Ser Gly Leu Leu Ile Gly Il Ser Ile Ala Ser Leu Cys Leu Val Val Ala Leu Leu Ala Leu Leu Cys His Leu Arg Lys Lys Gln Gly Ala Ala Arg Ala Lys Met Glu Tyr Lys Cys Ala Ala Pro Ser Lys Glu Val Val Leu Gln His Val Arg Thr Glu Arg Thr Peptide formed by binding of Pro Gln Arg Leu.

The peptide may contain the amino acid methionine as the N-terminal amino acid residue. The peptide may also have the N-terminal amino acid sequence, for example: May have a leader sequence having the amino acid sequence represented by Due to natural or artificial mutations, it is possible to change part of the structure of the peptide without significantly altering the activity of the peptide. The peptides of the invention may also contain one sugar residues even without small, it may not be contained. That is, in the present invention, it is shown that at least the amino acid sequence is a sequence described in the present specification, and is not particularly limited by a sugar residue.

The D required for the genetic engineering production of the peptide
The NA following formula (II): GTGGAGCCCG TGGACCCGTG CTTCAGAGCC AACTGCGAGT ACCAGTGCCA GCCCCTGAAC CAAACTAGCT ACCTCTGCGT CTGCGCCGAG GGCTTCGCGC CCATTCCCCA CGAGCCGCAC AGGTGCCAGA TGTTTTGCAA CCAGACTGCC TGTCCAGCCG ACTGCGACCC CAACACCCAG GCTAGCTGTG AGTGCCCTGA AGGCTACATC CTGGACGACG GTTTCATCTG CACGGACATC GACGAGTGCG AAAACGGCGG CTTCTGCTCC GGGGTGTGCC ACAACCTCCC CGGTACCTTC GAGTGCATCT G There is a DNA containing a base sequence represented by CGGGCCCGA CTCGGCCCTT GTCGCCACA TTGGCACCGA CTGT.

According to the degeneracy of the genetic code, at least one base in the base sequence of the gene can be replaced by another type of base without changing the amino acid sequence of the polypeptide produced from the gene. Therefore, the DN of the formula (II)
A can also contain a base sequence altered by substitution based on the degeneracy of the genetic code. in this case,
The amino acid sequence deduced from the nucleotide sequence obtained by the above substitution matches the amino acid sequence defined previously. The DN
A may contain the base sequence represented by the above formula (II) and at least one other base sequence bonded to its 5 'end and / or 3' end. Examples of the DNA containing the base sequence represented by the formula (II) and at least one other base sequence include the following DNAs (1) to (5).

(1) 5 'of the base sequence represented by the formula (II)
Following formula at the terminal: TGCAGCGTGG AGAACGGCGG CTGCGAGCAC GCGTGCAATG CGATCCCTGG GGCTCCCCGC TGCCAGTGCC CAGCCGGCGC CGCCCTGCAG GCAGACGGGC GCTCCTGCAC CGCATCCGCG ACGCAGTCCT GCAACGACCT CTGCGAGCAC TTCTGCGTTC CCAACCCCGA CCAGCCGGGC TCCTACTCGT GCATGTGCGA GACCGGCTAC CGGCTGGCGG CCGACCAACA CCGGTGCGAG GACGTGGATG ACTGCATACT GGAGCCCAGT CCGTGTCCGC AGCGCTGTGT CAACACACAG GGTGGCTTCG AGTGCC CTG CTACCCTAAC TACGACCTGG TGGACGGCGA DNA base sequence is bonded, represented by GTGT.

(2) 5 'of the base sequence represented by the formula (II)
Following formula at the terminal: GCACCCGCAG AGCCGCAGCC GGGTGGCAGC CAGTGCGTCG AGCACGACTG CTTCGCGCTC TACCCGGGCC CCGCGACCTT CCTCAATGCC AGTCAGATCT GCGACGGACT GCGGGGCCAC CTAATGACAG TGCGCTCCTC GGTGGCTGCC GATGTCATTT CCTTGCTACT GAACGGCGAC GGCGGCGTTG GCCGCCGGCG CCTCTGGATC GGCCTGCAGC TGCCACCCGG CTGCGGCGAC CCCAAGCGCC TCGGGCCCCT GCGCGGCTTC CAGTGGGTTA CGGGAGACAA CAACACCAGC TATAGCAGGT GGGCAC GCT CGACCTCAAT GGGGCTCCCC TCTGCGGCCC GTTGTGCGTC GCTGTCTCCG CTGCTGAGGC CACTGTGCCC AGCGAGCCGA TCTGGGAGGA GCAGCAGTGC GAAGTGAAGG CCGATGGCTT CCTCTGCGAG TTCCACTTCC CAGCCACCTG CAGGCCACTG GCTGTGGAGC CCGGCGCCGC GGCTGCCGCC GTCTCGATCA CCTACGGCAC CCCGTTCGCG GCCCGCGGAG CGGACTTCCA GGCGCTGCCG GTGGGCAGCT CCGCCGCGGT GGCTCCCCTC GGCTTACAGC TAATGTGCAC CGCGCCGCCC GGAGCGGTC AGGGGCACTG GGCCAGGGAG GCGCCGGGCG CTTGGGACTG CAGCGTGGAG AACGGCGGCT GCGAGCACGC GTGCAATGCG ATCCCTGGGG CTCCCCGCTG CCAGTGCCCA GCCGGCGCCG CCCTGCAGGC AGACGGGCGC TCCTGCACCG CATCCGCGAC GCAGTCCTGC AACGACCTCT GCGAGCACTT CTGCGTTCCC AACCCCGACC AGCCGGGCTC CTACTCGTGC ATGTGCGAGA CCGGCTACCG GCTGGCGGCC GACCAACACC GGTGCGAGGA CGTGGATGAC TGCATACTGG AGCCCAGTCC GTGTCCGCAG C CTGTGTCA ACACACAGGG TGGCTTCGAG TGCCACTGCT ACCCTAACTA CGACCTGGTG GACGGCGAGT DNA base sequence is bonded, represented by GT.

(3) 5 'of the base sequence represented by the formula (II)
End and 3 'end, respectively following formula: TGCAGCGTGG AGAACGGCGG CTGCGAGCAC GCGTGCAATG CGATCCCTGG GGCTCCCCGC TGCCAGTGCC CAGCCGGCGC CGCCCTGCAG GCAGACGGGC GCTCCTGCAC CGCATCCGCG ACGCAGTCCT GCAACGACCT CTGCGAGCAC TTCTGCGTTC CCAACCCCGA CCAGCCGGGC TCCTACTCGT GCATGTGCGA GACCGGCTAC CGGCTGGCGG CCGACCAACA CCGGTGCGAG GACGTGGATG ACTGCATACT GGAGCCCAGT CCGTGTCCGC AGCGCTGTGT CAACACACAG GGTGGC TTCG AGTGCCACTG CTACCCCTAAC TACGACCTGGG TGGACGGGCGA GTGT and GACTCCGGCA AGGTGGACGG TGGCGACAGC GGCTCTGGCGCG AGCCCCGCGCCGCCTCGCGCGCTCGCGCGCGCGCGTCC

(4) 5 'of the base sequence represented by the formula (II)
End and 3 'end, respectively following formula: GCACCCGCAG AGCCGCAGCC GGGTGGCAGC CAGTGCGTCG AGCACGACTG CTTCGCGCTC TACCCGGGCC CCGCGACCTT CCTCAATGCC AGTCAGATCT GCGACGGACT GCGGGGCCAC CTAATGACAG TGCGCTCCTC GGTGGCTGCC GATGTCATTT CCTTGCTACT GAACGGCGAC GGCGGCGTTG GCCGCCGGCG CCTCTGGATC GGCCTGCAGC TGCCACCCGG CTGCGGCGAC CCCAAGCGCC TCGGGCCCCT GCGCGGCTTC CAGTGGGTTA CGGGAGACAA CAACACCAGC TATAGC AGGT GGGCACGGCT CGACCTCAAT GGGGCTCCCC TCTGCGGCCC GTTGTGCGTC GCTGTCTCCG CTGCTGAGGC CACTGTGCCC AGCGAGCCGA TCTGGGAGGA GCAGCAGTGC GAAGTGAAGG CCGATGGCTT CCTCTGCGAG TTCCACTTCC CAGCCACCTG CAGGCCACTG GCTGTGGAGC CCGGCGCCGC GGCTGCCGCC GTCTCGATCA CCTACGGCAC CCCGTTCGCG GCCCGCGGAG CGGACTTCCA GGCGCTGCCG GTGGGCAGCT CCGCCGCGGT GGCTCCCCTC GGCTTACAGC TAATGTGCAC CGCGCCGC C GGAGCGGTCC AGGGGCACTG GGCCAGGGAG GCGCCGGGCG CTTGGGACTG CAGCGTGGAG AACGGCGGCT GCGAGCACGC GTGCAATGCG ATCCCTGGGG CTCCCCGCTG CCAGTGCCCA GCCGGCGCCG CCCTGCAGGC AGACGGGCGC TCCTGCACCG CATCCGCGAC GCAGTCCTGC AACGACCTCT GCGAGCACTT CTGCGTTCCC AACCCCGACC AGCCGGGCTC CTACTCGTGC ATGTGCGAGA CCGGCTACCG GCTGGCGGCC GACCAACACC GGTGCGAGGA CGTGGATGAC TGCATACTGG AGCCCAGTCC G TGTCCGCAG CGCTGTGTCA ACACACAGGG TGGCTTCGAG TGCCACTGCT ACCCTAACTA CGACCTGGTG GACGGCGAGT GT and GACTCCGGCA AGGTGGACGG TGGCGACAGC GGCTCTGGCG AGCCCCCGCC CAGCCCGACG CCCGGCTCCA CCTTGACTCC TCCGGCCGTG GGGCTCGTGC DNA base sequence is bonded, represented by ATTCGGGC.

(5) 5 ′ of the base sequence represented by the formula (II)
End and 3 'end, respectively following formula: GCACCCGCAG AGCCGCAGCC GGGTGGCAGC CAGTGCGTCG AGCACGACTG CTTCGCGCTC TACCCGGGCC CCGCGACCTT CCTCAATGCC AGTCAGATCT GCGACGGACT GCGGGGCCAC CTAATGACAG TGCGCTCCTC GGTGGCTGCC GATGTCATTT CCTTGCTACT GAACGGCGAC GGCGGCGTTG GCCGCCGGCG CCTCTGGATC GGCCTGCAGC TGCCACCCGG CTGCGGCGAC CCCAAGCGCC TCGGGCCCCT GCGCGGCTTC CAGTGGGTTA CGGGAGACAA CAACACCAGC TATAGC AGGT GGGCACGGCT CGACCTCAAT GGGGCTCCCC TCTGCGGCCC GTTGTGCGTC GCTGTCTCCG CTGCTGAGGC CACTGTGCCC AGCGAGCCGA TCTGGGAGGA GCAGCAGTGC GAAGTGAAGG CCGATGGCTT CCTCTGCGAG TTCCACTTCC CAGCCACCTG CAGGCCACTG GCTGTGGAGC CCGGCGCCGC GGCTGCCGCC GTCTCGATCA CCTACGGCAC CCCGTTCGCG GCCCGCGGAG CGGACTTCCA GGCGCTGCCG GTGGGCAGCT CCGCCGCGGT GGCTCCCCTC GGCTTACAGC TAATGTGCAC CGCGCCGC C GGAGCGGTCC AGGGGCACTG GGCCAGGGAG GCGCCGGGCG CTTGGGACTG CAGCGTGGAG AACGGCGGCT GCGAGCACGC GTGCAATGCG ATCCCTGGGG CTCCCCGCTG CCAGTGCCCA GCCGGCGCCG CCCTGCAGGC AGACGGGCGC TCCTGCACCG CATCCGCGAC GCAGTCCTGC AACGACCTCT GCGAGCACTT CTGCGTTCCC AACCCCGACC AGCCGGGCTC CTACTCGTGC ATGTGCGAGA CCGGCTACCG GCTGGCGGCC GACCAACACC GGTGCGAGGA CGTGGATGAC TGCATACTGG AGCCCAGTCC G TGTCCGCAG CGCTGTGTCA ACACACAGGG TGGCTTCGAG TGCCACTGCT ACCCTAACTA CGACCTGGTG GACGGCGAGT GT and GACTCCGGCA AGGTGGACGG TGGCGACAGC GGCTCTGGCG AGCCCCCGCC CAGCCCGACG CCCGGCTCCA CCTTGACTCC TCCGGCCGTG GGGCTCGTGC ATTCGGGCTT GCTCATAGGC ATCTCCATCG CGAGCCTGTG CCTGGTGGTG GCGCTTTTGG CGCTCCTCTG CCACCTGCGC AAGAAGCAGG GCGCCGCCAG GGCCAAGATG GAGTACAAGT GCGCGGCCCC TTCCAAGGA GTAGTGCTGC AGCACGTGCG GACCGAGCGG ACGCCGCAGA DNA base sequence is bonded, represented by GACTC.

The DNA of the present invention can be used to produce the above-mentioned peptide using recombinant DNA technology. The DNA of the present invention can be obtained as follows. (1) Using an antibody obtained from a rabbit, which is specific for a peptide derived from human lung and capable of promoting protein C activation of thrombin, a peptide that binds to the antibody is extracted from a cDNA library prepared from human lung. The encoding cDNA fragment is isolated, and the nucleotide sequence of the isolated cDNA fragment is analyzed. The resulting cDNA fragment encodes a portion of a human lung-derived peptide capable of promoting protein C activation of thrombin. That portion includes the C-terminus but not the N-terminus of the peptide.

(2) As described above, the obtained cDNA fragment does not encode the entire amino acid sequence of the peptide derived from human lung, and lacks the nucleotide sequence corresponding to the N-terminal amino acid sequence of the peptide. , The cDNA fragment encoding the N-terminal amino acid sequence was obtained using the cD
It is obtained as follows by an ordinary known primer extension method using an NA fragment. First, a part of the cDNA fragment corresponding to the N-terminal amino acid sequence of the peptide encoded by the cDNA fragment obtained in the above step (1) is organically synthesized. Next, cDNA obtained in the above step (1) from poly (A) ⁺ RNA prepared from human umbilical cord endothelial cells by a commonly known primer extension method using the synthesized DNA as a primer
A cDNA fragment having a base sequence upstream of the 5 'end of the fragment is obtained. By repeating the above primer extension, a cDNA fragment encoding the N-terminal amino acid sequence of the peptide derived from human lung is obtained.

(3) Next, the cDNA fragments obtained in the above steps (1) and (2) are ligated so as to encode the entire amino acid sequence of the desired peptide, thereby starting from the codon of the N-terminal amino acid at 1671. Base pairs (hereinafter "bp")
(Hereinafter abbreviated as "cDNA-A"). The nucleotide sequence of this open reading frame is D
It is the same as the nucleotide sequence of NA (5). This cDNA-A
To the base sequence represented by the formula (II) and the aforementioned DNA
CDNA having the same nucleotide sequence as each of the nucleotide sequences (1) to (4) can be prepared as follows.

(I) cDNA containing the same nucleotide sequence as that of DNA (4) is obtained from cDNA-A by 5 ′
It can be obtained by deleting the portion downstream from the 1495th base counted from the terminal by the position-specific mutation method. The resulting cDNA is a 1494 bp DNA containing an N-terminal amino acid codon at the 5 'end. (Ii) cDNA containing the same nucleotide sequence as that of DNA (3) can be obtained in the same manner as in (i) above.
6 corresponding to the N-terminal amino acid sequence from the 4 bp cDNA
It is obtained by deleting the 78 bp portion by the site-specific mutagenesis method. The obtained cDNA is 816 bp. (Iii) cDNA containing the same nucleotide sequence as that of DNA (2) can be obtained in the same manner as in the above (i).
1 corresponding to the C-terminal amino acid sequence from 4 bp cDNA
It is obtained by deleting the 08 bp portion by the site-specific mutagenesis method. The obtained cDNA is 1386 bp.

(Iv) cDNA containing the same nucleotide sequence as that of DNA (1) is obtained by the above-mentioned (iii) of 1386 bp.
678 corresponding to the N-terminal amino acid sequence
The bp part is deleted by the site-specific mutation method. Thus, a 708 bp cDNA having the same nucleotide sequence as that of DNA (1) is obtained. (V) A cDNA containing the same nucleotide sequence as the nucleotide sequence represented by the formula (II) can be obtained as follows. First of all, in the same way as described in (ii) above,
A 386 bp cDNA is obtained. Next, 1386b obtained
From the cDNA of p, 10 corresponding to the N-terminal amino acid sequence
The portion consisting of 32 bp is deleted by the site-specific mutation method.
Thus, a 354 bp cDNA having the same nucleotide sequence as that of the formula (II) is obtained.

The nucleotide sequence of each cDNA thus obtained is analyzed by a known method, and matches the nucleotide sequences of DNAs (1) to (5) and the nucleotide sequence represented by formula (II), respectively. Make sure that The above DNA can also be obtained by organic chemical synthesis. In addition, this DNA
Without the primer extension described above,
It can also be prepared from precursor DNA. Precursor DNA
Represents the DNA fragment obtained in the step (1) or its D
It can be obtained from a human chromosomal DNA library by a usual hybridization method using a synthetic DNA prepared based on the nucleotide sequence of the NA fragment as a probe.

The DNA containing the base sequence of the above formula (II) may further comprise, for example, the following formula: May be contained as a 5 'terminal nucleotide sequence encoding a leader sequence represented by The above DN
Complementary DNA and A also Ru be prepared. According degeneracy of leading frame Den encryption, it is possible to replace at least one nucleotide of the nucleotide sequence of the Yadenko other types of bases without changing the amino acid sequence of the polypeptide produced from the gene. Therefore, the DNA can also contain a base sequence altered by substitution based on the degeneracy of the genetic code. In this case, the amino acid sequence deduced from the nucleotide sequence obtained by the above substitution matches the amino acid sequence defined previously.

Furthermore, a replicable recombinant DNA comprising the DNA and a replicable expression vector can be prepared. The recombinant DNA allows the desired peptide to be expressed in the microorganism or cells transformed thereby, and the culture supernatant can be recovered from the culture . An example of a suitable vector is plasmid pBR3
22, pBR327, YRp7, pSV2-dhfr
(ATCC37146), pBPV-1 (9-1) (A
TCC 37111). It is necessary to select an expression vector suitable for a microorganism or cell used as a host.

Examples of microorganisms transformed with the above replicable recombinant DNA include Escherichia coli ( E.
strains of scherichia coli), for example, E. coli (E. coli) K12 strain 294 (ATCC 3
1446), E. coli ( E. coli ) B, E. coli ( E. coli ) X1776 (ATCC 3153).
7), E. coli (E coli) C600 and E. coli (E coli) C600 hfl and E. coli ^{(E coli) W3110 (F -} , λ -, prototropic Fick, ATCC 27375);... Bacillus subtilis (Bacillus subtilis) Strains of the genus Bacillus such as Salmonella
Salmonella typhim
urium) or Serratia Masesansu (Serr
Various strains of shoe Domo eggplant (Pseudomonas) genus;; atia marcesans) intestinal bacteria other than E. coli, such as and Saccharomyces cerevisiae (Sac
charomyces cerevisiae ). Examples of cells include VERO (ATCC C
CL-81) cells, HeLa cells, Chinese hamster ovary (CHO) cell line, WI38, BHK, COS
-7 and MDCK cell lines.

[0036] Furthermore, transformed microorganisms or cells, such as the present invention described above, preferably an animal cell, more preferably, using any one selected from COS cells, CHO cells, C ₁₂₇ cells, normal growth A method, preferably, as a culture method, culturing this using any one selected from dish-attachment culture method, bead-attachment culture method, and suspension culture method, and expressing any recombinant DNA encoding the above peptide the resulting culture supernatant, preferably to the culture supernatant <br/> liquid contained in a form desired peptide may be secreted.

[0037] The peptides secreted into the culture supernatant liquid is detected as active substance that promotes activation of protein C by thrombin, it is recovered. Usually this activity is
Samples were prepared using 0.15 M salt, 2.5 mM calcium chloride, 1
mg / ml serum albumin, pH 7.4
It is added to 0 mM Tris-HCl buffer in the presence of thrombin and protein C, and the amount of activated protein C produced is quantified and evaluated. Therefore, culture supernatant of the present invention,
The concentration of the peptide obtained by expressing any recombinant DNA encoding the peptide is a concentration detectable in the buffer, preferably at least 10 ng / ml.
In the concentration for the culture supernatant containing the peptide is soluble. It goes without saying that such a soluble peptide is extremely advantageous in the production method and pharmaceutical composition described below.

In vivo, a peptide that promotes the activation of protein C by thrombin is expressed on the vascular endothelial cell membrane in the following primary structure. Ala Pro Ala Glu Pro Gln Pro Gly Gly Ser Gln Cys Val Glu His Asp Cys Phe Ala Leu Tyr Pro Gly Pro Ala Thr Phe Leu Asn Ala Ser Gln Ile Cys Asp Gly Leu Arg Gly His Leu Met Thr Val Arg Ser Ser Val Ala Ala Asp Val Ile Ser Leu Leu Leu Asn Gly Asp Gly Gly Val Gly Arg Arg Arg Leu Trp Ile Gly Leu Gln Leu Pro Pro Gly Cys Gly Asp Pro Lys Arg Leu Gly Pro Leu Arg Gly Phe Gln Trp Val Thr Gly Asp Asn Asn Thr Ser Tyr Ser Arg Trp Ala Arg Leu Asp Leu Asn Gly Ala Pro Leu Cys Gly Pro Leu Cys Val Ala Val Ser Ala Ala Glu Ala Thr Val Pro Ser Glu Pro Ile Trp Glu Glu Gln Gln Cys Glu Val Lys Ala Asp Gly Phe Leu Cys Glu Phe His Phe Pro Ala Thr Cys Arg Pro Leu Ala Val Glu Pro Gly Ala Ala Ala Ala Ala Val Ser Ile Thr Tyr Gly Thr Pro Phe Ala Ala Arg Gly Ala Asp Phe Gln Ala Leu Pro Val Gly Ser Ser Ala Ala Val Ala Pro Leu Gly Leu Gln Leu Met Cys Thr Ala Pro Pro Gly Ala Val Gln Gly His Trp Ala Arg Glu Ala Pro Gly Ala Trp Asp Cys Ser Val Glu Asn Gly Gly Cys Glu His Ala Cys Asn Ala Ile Pro Gly Ala Pro Arg Cys Gln Cys Pro Ala Gly Ala Ala Leu Gln Ala Asp Gly Arg Ser Cys Thr Ala Ser Ala Thr Gln Ser Cys Asn Asp Leu Cys Glu His Phe Cys Val Pro Asn Pro Asp Gln Pro Gly Ser Tyr Ser Cys Met Cys Glu Thr Gly Tyr Arg Leu Ala Ala Asp Gln His Arg Cys Glu Asp Val Asp Asp Cys Ile Leu Glu Pro Ser Pro Cys Pro Gln Arg Cys Val Asn Thr Gln Gly Gly Phe Glu Cys His Cys Tyr Pro Asn Tyr Asp Leu Val Asp Gly Glu Cys Val Glu Pro Val Asp Pro Cys Phe Arg Ala Asn Cys Glu Tyr Gln Cys Gln Pro Leu Asn Gln Thr Ser Tyr Leu Cys Val Cys Ala Glu Gly Phe Ala Pro Ile Pro His Glu Pro His Arg Cys Gln Met Phe Cys Asn Gln Thr Ala Cys Pro Ala Asp Cys Asp Pro Asn Thr Gln Ala Ser Cys Glu Cys Pro Glu Gly Tyr Ile Leu Asp Asp Gly Phe Ile Cys Thr Asp Ile Asp Glu Cys Glu Asn Gly Gly Phe Cys Ser Gly Val Cys His Asn Leu Pro Gly Thr Phe Glu Cys Ile Cys Gly Pro Asp Ser Ala Leu Val Arg His Ile Gly Thr Asp Cys Asp Ser Gly Lys Val Asp Gly Gly Asp Ser Gly Ser Gly Glu Pro Pro Pro Ser Pro Thr Pro Gly Ser Thr Leu Thr Pro Pro Ala Val Gly Leu Val His Ser Gly Leu Leu Ile Gly Ile Ser Ile Ala Ser Leu Cys Leu Val Val Ala Leu Leu Ala Leu Leu Cys His Leu Arg Lys Lys Gln Gly Ala Ala Arg Ala Lys Met Glu Tyr Lys Cys Ala Ala Pro Ser Lys Glu Val Val Leu Gln His Val Arg Thr Glu Arg Thr Pro Gln Arg Leu

When the peptide is expressed by gene engineering in the above cells, it is expressed and concentrated on those cell membranes.
The peptide that promotes the activation of protein C by thrombin expressed in this manner can be obtained as an extract, for example, extracted in the presence of a surfactant, following the usual method for extracting a membrane protein. . However, in order to obtain a larger amount of the active substance, the desired peptide must be cultured .
Kiyoshi liquid is being desirably secretion and concentration in. Amino acid sequence of the peptide Sequence after the 59th position from the C-terminus: By removing, it can be detected and recovered as a soluble substance in the culture supernatant in a form that can be secreted. That is, the present invention
In the culture supernatant of the object or cell, to promote the activation of protein C by (a) thrombin
A peptide having the action of
In the acid sequence , Amino acid sequence that does not contain the amino acid sequence represented by
Comprising a nucleotide sequence encoding a peptide comprising
Is ligated to a replicable expression vector, and the DNA is
Replicable recombinant containing an expression vector that can be produced
Give the body DNA, (b) the microorganism or cell with said replication possible recombinant DNA
To form a transformant , and (c) transforming the transformant from the parent cell of the microorganism or cell.
Selected and cultured (d) is the transformant, the DN in transformant
A is expressed, and a culture supernatant is obtained from the culture.
A microorganism or cell containing the peptide,
Culture supernatant of vesicles . In a preferred embodiment,
The peptide has the following restriction map : DNA having the restriction enzyme site shown in
Amino acid sequence consisting of a partial sequence of the amino acid sequence
Culture supernatants of microorganisms or cells are included . In addition, above
The restriction map shown above is a part of FIG.
It is a thing . According to the restriction site in the restriction map
The peptides that the characterized DNA can encode are
Has the effect of promoting the activation of protein C by
In the present invention, the human peptide
A peptide consisting of a partial sequence of a amino acid sequence;
Contains a peptide that is soluble as described in
It is understood to be the culture supernatant .

That is, the present invention relates to a culture supernatant obtained by expressing any of the above-mentioned recombinant DNAs, preferably a secretable soluble thrombin protein C.
The concentration of the peptide that has the effect of promoting the activation of is a detectable concentration, more preferably at least 10 ng /
ml relates to a culture supernatant is the concentration of. More specifically, the culture supernatant of the present invention is used for culturing a microorganism or a cell as an intermediate in the genetic engineering production of a novel peptide having an action of promoting the activation of protein C of thrombin.
For the supernatant . Furthermore, the present invention is thrombolytic, has an anti-blood coagulation activity and platelet aggregation inhibiting action, intermediates, as microorganisms or on the cell culture in the genetic engineering production of useful peptides in the treatment of cardiovascular diseases Regarding the clear solution. The present invention also provides a blood anti-coagulation reaction, for example in mass assays, such as activated protein C production reaction or blood thinning reaction, it can be used as effector or positive standard height repeatability, the culture as a crude preparation of the peptide
For the supernatant .

According to the method of the present invention, the above-mentioned DNA is transcribed correctly and the resulting mRNA is correctly translated from the downstream of a DNA region such as a promoter of an expression vector capable of replicating the DNA. A recombinant replicable DNA having the DNA is incorporated into the recombinant DNA, and a microorganism or a cell is transformed with the obtained recombinant DNA to obtain a transformant containing the recombinant DNA.
The resulting transformant is isolated from the microorganism or the parent cell of the cultured cell depending on the phenotype given to the recombinant DNA. The obtained transformant is cultured to express the genetic information of the DNA, thereby producing a culture supernatant of the present invention.

The above-mentioned DNA and recombinant DNA were constructed.
DNA sequences required for construction, such as promoters and
To clone the origin of replication, etc.
It is preferable to use a host-vector system used as
No. Examples of prokaryotic cells include Escherichia coli (Esc
herichia coli) Strains such as e.
Li (E.coli) K12 strain 294 (ATCC 314)
46), Ecollie (E.coli) B, E coli
-(E.coli) X1776 (ATCC 3153)
7), Ecollie (E.coli) C600 and A
ー Collie (E.coli) C600hfl and e
 Collie (E.coli) W3110 (F^-, Λ^-,Professional
Totrofic, ATCC 27375);
Subtilis (Bacillus subtilis)of
Bachiras (likeBacillus) A strain of the genus;
La Chifimurium (Salmonella typh
imurium) Or Serratia Mercense (Se
rratia marcesans) Etc. other than E. coli
Intestinal bacteria; Pseudomonas (Pseudomona
sVarious strains of the genus; and Saccharomyces cerevisiae
D (Saccharomyces cerevisia
e). Escherich out of these bacteria
A coli (E.coli) K12 strain 294 is most preferred
No.

When the above microorganisms are used as a host, a plasmid vector suitable for these microorganisms is
A is commonly used as a replicable expression vector.
For example, plasmids pBR322 and pBR327 can be used as plasmid vectors for transforming Escherichia coli. Plasmid vectors usually contain an origin of replication, a promoter, and a marker gene that provides the recombinant DNA with a phenotype useful for selecting cells transformed with the recombinant DNA. Examples of the promoter include a β-lactamase and lactose promoter, a tryptophan promoter and the like. Examples of the marker gene include an ampicillin resistance gene and a tetracycline resistance gene.

On the other hand, in order to produce the culture supernatant of the present invention by expressing the above DNA, a host-vector system using the above prokaryotic cells as a host and eukaryotic cells such as vertebrate cells are used. A host-vector system used as a host cell can be used. Examples of eukaryotic cells include cells such as the animal cell lines described above. The above DN
In order to express A in the above-described eukaryotic cells, the recombinant DNA of the present invention generally contains functional sequences for controlling gene expression, such as a replication origin, a promoter to be located upstream of the DNA, a ribosome binding site. Contains a polyadenylation site and a transcription termination sequence. Such functional sequences that can be used to express the DNA in eukaryotic cells can be obtained from viruses and viral substances.

For example, promoters that can be used in the present invention include adenovirus 2, polyoma virus,
It can be obtained from Simian virus 40 (SV40) or the like. In particular, the main late promoter of adenovirus 2 and the early and late promoters of SV40 are preferred.
In addition, a promoter originally present at a position upstream of a gene encoding a peptide derived from human lung, which has an action to promote the activation of protein C of thrombin, is also used in the above-described host-
Any suitable for use in a vector system can be used.

With respect to the origin of replication, foreign sources such as adenovirus, polyoma, SV40, vesicular stomatitis virus (VSV), bovine papilloma virus (BP)
A replication origin derived from a virus such as V) can be used. When a vector having the property of being integrated into the host chromosome is used as the expression vector, the origin of replication of the host chromosome can be used. Microorganisms or cells transformed with the replicable recombinant DNA are selected from the remaining parent cells that have not been transformed by the at least one phenotype given to the recombinant DNA, as described above. The phenotype can be conferred by inserting at least one marker gene into the recombinant DNA.

Further, a marker gene originally contained in a replicable expression vector can also be used. Examples of marker genes include, for example, drug resistance genes such as neomycin resistance, and genes encoding dihydrofolate reductase (hereinafter referred to as "DHFR"). In this regard, when the DHFR gene is used as a marker gene, since there are various types of DHFR, the DHFR encoding the marker gene used is
The host to be used must be selected according to the type of FR. For example, as a marker gene, wild-type DHF
When a gene encoding R is used, DH is used as a host.
It is preferable to use an FR-deficient strain. Since DHFR-deficient strains require hypoxanthine, glycine and thymidine,
It cannot grow in a medium without hypoxanthine, glycine and thymidine. However, when a DHFR-deficient strain is transformed with a recombinant DNA containing the DHFR gene, the strain no longer requires hypoxanthine, glycine and thymidine and can grow in a medium free of hypoxanthine, glycine and thymidine. it can. Therefore, the transformed cells can be easily selected from the cells which have not been transformed and are determined based on the auxotrophy for hypoxanthine, glycine and thymidine.

On the other hand, when a gene encoding a mutant DHFR having low affinity for methotrexate (MTX) (hereinafter referred to as “MTX-resistant DHFR gene”) is used as a marker gene, the host cell can be treated with normal DHF.
DHFR need not be deleted as long as it has a gene encoding R. The reason is as follows. Because normal DHFR is inhibited by MTX, host cells containing the gene encoding normal DHFR are
It requires hypoxanthine, glycine and thymidine in the presence of TX. However, when the host cell is transformed with the recombinant DNA containing the MTX resistant DHFR gene, the transformed cells no longer require hypoxanthine, glycine and thymidine, even in the presence of MTX. Thus, transformed cells can be selected from untransformed cells using auxotrophy for hypoxanthine, glycine and thymidine in the presence of MTX as a criterion. In this regard, the MTX-resistant DHFR gene is convenient to use as a marker gene because the majority of eukaryotic cells are MTX-sensitive.

Saccharomyces cerevisiae ( Sacch)
Yeast such as Aromyces cerevisiae can also be used as a host for expressing DNA. To express DNA in yeast, for example, plasmid YRp7 can be used as a replicable expression vector. Plasmid YRp7 contains a trpl gene, and this trpl gene can be used as a marker gene. Examples of promoters for expression vectors for yeast cells include 3-phosphoglycerate kinase or enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, Kinases, promoters of enzymes involved in glycolysis such as glycodehydrogenase 2, isocytochrome C, acid phosphatase, enzymes involved in nitrogen metabolism,
Examples include promoters of genes of enzymes involved in maltose and lactose utilization. Among these, the promoters of the genes for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, enzymes involved in nitrogen metabolism, glyceraldehyde-3-phosphate dehydrogenase, and enzymes involved in the use of maltose and lactose are Is advantageously controlled by changing the culture conditions of the host.

As the origin of replication, termination codons and other DNA sequences for controlling transcription and translation in yeast cells, those known in the art which are suitable for yeast cells can be used.
Arrays can be used. The transformed microorganism or cell is cultured in a usual nutrient medium by a usual well-known method.
And by Rukoto to obtain a culture supernatant from a culture obtained, it is possible to produce a culture supernatant containing the desired peptide. After culturing, the peptide of the present invention can be isolated from the main culture supernatant by a known method, for example, column chromatography. The peptide thus obtained may contain at least one sugar chain of various types and lengths. Whether or not the obtained peptide contains a sugar chain depends on the type of host cell used. In addition, when the peptide contains a sugar chain, the type and length of the sugar chain also differ depending on the type of host cell used.

As described above, using the culture supernatant of the present invention as an intermediate, a peptide having an action to promote the activation of protein C by thrombin can be produced by a method using recombinant DNA technology. Protein C is a vitamin K-dependent protein that plays an important role in the blood coagulation / fibrinolysis mechanism and is activated by the action of thrombin. Activated protein C inactivates the activated coagulation coenzyme cofactors active V and VIII in vivo and is involved in the production of plasminogen activator having a thrombolytic action It is known. [Koji Suzuki, History of Medicine, Vol. 125, 901
Shin, (1983)]. The peptide promotes the activation of protein C by thrombin and produces a large amount of active protein C having an anticoagulant effect and a thrombolytic effect. Therefore, the peptide greatly contributes to anticoagulation and thrombolysis in a living body.

As described above, the peptide has an anticoagulant effect, a platelet aggregation inhibitory effect, and a thrombolytic effect.
Obstructive atherosclerosis, intravascular coagulation syndrome (DIC),
It can be used for the treatment and prevention of diseases such as angina pectoris, transient ischemic attacks, preeclampsia and the like. The present invention will be described in more detail by reference examples and examples to describe the present invention in more detail, but the present invention is not limited to these examples.

[0053]

【Example】

REFERENCE EXAMPLE 1 (Measurement of Action to Promote Protein C Activation) The measurement of the action of promoting the protein C activation of a peptide having an action to promote the activation of protein C by thrombin was performed using a synthetic substrate Boc-Leu-Ser-Thr. -Arg-MC
A (Boc and MCA are abbreviations of t-butoxycarbonyl group and 4-methylcoumaryl-7-amide, respectively) [A known method for measuring protein C [Y. Ohno, et al., The Journal of Biochemistry (J. Biochem.) 90 volumes, 1387
(1981)]. That is, 5 μl of an aqueous solution containing the peptide (0 to 0.01 A ₂₈₀ / ml) was added to 5 μl of an aqueous solution containing protein C (final concentration 0.5 μM) and thrombin (final concentration 80 nM), and NaCl and CaCl ₂ were added thereto. , Serum albumin and Tris-HCl buffer (pH 7.4) at final concentrations of 0.15 M, 2.5 mM, 1 mg / ml and 20 mM, respectively.
And a total volume of 30 μl was added.

The obtained mixture was reacted at 37 ° C. for 15 minutes to activate protein C, and then 10 μl of 2 μM antithrombin III and 10 μl of an aqueous solution containing 10 units / ml heparin were added, and the mixture was added at 37 ° C. for 15 minutes. The reaction was stopped by heating. The obtained reaction mixture was added to the above-mentioned synthetic substrate Boc-Leu-Ser-Thr-Arg-
MCA (Protein Research Foundation Peptide Research Group (P
(Eptide Institute), Japan) 200
20 mM Tris-HCl buffer (pH 7.4) containing μM 2
After adding 50 μl and reacting at 37 ° C. for 10 minutes, 20 μl was added.
% Acetic acid was added to stop the reaction, and the concentration of the released AMC (7-amino-7-methyl-coumarin) was measured at an excitation wavelength of 380 nm and an emission wavelength of 440 nm using a fluorescence spectrophotometer RF-540 (Shimadzu Corporation). Manufactured in Japan).

The amount of released AMC was determined by comparing the obtained fluorescence intensity with the fluorescence intensity of AMC at a known concentration. Value is 1
Expressed as the amount of AMC generated per minute. The value obtained by subtracting the amount of AMC when the aqueous solution not containing the peptide was added from the amount of AMC was the protein C by the thrombin of the sample.
Shows the strength to promote activation. Here, protein C was obtained from human plasma by the method of Suzuki et al. [Suzuki et al.
The Journal of Biological Chemistry (J. Biol. Chem.), 258, 1914
(1983)]. Human thrombin was prepared by the method of Lundblad et al. [Lundblad et al., Biochemical and Biophysical Research Communication (Biochem. Biophys. Res.
Commun. 66, 482, (1975)]
Was purified.

[0056] Reference Example 2 (1): human lung cDNA library obtained) lung poly (A) ⁺ bacteriophage .lambda.gt ₁₁ cDNA library prepared from RNA of human, USA, Clontech (Clontech LABORATOR
ies, Inc. , 922 Industrial, A
ve. Palo Alto, CA94303) (catalog number HL1004).

(2): (Protein C by thrombin
Purification of Glycopeptides that Promote Activation) Glycopeptides that have the activity of promoting protein C activation were obtained by extraction from human lung as follows. About 800g of human lung specimen provided by public hospital with scissors about 1
After shredding to a size of about 1 cm square, 1 mM DFP (Diisopropyl fluo) was added to the obtained tissue piece.
500 cooled to 4 ° C.
Then, ml of physiological saline was added, and homogenized at 4 ° C. for 5 minutes using an acehomogenizer AM-1 (manufactured by Nippon Seiki Co., Ltd., Japan) as a waring blender. After homogenization, the mixture was cooled in ice for 5 minutes.

The mixture was then homogenized at 4 ° C. for 5 minutes and cooled in ice for 5 minutes. The above homogenization and cooling operations were repeated three more times. The obtained homogenate is centrifuged at 12,000 g at 4 ° C. for 30 minutes to separate the supernatant and the pellet, and the pellet is collected. 0.5% (v / v) Triton X-100, 0.25
M sucrose, 1 mM benzamidine hydrochloride, 0.5 mM Ca
0.02 M Tris-HCl buffer containing Cl ₂ (pH 7.
5) The cells were suspended in 100 ml and homogenized 5 times at 4 ° C. for 5 minutes using a Waring blender to obtain a cell extract.

The obtained extract was weighed at 35,000 g and 10 ° C.
The supernatant was collected by centrifugation at for 60 minutes. NL Esmon et al. [The Journal of Biological Chemistry (J. Bio)
l. Chem. , 257, 859 (1982)
Year)) and DIP-thrombin [diisopropylphosphorothrombin (diisopro).
pyrphosphoro-thrombin)]
P. Cuatrecasas
[The Journal of Biological Chemistry (J. Biol. Chem.), 245, 3
059 (1970)] to produce DIP-thrombin-agarose.

Next, DIP-thrombin-agarose was packed in a column having a size of 2.5 cmφ × 10 cm, and
An IP-thrombin-agarose column was prepared and at room temperature 0.1 M NaCl, 0.5 mM CaCl ₂ , 1 mM
Benzamidine hydrochloride, 0.5% (v / v) Lubrol
The column was equilibrated with a 0.02 M Tris-HCl buffer (pH 7.5) containing PX (manufactured by Hanoi Chemicals, Japan). Next, the above-mentioned extracted supernatant was applied to a column. The column was filled with 0.3 M NaCl, 0.5 mM CaCl ₂ , 1 mM
Benzamidine hydrochloride, 0.5% (v / v) Lubrol
0.02 M Tris-HCl buffer containing PX (pH 7.
After washing in 5), 1 M NaCl, 0.1 mM ED
TA, 1 mM benzamidine hydrochloride 0.5% (v / v) L
The fraction was eluted with a 0.02 M Tris-HCl buffer (pH 7.5) containing ubrol PX, and 2.0 ml fractions were collected. For each fraction obtained by elution, the ability of thrombin to promote protein C activation was measured by the method described above. At the same time, the absorbance (A ₂₈₀ ) of each fraction at a wavelength of 280 nm was measured using a spectrophotometer UV-240 manufactured by Shimadzu (Japan).

The fraction having the ability to activate protein C was collected, and 0.1 M NaCl, 0.5 mM CaCl ₂ , 0.1.
0.02M with 05% (v / v) Lubrol PX
It was dialyzed against Tris-HCl buffer (pH 7.5). The obtained dialysate was subjected to a second DIP-thrombin-agarose column chromatography. That is, the dialysis solution is 1.5 times
The sample was applied to a DIP-thrombin-agarose column having a size of cmφ × 10 cm, and 0.4 M NaCl, 0.5 mM
After washing with 0.02 M Tris-HCl buffer (pH 7.5) containing CaCl ₂ and 0.1% (v / v) LubrolPX, 0.4 M NaCl and 0.1 mM EDT were further added.
A, containing 0.1% (v / v) Lubrol PX.
Wash with 02 M Tris-HCl buffer (pH 7.5), then 1 M NaCl, 0.5 mM EDTA, 0.1%
(V / v) Elution was carried out with a 0.02 M Tris-HCl buffer (pH 7.5) containing Lubrol PX. The fraction capable of activating protein C was collected, and further 0.1 M NaCl,
0.0 with 0.05% (v / v) Lubrol PX
It was dialyzed against 2M Tris-HCl buffer (pH 7.5). The obtained dialysate was subjected to a third DIP-thrombin-agarose column chromatography.

The column size, washing conditions and elution conditions were exactly the same as the conditions for the second DIP-thrombin-agarose column chromatography. In addition,
Fractions obtained by elution were collected in 2 ml portions. The fraction having the ability to activate protein C was collected, and 0.1 M N
After dialysis against a 0.02 M Tris-HCl buffer (pH 7.5) containing aCl and 0.05% (v / v) Lubrol PX, a fourth DIP having a size of 0.9 cmφ × 8 cm was performed.
-Thrombin-agarose column chromatography. 0.35 M NaCl, 0.5 mM CaCl
₂ , containing 0.1% (v / v) Lubrol PX.
After washing with 02M Tris-HCl buffer (pH 7.5), 1M
NaCl, 0.5 mM EDTA, 0.1% (v /
v) Elution was carried out with 0.02 M Tris-HCl buffer (pH 7.5) containing Lubrol PX. Fractions obtained by elution were collected in 1.9 ml portions.

FIG. 1 shows the elution pattern of the fourth DIP-thrombin-agarose column chromatography.
Shown in Fraction numbers 48 to 56 were collected. From the absorbance of the fraction purified in this manner, the molecular extinction coefficient of the obtained purified product was determined to be 10.0 (E ^1% _{1 cm}
280 nm = 10.0), and the amount of the purified product was calculated based on the definition and found to be about 500 μg. The purified fraction obtained was subjected to polyacrylamide gel concentration of 5-1.
SDS-polyacrylamide gel electrophoresis using a 0% gradient was performed at a voltage of 50 V for 2 hours, and the band was observed by silver staining. Only a single band was confirmed.

Further, about 10 μg of this purified protein was added to 20
50 mM Tris-HCl buffer containing 0 mM NaCl (p
H7.5), and then ConA equilibrated with the same buffer.
Sepharose (Pharmacia, catalog number 17-
0440) (resin amount: about 1 ml) and sufficiently washed with the same buffer. This protein was adsorbed on ConA Sepharose and was not eluted in the washing solution.
Then 0.5M methyl-α-D-mannopyranoside (Methyl-α-D-mannopyranosi).
de) (manufactured by Sigma, USA, catalog number M-6882)
The protein was eluted when the same buffer was passed as above except that it contained. Therefore, this protein was found to be a so-called glycopeptide containing sugar.

(3): (Analysis of amino acid sequence of glycopeptide that promotes activation of protein C of thrombin) The amino acid sequence of this glycopeptide was analyzed as follows. 0.1% (v / v) of purified glycopeptide
16% aqueous solution of sodium lauryl sulfate (SDS) at room temperature
The sample is dialyzed for a time to prepare a sample for amino acid sequence analysis. Using an amino acid sequencing analyzer (Model 470A) manufactured by Applied Biosystems (USA),
RM Hewick et al. [The Journal of Biological Chemistry (vol. 256), 7990]
(1981)], and Edman analysis was performed sequentially from the N-terminal side. Free phenylthiohydantoin amino acids are released from Spectrophysics (USA)
High Performance Liquid Chromatography Equipment (SP8100)
Analysis was performed using a Zorbax ODS column manufactured by DuPont, USA, and the amino acid sequence was determined. As a result, a part of the amino acid sequence became clear,
It was found that up to the first amino acid had the following amino acid sequence. Ala-Pro-Ala-Glu-Pro-Gln-P
ro-Gly-Gly-Ser-Gln

(4): (Preparation of DNA probe coding for N-terminal amino acid sequence) Considering the frequency of use of bases encoding amino acids in human-derived genes, [Nucleic Acid Res., Vol. 9, R4
3 (1981)], as a base sequence encoding an amino acid sequence from the N-terminus, 5'CTGGGG AGCC
G CCGGGG CTGGG GCTCG GCGGG
The 33mer of GGC 3 'was also described by Otsuka et al. [E. Ohtsuka, et al.
l. ), The Journal of Biological Chemistry (J. Biol. Chem.) 260, 26
05, 1985], using deoxyinosine (indicated by "I") instead of thymidylate as the base sequence encoding the amino acid sequence from the N-terminus:

(1) 5′GCICC IGCIG AACCI CAGCCCIGG3 ′ (2) 5′GCICC IGCIG AGCCI CAACCIGG3 ′ (3) 5′GCICC IGCIG AGCCI CAGCCCIGG3 ′ (4) 5′GCICCC IGGCIG AACCIGACCACC 23mer from Applied Biosystems, USA
The DNA was synthesized using a type 80A DNA synthesizer, purified according to the manufacturer's manual, and described in an experimental book [Maniatis EF, et al., Molecular Cloning (Molecular Clon)].
ing), 122 pp., as described in 1982), were labeled with T ₄ DNA kinase, and γ- ³² P-ATP.

(5): (Protein C by thrombin
Rabbit antibody against a glycopeptide having an action of promoting the activation of protein C of thrombin has a function of promoting the activation of protein C by thrombin purified as described above. Using a glycopeptide derived from a certain human lung,
Book [El Hudson et al. (L. Hudson et a
l. ), Practical Immunology (Practi)
cal Immunology), page 9 (1976).
Year), Blackwell Scientific Publications (Blackwell Scientific)
c Publications)].

This antibody was used for protein C by thrombin.
It was confirmed as follows that it reacts with a glycopeptide derived from human lung which has an activity of promoting activation. That is, about 10 ng of the purified protein obtained by the method described in Reference Example 2- (2) is spotted on a nitrocellulose filter. After air-drying well, this antibody was reacted as a primary antibody with a protein on a nitrocellulose filter, and then a biotinylated anti-rabbit IgG prepared by a goat (Zymet Laboratory, USA, Catalog No. 62-1)
840) as a secondary antibody, and then avidin / biotinylated horseradish peroxidase (manufactured by Amersham Japan, Japan, catalog number RPN.105).
When the color was developed by the method of 1), a black-brown spot was obtained.

(6): (Collection and culture of human umbilical cord endothelial cells)
Using the method of Mano et al. [Japan]
no, et al. ), Experin Encia (Exp)
erientia), Vol. 39, p. 1144, (19
1983)] and collected from a vein obtained from a fresh human umbilical cord provided by a private hospital and cultured.

Reference Example 3 (Acquisition of Recombinant DNA) (1): (Preparation of poly (A) ⁺ RNA) The method of Chargin et al.
Chargin (Chirgwin, JM et a)
l. ), Biochemistry (Biochemistry)
y), Vol. 18, p. 5294 (1979)], to prepare poly (A) ⁺ RNA.

[0072] (2): human lung cDNA screening of from library) human lung poly (A) ⁺ cDNA library of the cDNA prepared from RNA was incorporated into bacteriophage λgt ₁₁ (Clontech, USA) that E. coli Y1090 (manufactured by Clontech, USA) was inoculated according to the manual and transplanted onto an LB medium plate so as to have about 100,000 plaques per 15 cm diameter plate. After culturing at 42 ° C for 3.5 hours, 10 mM IPTG (isopropy
l-β-D-thiogalactopyranosi
de) and then dried and placed on a plate.
Incubate for 3.5 hours at 7 ° C. to convert the peptide to IP
Induced expression with TG and transfer on nitrocellulose filter.

Reference Example 2- (5) in which this nitrocellulose filter was used to promote the activation of protein C of thrombin prepared in rabbits according to the manual.
After reacting the antibody against the glycopeptide obtained in the above as a primary antibody, and then reacting a biotinylated anti-rabbit IgG (manufactured by Zymed Laboratories, US Cat. No. 62-1840) prepared with goat as a secondary antibody, Color was developed with biotinylated horseradish-derived peroxidase (Amersham Japan, Japan, catalog number RPN.1051), and positive clones were isolated. Recombinant c possessed by this positive clone
The cDNA fragment contained in DNA / .lambda.gt ₁₁ was designated TM13.

(3): (Hybridization with DNA probe encoding N-terminal amino acid sequence) The DNA fragment TMI3 obtained in Reference Example 3- (2) was prepared using the N-terminal amino acid prepared in Reference Example 2- (4). An experimental report [Silhavy et al., Experiments with Gene Fusions] determined whether or not to hybridize with a DNA probe encoding a sequence.
ents With Gene Fusions), 1
91, (1984) Cold Spring Harbor Laboratory
r Laboratory)]. DN
It was found that the A fragment TM13 did not hybridize with any of the DNA probes encoding the N-terminal amino acid sequence.

(4): (Base sequence of TM13) DNA contained in the clone obtained in Reference Example 3- (2)
The nucleotide sequence of fragment TM13 was determined by the method of Sanger et al. (Sanger, F. et al., Proceeding of National Academy of
Science USA (Proc. Natl. Ac
ad. Sci. USA), 74, 5463 (197)
7 years). The results are shown in FIGS.

[0076] (5) :( DNA fragment TM13 human lung cDNA library screening as a probe) restriction DNA fragment TM13 enzymes Kpn I and Pvu II
To obtain a DNA fragment of about 440 base pairs, which was labeled with ³² P by the nick translation method. This DN
Plasmid hybridization was performed from a human lung cDNA library using the A fragment as a probe to screen for positive clones. That is, a conventional method of DNA clones TM13 was digested with Kpn I and Pvu II were separated by polyacrylamide gel electrophoresis according to, extraction,
Purification yielded about 500 ng of a purified fragment of about 440 bp.
This DNA was converted to a nick translation kit (catalog number N.5) manufactured by Amersham Japan (Japan).
000) and labeled with α- ³² P-dCTP according to the user manual attached to it.

Using this ³² P-labeled DNA fragment as a probe, an experimental report [Maniatis et al.
Molecular cloning (Molecular C
ling, p. 320, 1982, Cold Spring Harbor Laboratory.
ing Harbor Laboratory)] to perform plaque hybridization of the human lung cDNA library. Positive clones were isolated and the recombinants contained in the clones were analyzed with various restriction enzymes. It was found that a likely DNA fragment of about 2400 bp was incorporated. This DNA fragment was designated as TM137.

(6): (Base sequence of DNA fragment TM137) The base sequence of DNA fragment TM137 obtained in (5) was determined in the same manner as in the method described in Reference Example 3- (4). The results are shown in FIGS. From this result, it was found that the DNA fragment TM137 did not contain the nucleotide sequence encoding the N-terminal amino acid sequence described in Reference Example 2- (3).

(7): (Primer extension) In the base sequence of the DNA fragment obtained in Reference Example 3- (4), three types of synthetic DNA were used based on the N-terminal sequence of DNA fragment TM13. It was created in the same manner as described in 2- (4), and was named HTM131, HTM132, and HTM133. In designing the synthetic DNA, the base sequence that hybridizes with mRNA prepared from human umbilical cord endothelial cells was used. The base sequence of each synthetic DNA is as follows, and the corresponding DNA
The position in the A fragment TM13 is shown in FIG. 2, and the position in the TM137 is shown in FIG.

HTM131: 5'GACGCAGAGGTAGCTAGTTTT 3 '(20mer) HTM132: 5'AACATCTGGCCACCTG 3' (15mer) HTM133: 5'GACAGGGCAGCTGGGTTGCAA 3 '(20mer)
Using poly (A) ⁺ RNA prepared from human umbilical cord endothelial cells obtained by the method described in (1), a so-called primer extension (Primer Extension) was used.
on) to synthesize a further 5 'upstream portion of the DNA fragment TM137.

That is, about 1 μg / μl of poly (A) ⁺
About 27 ng / μl of HTM133 solution 2 in 5 μl of RNA
Add 0 μl, heat at 65 ° C for 20 minutes, and bring
Cooled down over time. Thereafter, the cDNA synthesis system (Amersham Japan, Japan, catalog number RP
N1256) according to the manual.
A was synthesized. However, HTM133 was used in place of the oligo (dT) primer included in the cDNA synthesis system.

The synthesized cDNA was prepared according to the manual (Maniatis et al., Molecular Cloning, 241).
Page, 1982, Cold Spring Harbor Laboratory.
Laboratory)], pBR322 (ATC) with a C tail at both ends and a G tail at both ends.
C37017), and after heating at 65 ° C. for 5 minutes, 57
C. After heating for 2 hours at room temperature and slowly returning to room temperature, E. coli K12MC1061 (obtained from Beckman City of Hope Medical Institute, USA)
Was transformed. Specifically, E. coli K12MC1061
Strain colonies were cultured in LB medium until the absorbance at 550 nm reached 0.3. 50 ml of the culture
And collect 25 mM 10 mM RbCl in 10 mM
3- (N-morpholino) propane-sulfonic acid (MO
PS) (pH 7.0) solution, then 50 mM
25 ml of 0.1 ml containing CaCl ₂ , 10 mM RbCl.
Resuspended in 1M MOPS (pH 6.5).

The resulting suspension was cooled on ice for 30 minutes, centrifuged, the supernatant was removed, and 30 μl of DMSO and 50 mM
The suspension was suspended in a mixture of 2.0 ml of 0.1 M MOPS (pH 6.5) containing CaCl ₂ and 10 mM RbCl. The suspension was dispensed in 200 μl portions, and 10 μl of the above plasmid DNA solution was added to each. The mixture was ice-cooled for 30 minutes, and then heat-shocked at 44 ° C. for 60 seconds.
1 LB medium was added. After culturing this solution at 37 ° C. for 1 hour, each solution was centrifuged and the supernatant was removed to obtain a cell pellet. An LB medium was added to the cell pellet, and stirred to form a suspension. The suspension was spread on an LB agar plate containing 5 μg / ml of tetracycline and cultured at 37 ° C. overnight.

From the cDNA bank obtained in this manner, colony hybridization was performed using HTM131 and HTM132 labeled at their 5 'ends with ³² P as probes according to the method described in Reference Example 2- (4). It carried out by the method similar to (3). Colony
By screening about 70,000 transformants by hybridization, 6 clones were obtained which reacted with both the HTM131 and HTM132 probes. From these 6 clones, an experimental book [Maniatis (Ma
niatis) et al., Molecular Cloning (Mo)
(Regular Cloning), p. 366, 198
Two years, Cold Spring Harbor Laboratory
attorney)] according to the plasmid DNA (this
TMP5 ″) was prepared, cleaved with various restriction enzymes, and analyzed by electrophoresis. The plasmid DNAs obtained from the six clones were all the same, and
It was found that the vector consisted of a DNA fragment of bp size and a vector. This DNA fragment was named TMP5.

(8): (Hybridization of DNA fragment TMP5 with N-terminal amino acid sequence-encoding DNA probe) In the same manner as described in Reference Example 3- (3), the DNA fragment TMP5 has the N-terminal amino acid sequence. Was hybridized with a DNA probe encoding the DNA probe. It was found that the DNA fragment TMP5 did not hybridize with any of the N-terminal DNA probes, that is, did not encode the N-terminal amino acid sequence portion.

(9): (Base sequence of DNA fragment TMP5) The base sequence of DNA fragment TMP5 was determined in the same manner as in the method described in Reference Example 3- (4). The results are shown in FIGS.

(10): (Second Primer Extension) HTM134, HTM based on the nucleotide sequence of DNA fragment TMP5 in the same manner as described in Reference Example 3- (7)
135, three 20mer synthetic DNAs of HTM136
Is prepared. FIG. 8 shows the positions in the DNA fragment TMP5 corresponding to these synthetic DNAs. Reference Example 3- (7)
The primer extension was performed using HTM136 as a primer and HTM134 and HTM135 as probes in the same manner as described in the above section. About 5000
From 0 transformants, HTM134 and HTM13
One transformant hybridizing with No. 5 was obtained.
DN contained in the recombinant carried by this transformant
The A fragment was named TMP26.

(11): (Hybridization of DNA fragment TMP26 with DNA probe encoding N-terminal amino acid sequence) DNA fragment TMP26 was converted to N-terminal amino acid in the same manner as described in Reference Example 3- (3). It was examined whether it hybridized with the DNA probe encoding the sequence. As a result, the DNA fragment TMP26 was a DNA encoding the 33-mer N-terminal amino acid sequence synthesized in Reference Example 2- (4).
Hybridized with the probe and a mixed probe of four 25-mer probes. That is, the DNA fragment TM
P26 was found to encode the N-terminal amino acid sequence portion.

(12): (Base sequence of DNA fragment TMP26) The base sequence of DNA fragment TMP26 was determined in the same manner as in the method described in Reference Example 3- (4). DNA fragment TMP
FIG. 10 shows the nucleotide sequence of about 540 bases from the 26 carboxyl terminus.

(13): (DNA fragment TMP26, TM
Conjugation of P5 and TMP137) Four DNA fragments (TM13, TM137, TMP) obtained in Reference Examples 3- (1) to (12) and determined in base sequence
5 and TMP26) in their base sequences and a simple restriction map are shown in FIG. As shown in FIG. 11, when an open reading frame is assembled from the first ATG upstream of the nucleotide sequence encoding the N-terminal amino acid sequence contained in the DNA fragment TMP26,
It was found that it consisted of 1725 bp that passed through MP26 and TMP5 and continued halfway through TM137. This DNA
DNA fragment TMP26, TM to obtain a DNA fragment encoding the open reading frame spanning the fragment
P5 and TM137 were spliced together in the usual manner as follows.

(13-1) (DNA fragment TM137 and T
MP5 of seaming) First, DN, which is inserted into the Eco RI site of λgt ₁₁
The A fragment TM137 was isolated and the plasmid pUC18 (Pharmacia, Sweden, catalog number 27-4)
It was inserted into the Eco RI site of 949-01) to obtain plasmid pUC18TM137 by. Next, the plasmid pUC
18TM137 was digested with restriction enzymes Hinc II and Eco RI and separated by 4% (v / v) polyacrylamide gel electrophoresis.
X-YIELD ^R) to collect a DNA fragment of about 2300bp was used to purify by performing ethanol precipitation.

On the other hand, the TMP obtained in Reference Example 3- (7)
5 into plasmid pBR322
After complete digestion of TMP5 with Dde I,
E. coli DNA polymerase (Klenow Po
about 800 bp of DN
A fragment was recovered, and this DNA fragment was digested with Sma of pUC18.
The plasmid was inserted into the I site to obtain a plasmid pUC18TMP5. Next, this plasmid pUC18TMP5 was replaced with a restriction enzyme.
Complete digestion with Bam HI and Hinc II results in approx.
A Bam HI- Hinc II fragment of p was obtained. Approximately 23 prepared from plasmid pUC18TM137 as described above.
00 bp DNA fragment and plasmid pUC18TM
A DNA fragment of about 600 bp prepared from P5 was inserted into a vector prepared by digesting plasmid pUC18 with Bam HI and Eco RI, and the plasmid pUC18TMJ was inserted.
1 was obtained. This step is shown in FIG.

(13-2) (DNA fragments TMJ1 and TMJ
Plasmid pUC18TMJ1 was replaced with restriction enzymes DdeI , K
Complete digestion with pn I and Bam HI yielded fragments of about 950 bp and about 1500 bp. On the other hand, DNA fragment T
MP26 was converted to plasmid pUC13 (Pharmacia,
Sweden, catalog no. 27-4954-01) and inserted into the Pst I site of the restriction enzyme to form plasmid pUC1.
3TMP26 was obtained. This was digested to completion with BBE I, the digested ends were blunt ended using T ₄ DNA polymerase, about was completely digested further with the restriction enzyme Bgl II 17
A 0 bp DNA fragment was obtained. Furthermore, plasmid p
The UC13TMP26 obtain complete digestion to about DNA fragment of 280bp in Bgl II and Dde I.

Next, about 170 bp, about 280 bp,
Seaming a DNA fragment of about 950bp with T ₄ DNA ligase, was digested with restriction enzymes Kpn I, 50 V
Purified and isolated by 1.3% low melting point agarose gel electrophoresis at 4 ° C. for 2 hours at a voltage of about 1400 bp of DNA.
A fragment was obtained. Separately, plasmid pUC18 is replaced with Sph
After complete digestion with E.I. After making the cut end blunt with E. coli DNA polymerase, the vector was prepared by complete digestion with Bam HI. This vector contains about 14
T ₄ DNA and a DNA fragment of 00bp and approximately 1500bp
Plasmid pUC18TM was inserted using ligase.
J2 was obtained. This step is shown in FIG.

Reference Example 4 (Screening of target gene from human chromosome) Screening of a target gene from a human chromosome library was carried out as follows. phage vector E
The human chromosome library in MBL-3 is available from Clontech Laboratories, USA.
es, Inc. 922 Industrial Ave.
Palo Alto, CA94303) (catalog number HL1006). Screening was carried out from this library using the DNA fragment TM13 obtained in Reference Example 3- (2) as a probe in the same manner as in Reference Example 3- (5). One clone was obtained.

This chromosomal clone was cloned into the restriction enzyme Bam HI.
After completion of the digestion with 1.0% agarose gel, electrophoresis was performed.
oning), p. 382, 1982, Cold Spring Harbor Laboratory.
ng Harbor Laboratory)]. Southern blot hybridizations were performed using the same probe. As a result, about 4000 bp of D
Because to give a strong positive band in NA fragment was isolated fragments thereof in accordance with a conventional method, it was subcloned into the Bam HI site of the plasmid pUC18. This about 4000bp of D
The nucleotide sequence of NA was determined.
It was found that the nucleotide sequence completely matched the nucleotide sequence of the DNA fragment inserted into the plasmid pUC18TMJ2 prepared in 2).

Example 1 (Plasmids pSV2TMJ2, pSV2TMD1, p
SV2TMD2, pSV2TMD4 and pSV2TM
Preparation of D5) (1) Construction of plasmid pSV2TMJ2 Plasmid pSV2-dhfr (ATCC37146)
Was completely digested with HindIII and BglII to obtain a vector having an SV40 early transcription promoter and an SV40 transcription terminator. Next, Reference Example 2- (13-2)
Hind II plasmid pUC18TMJ2 that was created in
After partial digestion with I and complete digestion with Bam HI, about 290
A 0 bp DNA fragment was isolated. This DNA fragment was converted to TM
J2. The 2900 bp DNA fragment and the vector prepared as described above were spliced together using T4 DNA ligase to obtain plasmid pSV2TMJ2. The steps for constructing plasmid pSV2TMJ2 are shown in FIG. The obtained plasmid pSV2TMJ2 was deposited with the American Type Culture Collection (ATCC) under the deposit number 672 in accordance with the provisions of the Budapest Treaty.
No. 83 has been deposited.

(2) Construction of plasmid pSV2TMD1 (a) Preparation of DNA fragment TMD1 Plasmid pSV2TMJ2 obtained in the above step (1)
It was completely digested with Nco I, E the digested ends. coli
The ends were made blunt using DNA polymerase. Then H
Complete digestion with ind III gave a DNA fragment of about 1900 bp. The obtained DNA fragment was designated as TMJ3. On the other hand, a vector was prepared by digesting phage M-13mp19 (manufactured by Takara Shuzo, Japan, catalog number 3119) with HindIII and HincII . The DNA fragment TMD3 was inserted into this vector and the recombinant plasmid M-13mp was inserted.
19TMJ3 was obtained.

Separately, a DNA probe for deletion having the following base sequence [hereinafter referred to as “delete (delete)
r) "] was synthesized organically: 5'-GGAGGCCGCTCAGCCCCGAATGC
ACG-3 '(25 mer). The synthetic releaser was designated TMD. Using the deleter TMD created in this way, Method in Enzymology (Method in Enzymol)
100, p. 468 (1983), and a recombinant plasmid M-13mp19TMJ1 obtained as described above by site-directed mutagenesis according to the method described in Academic Press.
The portion consisting of 3 177 bases was deleted.

That is, 25 pmol of the deleter TMD
And M13 primer M3 of 10 pmol (universal primer, Takara Shuzo, Catalog No. 3831) after the 5 'end of the phosphorylated using of T ₄ kinase, 0.
5 pmol of recombinant plasmid M13mp19TMJ
The single strand DNA No. 3 was added, heated at 95 ° C. for 5 minutes, and then cooled to room temperature. Then 5 units of E. co
li DNA polymerase 1 (Klenow Fragm
ent), and to produce a recombinant plasmid incubated with a mixture of 10 units T ₄ DNA ligase was added to the mixture 37 ° C. for 30 minutes. The resulting mixture E. coli (E. Coli) JM105 was added (manufactured by Pharmacia, Sweden, Cat. No. 27-1550) to. This E. coli was then transfected with the recombinant plasmid. The plaque on the agar medium formed by culturing overnight at 37 ° C. was transferred to a nitrocellulose filter, heated at 80 ° C. for 2 hours, and prehybridized.

Pre-hybridization was performed with 6 × SET
[0.9 M NaCl, 180 mM Tris buffer (pH
8.0), 6 mM EDTA], 5 × Denhart
s' [0.1% (w / v) Ficoll (Ficol
l), 0.1% (w / v) polyvinylpyrrolidone, 0.1%
1% (w / v) bovine serum albumin (BSA)].
The test was performed by heating at 55 ° C. for 2 hours in a solution containing 1% SDS and 100 μg / ml denatured salmon sperm DNA. Next, a hybridization reaction was carried out at 55 ° C. for 2 hours using a solution in which TMD labeled with ³² P was added instead of the denatured salmon sperm DNA in the above solution. Next, the nitrocellulose filter was washed with 6 × SSC (0.9 M salt, 0.09 M aqueous solution of trisodium citrate).

After washing twice at room temperature for 5 minutes,
The temperature was gradually increased to 5 ° C., 65 ° C., and 75 ° C., and each was washed twice for 5 minutes. X-ray film XA
R-5 (manufactured by Eastman Kodak Company, USA) was brought into close contact with the obtained nitrocellulose filter at -80 ° C,
After exposure overnight, several tens of black spots that were strongly exposed on the X-ray film were detected. Each spot corresponds to a clone infected with the recombinant plasmid. Among them, 6 clones were selected, and the recombinant plasmids of each clone were isolated and subjected to restriction enzyme analysis and nucleotide sequence analysis. The recombinant plasmids possessed by these clones had restriction sites and nucleotide sequences. Each was found to be identical. The resulting recombinant plasmid was designated as M13-TMD1.

Further, the recombinant plasmid M13-T
MD1 has an initiation codon (ATG) and 498 downstream thereof.
It was found to have a DNA fragment containing a nucleotide sequence containing a nucleotide sequence encoding a peptide consisting of a number of amino acids. The DNA fragment contained in the recombinant plasmid M13-TMD1 was called TMD1. FIG. 15 shows the recombinant plasmid M-13mp19TMJ3 and Dirita-T.
This shows that the DNA hybridizes with MD and a part of the DNA region corresponding to the DNA fragment TMJ3 is deleted.

[0104] (b) Construction Example of plasmid pSV2TMD1 1- (2) - (a ) was completely digested recombinant plasmid M13-TMD1 prepared in Hind III and Bam H1 at by single approximately 1900 bp DNA fragment TMD1 Released. On the other hand, plasmid pSV2-dhfr (AT
CC 37146) was completely digested with HindIII and BglII to obtain a vector. This vector and DNA fragment T
Seaming the MD1 using T ₄ DNA ligase to obtain a plasmid PSV2TMD1.

(3) Construction of plasmid pSV2TMD2 (a) Preparation of DNA fragment TMD2 The following nucleotide sequence: 5'-CTCCACGCTGCAGGGGGAACCCC
AGG-3 '(25 mer) Example except for using as the deletion DNA probe for 1- (2) instead deleter over TMd ₂ of deleter over TMD having - repeat substantially the same manner as in (a), TMD2 A recombinant plasmid M13-TMD2 containing a DNA fragment called D
The NA fragment TMD2 is a 678 bp DN from the 5 'end of the DNA fragment TMD1 obtained in Example 1- (2)-(a).
A has a deleted structure. This DNA fragment TMD2
Had a base sequence including an initiation codon (ATG) and a base sequence encoding a peptide consisting of 272 amino acids downstream thereof. FIG. 16 shows the recombinant plasmid M13.
Hybridized with -TMD1 and deleter over TMd ₂ indicates the place where a part of the DNA region corresponding to the DNA fragment TMD1 is deleted.

[0106] (b) Construction Example 1- (3) of plasmid PSV2TMD2 - isolation complete digestion to about 1200bpDNA fragment TMD2 the recombinant plasmid M13-TMD2 prepared in Hind III and Bam HI in (a) did. On the other hand, plasmid pSV2-dhfr (ATC
C 37146) was completely digested with HindIII and BglII to obtain a vector. This vector and DNA fragment TM
Seaming and D2 using T ₄ DNA ligase to obtain a plasmid PSV2TMD2.

(4) Preparation of plasmid pSV2TMD4 (a) Preparation of DNA fragment TMD3 Plasmid pSV2TMJ2 obtained in the above step (1)
It was completely digested with Nco I, E the digested ends. coli
The ends were made blunt using DNA polymerase. Then H
Complete digestion with ind III gave a DNA fragment of about 1900 bp. The obtained DNA fragment was designated as TMJ3. On the other hand, a phage M-13mp19 (manufactured by Takara Shuzo Co., Ltd., Japan, catalog No. 3119) was prepared vector was digested with Hind III and Hinc II of. The DNA fragment TMJ3 was inserted into this vector and the recombinant plasmid M-13mp was inserted.
19TMJ3 was obtained.

Separately, a deleter having the following nucleotide sequence was organically synthesized: 5'-GGAGGCCGCTCAACAGTCGGTG
CCA-3 '(25 mer). Synthesis deleter chromatography was designated TMd _3. Using the deleter TMd ₃ thus prepared, Method in Enzymology (Method in Enzymology)
100), p. 468, (1983).
Year), Academic Press (Academic Pre)
ss), the recombinant plasmid M-13mp19 obtained as described above by site-directed mutagenesis according to the method described in ss).
The 285 bp portion of TMJ3 was deleted.

That is, 25 pmol of the deleter T
Md ₃ and M13 primer M3 (universal primer of 10pmol, Takara Shuzo Co., Ltd., catalog number 383
After the 5 'end of the 1) was phosphorylated with of T ₄ kinase, 0.5 pmol recombinant plasmid M13mp19
A single-strand DNA of TMJ3 was added, heated at 95 ° C. for 5 minutes, and then cooled to room temperature. Then 5 units
E. coli DNA polymerase 1 (Klenow F
ragment), and to produce a recombinant plasmid incubated with a mixture of 10 units T ₄ DNA ligase was added to the mixture 37 ° C. for 30 minutes.

The obtained mixture was used for E. coli ( E. col) .
i ) Added to JM105 (Pharmacia, Sweden, Catalog No. 27-1550). Thereby, E. coli was transfected with the recombinant plasmid. The plaque formed on the agar medium obtained by culturing overnight at 37 ° C. was transferred to a nitrocellulose filter, and the plaque was incubated at 80 ° C. for 2 hours.
After heating for an hour, prehybridization was performed. Prehybridization was performed in 6 × SET [0.9M N
aCl, 180 mM Tris buffer (pH 8.0), 6 m
M EDTA], 5 × Denhards' [0.1%
(W / v) Ficoll, 0.1% (w
/ V) polyvinylpyrrolidone, 0.1% (w / v) bovine serum albumin (BSA)], 0.1% SDS, 100
55 ° C. in a solution containing μg / ml denatured salmon sperm DNA,
Heating was performed for 2 hours.

Next, denatured salmon sperm DN in the above solution
The hybridization reaction was carried out at 55 ° C. for 2 hours using a solution containing TMd ₃ labeled with ³² P instead of A. Then 6 × SSC (0.9M salt, 0.09M
The nitrocellulose filter was washed using an aqueous solution of trisodium citrate). Wash at room temperature for 5 minutes, 2
After washing twice, the temperature was gradually increased to 55 ° C, 65 ° C, and 75 ° C, and each was washed twice for 5 minutes. When X-ray film XAR-5 (manufactured by Eastman Kodak Company, USA) was brought into close contact with the obtained nitrocellulose filter and exposed at -80 ° C overnight, several tens of black spots were strongly exposed on the X-ray film. was detected.

Each spot corresponds to a clone infected with the recombinant plasmid. Among them, 6 clones were selected, and recombinant plasmids of each clone were isolated and analyzed for control enzyme and nucleotide sequence. Recombinant plasmids of these clones had restriction sites and nucleotide sequences. Each was found to be identical. The resulting recombinant plasmid was designated as M13-TMD3. Further, the recombinant plasmid M13-TMD3
Was found to have a DNA fragment containing a start codon (ATG) and a base sequence encoding a peptide consisting of 426 amino acids downstream thereof. The DNA fragment contained in the recombinant plasmid M13-TMD3 was
D3. FIG. 17 shows the recombinant plasmid M-13m.
hybridized with p19TMJ3 and deleter over TMd ₃ indicates the place where a part of the DNA region corresponding to the DNA fragment TMJ3 is deleted.

[0113] (b) Example 1- (3) in place of the deleter over TMd ₃ using techniques produce site-specific mutations in DNA fragments TMD4 - Example except for using deleter over TMd ₂ obtained in (a) 1- (4)-(a)
In substantially the same manner as described above, a part of the recombinant plasmid M13-TMD3 obtained as described above was deleted, and TMD
Recombinant plasmid M13 containing a DNA fragment designated
-TMD4 was obtained. The DNA fragment TMD4 was prepared in Example 1
(4) It has a structure in which 678 bp of DNA is deleted from the 5 'end of the DNA fragment TMD3 obtained in (a).

This DNA fragment TMD4 has an initiation codon (A
TG) and a base sequence including a base sequence encoding a peptide consisting of 236 amino acids downstream thereof. Figure 18 two sets recombinant plasmid M13-TMD3 with the deleter over TMd ₂ is hybridize, indicating the place where a part of the DNA region corresponding to the DNA fragment TMD3 is deleted.

[0115] (c) Construction Example of plasmid pSV2TMD4 1- (4) - (b ) isolating the complete digestion to about 1100bpDNA fragment TMD4 the recombinant plasmid M13-TMD4 prepared in Hind III and Bam HI in did. On the other hand, plasmid pSV2-dhfr (ATC
C 37146) was completely digested with HindIII and BglII to obtain a vector. This vector and DNA fragment TM
Seaming and D4 using T ₄ DNA ligase to obtain a plasmid PSV2TMD4.

(5) Construction of plasmid pSV2TMD5 (a) Preparation of DNA fragment TMD5 The following base sequence: 5'-CACGGGCTCCACGGGGGAACCCC
AGG-3 '(25 mer). The releaser TMd ₄ with the releaser TMd ₂
Except that it is used as a DNA probe for deletion instead of
By substantially the same method as in Example 1- (4)-(b), a recombinant plasmid M13-TMD5 containing a DNA fragment called TMD5 was obtained. DNA fragment TMD5
To the DNA fragment TM obtained in Example 1- (4)-(a).
It has a structure in which 1032 bp DNA has been deleted from the 5 'end of D3. This DNA fragment TMD5 had a base sequence including a start codon (ATG) and a base sequence encoding a peptide consisting of 118 amino acids downstream thereof. Figure 19 two sets recombinant plasmid M13-TMD3 with the deleter over TMd ₄ is hybridized, showing the place where a part of the DNA region corresponding to the DNA fragment TMD3 is deleted.

[0117] (b) Construction Example of plasmid pSV2TMD5 1- (5) - (a ) was completely digested recombinant plasmid M13-TMD5 prepared in Hind III and Bam HI in with a single approximately 740 bp DNA fragment TMD5 Released. On the other hand, plasmid pSV2-dhfr (ATC
C 37146) was completely digested with HindIII and BglII to obtain a vector. This vector and DNA fragment TM
And D5 faded splicing using T ₄ DNA ligase to obtain plasmid pSV2TMD5.

Example 2 (Transformation of COS-1 cells with plasmid pSV2TMD5) COS-1 cells (ATCC CRL165
Dulbecco's minimum essential medium (hereinafter abbreviated as "MEM") supplemented with 10% (v / v) fetal bovine serum (hereinafter abbreviated as "FCS") in an incubator (US, Flow) 5 at 37 ° C. using a laboratory (Flow Laboratories), catalog number 10-331.
Cultured in logarithmic growth phase in 0.1% carbon dioxide incubator, 0.1% trypsin and 0.02% EDTA.
The cells adhered and grown on the incubator were removed from the incubator using a Hanks balanced salt solution [Flow Laboratories, USA, Catalog No. 17-101-22], about 1 × 10 ⁷ cells / ml. To a concentration of 1.

The plasmid p obtained in Example 1- (5)
SV2TMD5 was suspended in 1 mM Tris-HCl buffer (pH 8.0) to a concentration of about 2 μg / μl. About 10μg
5 μl of the obtained plasmid suspension containing the plasmid pSV2TMD5 was placed in a 1.5 ml Eppendorf-type test tube, and the COS-
200 μl of a cell suspension of one cell was added and left at 0 ° C. for 10 minutes. The suspension in the test tube was prepared according to U.S.A. E. FIG. P. SYS
The cells were transferred to a cuvette of a cell fusion device FPH1001 manufactured by TEM, and electric pulses were applied twice at 40 ksec at 1.2 kV. Thereafter, the suspension was transferred again to the original Eppendorf type test tube, left at 0 ° C. for 5 minutes, and then 10% (v / v).
v) 10 ml of Dulbecco's MEM containing FCS was transferred to a 10 cm diameter tissue culture plate using the following method. That is, Dulbecco's MEM containing a small amount of 10% (v / v) FCS was added to the suspension, and the mixture was transferred to a tissue culture plate. The tubes were then washed several times with the remaining Dulbecco's MEM and the wash was added to the same plate. The plates were then incubated at 37 ° C in the presence of 5% CO ₂ for 24 hours.
Cultured for hours.

(Confirmation of Action to Promote Activation of Protein C by Thrombin) After completion of the culture, the medium in the plate was
The cells were replaced with Dulbecco's MEM containing no CS and cultured for 48 hours. 5 μl of the culture supernatant was collected, and this was used as a sample to measure the effect of promoting protein C activation by the method described in Reference Example 1. Further, cells for one tissue culture plate having a diameter of 10 cm were transferred to a US Coaster (Coaster).
r) Cell Scraper (Cell Scrape)
r) Scrape and collect using (catalog number 3010) and collect by centrifugation at 800 rpm for 10 minutes. Using this pellet as a sample, the effect of promoting the activation of protein C was measured by the method described in Reference Example 1. As a control, plasmid pSV2-d
Culture supernatants and cell pellets of hfr-transformed COS-1 cells were used as samples. Table 1 shows the results. The values of the absorbance shown in the table are obtained by converting the absorbance of the sample into the value of one tissue culture plate.

[0121]

[Table 1]

Example 3 (Transformation of COS-1 Cells with Plasmid pSV2TMD4 and Measurement of the Promoting Effect of Thrombin on Protein C Activation of Peptides Produced by the Transformed Cells) The same procedure as in Example 2 was carried out except that plasmid pSV2TMD4 was used. The operation was performed to measure the promoting effect of thrombin on the activation of protein C by the peptide produced by the cells transformed with the plasmid pSV2TMD4. Table 2 shows the results. The values of the absorbance shown in the table are obtained by converting the absorbance of the sample into the value of one tissue culture plate.

[0123]

[Table 2]

Example 4 (Transformation of COS-1 Cells by Plasmid pSV2TMD2 and Measurement of Promoting Effect of Thrombin on Protein C Activation of Peptide Produced by Transformed Cells) The same procedure as in Example 2 was carried out except that plasmid pSV2TMD2 was used. The operation was performed to measure the promoting effect of thrombin on the activation of protein C by the peptide produced by the cells transformed with the plasmid pSV2TMD2. Table 3 shows the results. The values of the absorbance shown in the table are obtained by converting the absorbance of the sample into the value of one tissue culture plate.

[0125]

[Table 3]

Example 5 (Transformation of COS-1 Cells by Plasmid pSV2TMD1 and Measurement of Promoting Effect of Thrombin on Protein C Activation of Peptide Produced by Transformed Cells) The same procedure as in Example 2 was carried out except that plasmid pSV2TMD1 was used. The operation was performed to measure the effect of the peptide produced by the cells transformed with the plasmid pSV2TMD1 on the activation of protein C by thrombin. Table 4 shows the results. The values of the absorbance shown in the table are obtained by converting the absorbance of the sample into the value of one tissue culture plate.

[0127]

[Table 4]

Example 6 (Transformation of COS-1 Cells with Plasmid pSV2TMJ2 and Measurement of the Effect of Thrombin on the Activation of Protein C by Peptides Produced by the Transformed Cells) The same procedure as in Example 2 was carried out except that plasmid pSV2TMJ2 was used. The operation was performed to measure the promoting effect of thrombin on the activation of protein C by the peptide produced by the cells transformed with the plasmid pSV2TMJ2. As a result, the cell pellet sample exhibited a strong protein C activation promoting action, and the amount of generated protein C was about 300 ng.
Met. On the other hand, this activity was not detected in cells transformed with the plasmid pSV2-dhfr used as a control.

Example 7 (Transformation of CHO Cells by Plasmid pSV2TMD5 and Expression in Transformed Cells) About 4 μg of plasmid pSV-2-neo (ATCC 37150) and about 20 μg of Example 1- (5) were used. Plasmid p
SV2TMD5 was mixed and ethanol precipitated. After air-drying the precipitate, the precipitate was dissolved in 450 μl of TE (pH 7.9, 1 mM Tris-HCl buffer, 0.1 mM EDTA), and dissolved in 50 μl of TE.
0 μl of 2 × HBS (50 mM HEPES, 280 m
M NaCl, 1.5 mM Na ₂ HPO ₄ , pH7.
12) was added. Then, 50 μl of 2.5 M CaC
l ₂ was added dropwise and left at room temperature for 10 minutes.

On the other hand, 10% (v / v) FCS and 1 v / v
Diameter using Ham's F-12 culture medium (manufactured by Flow Laboratories, USA, catalog number 10-421-20) containing v% penicillin-streptomycin (manufactured by Flow Laboratories, USA, catalog number 16-700-49) A CHO-KI strain (A) in which about 5 × 10 ² cells were seeded per plate on a 6 cm tissue culture plate.
TCC CCLD 61) was cultured overnight, the medium was replaced with fresh medium, and the cells were further cultured for 3 hours. This CHO-KI
Was overlaid with the above-described plasmid DNA solution to which CaCl ₂ was added dropwise, and cultured at 37 ° C. for about 8 hours. 5 ml of PBS
(−) (Cat. No. 28-103-05, manufactured by Flow Laboratories, USA)
After washing with ml of the above-mentioned medium, fresh medium was added to the medium for about 16 minutes.
The cells were further cultured for hours.

The cells adhered to the plate were peeled off using a 0.25% trypsin, 0.02% EDTA solution, spread on four tissue culture plates having a diameter of 10 cm, and cultured. 2
After 4 hours, the medium was changed to a selective medium. The composition of the selection medium is such that Geneticin C-418 (manufactured by GIBCO, USA, catalog number 8) is added to the above-mentioned medium so as to have a concentration of 400 μg / ml.
60-1811). The cells were cultured for about 2 weeks while changing the medium every 3 to 4 days, and the transformed cells were cloned. The clones of the cells obtained by this operation were grown on tissue culture plates each having a diameter of 10 cm until they became confluent. On the way,
The culture was switched to a medium in which the FCS concentration of the medium was reduced from 10% to 1%. When 50 μl of a culture solution cultured in the FCS-containing selective medium was taken, and the activity of promoting protein C activation was measured by the method described in Reference Example 1, a strong activity of promoting protein C activation was observed. . On the other hand, the plasmid pSV used as a control
This activity was not detected in cells transformed with 2-neo alone.

Example 8 (Transformation of CHO Cells by Plasmid pSV2TMD4 and Measurement of the Effect of Thrombin on the Activation of Protein C by Peptide Produced by the Transformed Cells) The same operation as in Example 7 was carried out except that plasmid pSV2TMD4 was used. Then, thrombin-stimulated protein C activation promoting action of the peptide produced by the cells transformed with the plasmid pSV2TMD4 was measured, and a strong protein C activation promoting action was observed. On the other hand, this activity was not detected in cells transformed only with the plasmid pSV2-neo used as a control.

Example 9 (Transformation of CHO cells by plasmid pSV2TMD2 and measurement of the effect of promoting the protein C activation of the peptide produced by the transformed cells by thrombin) The same operation as in Example 7 was carried out except that plasmid pSV2TMD2 was used. Then, thrombin-stimulated protein C activation of peptide produced by cells transformed with the plasmid pSV2TMD2 was measured, and a strong protein C activation-stimulating effect was observed. On the other hand, this activity was not detected in cells transformed only with the plasmid pSV2-neo used as a control.

Example 10 (Measurement of CHO cell transformation by plasmid pSV2TMD1 and measurement of the effect of thrombin on the promotion of protein C activation of the peptide produced by the transformed cell) The same operation as in Example 7 was carried out except that the plasmid pSV2TMD1 was used. Then, thrombin-stimulated protein C activation promoting action of the peptide produced by the cells transformed with the plasmid pSV2TMD1 was measured. As a result, a strong protein C activation promoting action was observed. On the other hand, this activity was not detected in cells transformed only with the plasmid pSV2-neo used as a control.

The peptide was confirmed in the culture solution by Western blotting. After non-reduced SDS polyacrylamide electrophoresis, the protein was transferred to a nitrocellulose filter, and colored using the rabbit anti-human TM antibody, HRP-labeled anti-rabbit IgG and substrate solution (orthophenylenediamine) prepared in Reference Example. A band having a molecular weight of about 70,000 mainly showing tailing on the polymer side was confirmed.

Example 11 (Measurement of Transformation of CHO Cells by Plasmid pSV2TMJ2 and Measurement of Promoting Effect of Thrombin on Protein C Activation of Peptide Produced by Transformed Cells)
Plasmid pSV-2-neo (ATCC 3715)
0) and about 20 μg of the plasmid pSV2TMJ2 prepared in Example 1 were mixed and precipitated with ethanol. After air-drying the precipitate, the precipitate was dissolved in 450 μl of TE (pH 7.9, 1 mM Tris-HCl buffer, 0.1 mM EDTA), and
00 μl of 2 × HBS (50 mM HEPES, 280
mM NaCl, 1.5 mM Na ₂ HPO ₄ , pH
7.12) was added. Then 50 μl of 2.5 M Ca
Cl ₂ was added dropwise and left at room temperature for 10 minutes.

On the other hand, 10% (v / v) FCS and 1 v / v
Diameter using Ham's F-12 culture medium (manufactured by Flow Laboratories, USA, catalog number 10-421-20) containing v% penicillin-streptomycin (manufactured by Flow Laboratories, USA, catalog number 16-700-49) A CHO-KI strain (A) in which about 5 × 10 ² cells were seeded per plate on a 6 cm tissue culture plate.
TCC CCLD 61) was cultured overnight, the medium was replaced with fresh medium, and the cells were further cultured for 3 hours. This CHO-KI
Was overlaid with the above-described plasmid DNA solution to which CaCl ₂ was added dropwise, and cultured at 37 ° C. for about 8 hours. 5 ml of PBS
(−) (Cat. No. 28-103-05, manufactured by Flow Laboratories, USA)
After washing with ml of the above-mentioned medium, fresh medium was added to the medium for about 16 minutes.
The cells were further cultured for hours.

The cells adhered to the plate were peeled off using a 0.25% trypsin, 0.02% EDTA solution, spread on four tissue culture plates having a diameter of 10 cm, and cultured. 2
After 4 hours, the medium was changed to a selective medium. The composition of the selection medium is such that Geneticin C-418 (manufactured by GIBCO, USA, catalog number 8) is added to the above-mentioned medium so as to have a concentration of 400 μg / ml.
60-1811). The cells were cultured for about 2 weeks while changing the medium every 3 to 4 days, and the transformed cells were cloned. The clones of the cells obtained by this operation were grown on tissue culture plates each having a diameter of 10 cm until they became confluent. On the way,
The culture was switched to a medium in which the FCS concentration of the medium was reduced from 10% to 1%.

[0139] Thrombin-stimulating effect of peptide produced by cells transformed by plasmid pSV2TMJ2 on protein C activation was measured using a cell pellet as a sample. As a result, a strong effect of protein C activation was observed. On the other hand, this activity was not detected in the cells transformed with only the plasmid pSV2-neo used as a control and in the culture supernatant thereof.

Example 12 (Transformation of C ₁₂₇ I cells with plasmid pSV2TMD5 and measurement of the promoting effect of peptide produced by the transformed cells on protein C activation by thrombin) Plasmid pSV2TMD5 prepared in Example 1- (5) To
Was completely digested with Hind III, the cut ends were blunted using DNA polymerase, by the action of T ₄ DNA ligase, a plasmid pSV2TMD5 Hind III
The plasmid pSV2TMD5-1 lacking the site was obtained. Next, this plasmid pSV2TMD5-1 was transferred to Pv
D of about 1700bp was completely digested with u II and Bam HI
An NA fragment was obtained. Hin of this plasmid pUC18
resulting in plasmid pUCTMD5-1 inserted completely digested vector c II and Bam HI.

On the other hand, plasmid pBR322 (ATCC
37017) to the method of Kobasrubias et al. [L. Covasrubias et al.
l), Gene, 13, 25, (1981)
Year), plasmid pBR327 was prepared. Bam HI and Hind The resulting plasmid pBR327
The DNA fragment of about 2960bp obtained by digestion with III, Bam HI and Hind plasmid pUCTMD5-1
A DNA fragment of about 2600 bp obtained by complete digestion with III was inserted to obtain a plasmid pBRTMD5-1. This plasmid pBRTMD5-1 was completely digested with HindIII and plasmid pBPV-1 (9-1) (ATC
In the footsteps C37111) and fragment obtained by complete digestion with Hind III using T ₄ DNA ligase to obtain plasmid pdBPVTMD5-1 for C ₁₂₇ cell expression. The above steps are shown in FIGS.

Next, according to the method described in Example 7, pd
C ₁₂₇ I cell with BPVTMD5-1 (ATCC CRL
1616). 10% FCS and 1 (v / v)% penicillin-streptomycin (Flow Laboratories, USA, catalog number 16-70)
0-49), the cells were cultured for about 3 weeks in Dulbecco's MEM. As a result, six cells forming focus were obtained.
The cells were grown to confluence on a 0 cm tissue culture plate. Thereafter, the medium was replaced with a medium not containing FCS and cultured. Culture medium 50 cultured for 1 day in this medium
An aliquot was used to measure the promoting effect of protein C activation by the method described in Reference Example 1, and a strong activity was observed. On the other hand, this activity was not detected in cells transformed only with the plasmid pBV-1 (9-1) used as a control.

Example 13 (Transformation of C ₁₂₇ I cells with plasmid pSV2TMD4 and measurement of the promoting effect of peptide produced by the transformed cells on protein C activation by thrombin) Plasmid pSV2TMD4 prepared in Example 1- (4) To
Was completely digested with Hind III, the cut ends were blunted using DNA polymerase, by the action of T ₄ DNA ligase, a plasmid pSV2TMD4 Hind III
The plasmid pSV2TMD4-1 lacking the site was obtained. Next, this plasmid pSV2TMD4-1 was transferred to Pv
to obtain a fragment of about 2100bp was completely digested with u II and Bam HI. This was inserted into a vector obtained by completely digesting plasmid pUC18 with Hinc II and Bam HI to obtain plasmid pUCTMD4-1.

On the other hand, plasmid pBR322 (ATCC
 37017) and the method of Kobas Rubias et al.
Baslubias et al. (L. Covasrubias et al.
al), Gene, 13, 25, (1981)
Year), plasmid pBR327 was prepared. Get
Plasmid pBR327BamHI andHind
The DNA fragment of about 2960 bp obtained by digestion with III
Rasmid pUCTMD4-1BamHI andHind
Approximately 3000 bp DNA fragment obtained by complete digestion with III
Insertion yielded plasmid pBRTMD4-1. This
Rasmid pBRTMD4-1HindComplete digestion with III
And the plasmid pBPV-1 (9-1) (ATC
C 37111)HindThe digest obtained by complete digestion with III
T with a piece _FourUsing DNA ligase, C₁₂₇cell
The plasmid pdBPVTMD4-1 for expression was obtained. Less than
The above steps are shown in FIGS.

Next, Example 7 was performed using pdBPVTMD4-1.
C ₁₂₇ I cells (ATCC CRL) according to the method described in
1616). 10% FCS and 1 (v / v)% penicillin-streptomycin (Flow Laboratories, USA, catalog number 16-70)
0-49), the cells were cultured for about 3 weeks in Dulbecco's MEM. As a result, six cells forming focus were obtained.
The cells were grown to confluence on a 0 cm tissue culture plate. Thereafter, the medium was replaced with a medium not containing FCS and cultured. Culture medium 50 cultured for 1 day in this medium
An aliquot was used to measure the promoting effect of protein C activation by the method described in Reference Example 1, and a strong activity was observed. On the other hand, this activity was not detected in cells transformed only with the plasmid pBPV- (9-1) used as a control.

Example 14 (Transformation of C ₁₂₇ I Cells with Plasmid pSV2TMD2 and Measurement of the Promoting Effect of Thrombin on Protein C Activation of Peptides Produced by the Transformed Cells) Plasmid pSV2TMD2 prepared in Example 1- (3) To
Was completely digested with Hind III, the cut ends were blunted using DNA polymerase, by the action of T ₄ DNA ligase, a plasmid pSV2TMD2 Hind III
A plasmid pSV2TMD2-1 lacking the site was obtained. Next, this plasmid pSV2TMD2-1 was transferred to Pv
D of about 2200bp was completely digested with u II and Bam HI
An NA fragment was obtained. Hin of this plasmid pUC18
resulting in plasmid pUCTMD2-1 inserted completely digested vector c II and Bam HI.

On the other hand, plasmid pBR322 (ATCC
 37017) and the method of Kobas Rubias et al.
Baslubias et al. (L. Covasrubias et al.
al), Gene, 13, 25, (1981)
Year), plasmid pBR327 was prepared. Get
Plasmid pBR327BamHI andHind
The DNA fragment of about 2960 bp obtained by digestion with III
Rasmid pUCTMD2-1BamHIas well asHind
The DNA fragment of about 3070 bp obtained by complete digestion with III
Insertion resulted in plasmid pBRTMD2-1. this
Plasmid pBRTMD2-1HindCompletely erased with III
And plasmid pBPV-1 (9-1) (AT
CC 37111)HindObtained by complete digestion with III
Fragment and T _FourUsing DNA ligase, C₁₂₇For cell expression
The plasmid pbBPVTMD2-1 was obtained. More than
The process is shown in FIGS.

Next, C ₁₂₇ I cells (ATCC CRL1) were treated in pdBPVTMD2-1 according to the method described in Example 7.
616) was transformed. 10% FCS and 1
(V / v)% penicillin-streptomycin (US,
Catalog No. 16-700-, manufactured by Flow Laboratory
When the cells were cultured in Dulbecco's MEM containing about 49 weeks for about 3 weeks, 6 cells forming a focus were obtained.
m of tissue culture plates until confluent. Thereafter, the medium was replaced with a medium not containing FCS and cultured. 50 μl of culture solution cultured for 1 day in this medium
The activity of promoting protein C activation was measured by the method described in Reference Example 1 and found to be strong. On the other hand, this activity was not detected in cells transformed only with the plasmid pBPV-1 (9-1) used as a control.

Example 15 (Transformation of C ₁₂₇ I Cells with Plasmid pSV2TMD1 and Measurement of Promoting Effect of Thrombin on Protein C Activation of Peptide Produced by Transformed Cells) Plasmid pSV2TMD1 prepared in Example 1- (2) To
Was completely digested with Hind III, the cut ends were blunted using DNA polymerase, by the action of T ₄ DNA ligase, a plasmid pSV2TMD1 Hind III
The plasmid pSV2TMD1-1 from which the site was deleted was obtained. Then, this plasmid pSV2TMD1-1 Pv
digested with u II and Bam HI to obtain about 3100 bp of D
An NA fragment was obtained. Hin of this plasmid pUC18
resulting in plasmid pUCTMD1-1 inserted completely digested vector c II and Bam HI.

On the other hand, plasmid pBR322 (ATCC
37017) to the method of Kobasrubias et al. [L. Covasrubias et al.
al), Gene, 13, 25, (1981)
Year), plasmid pBR327 was prepared. Bam HI and Hind The resulting plasmid pBR327
The DNA fragment of about 2960bp obtained by digestion with III, Bam HI and Hind plasmid pUCTMD1-1
The DNA fragment obtained by complete digestion with III was inserted to obtain plasmid pBRTMD1-1. This plasmid pBRT
MD1-1 was completely digested with HindIII and plasmid pBPV-1 (9-1) (ATCC 37111) was used.
And a fragment obtained by complete digestion with Hind III in the footsteps using T ₄ DNA ligase to obtain plasmid pdBPVTMD1-1 for C ₁₂₇ cell expression. The above steps are shown in FIG.
As shown in FIG.

Next, Example 7 was performed using pdBPVTMD1-1.
C ₁₂₇ I cells (ATCC CRL) according to the method described in
1616). 10% FCS and 1 (v / v)% penicillin-streptomycin (Flow Laboratories, USA, catalog number 16-70)
0-49), the cells were cultured for about 3 weeks in Dulbecco's MEM. As a result, six cells forming focus were obtained.
The cells were grown to confluence on a 0 cm tissue cell plate. Thereafter, the medium was replaced with a medium not containing FCS and cultured. Culture medium 50 cultured for 1 day in this medium
An aliquot was used to measure the promoting effect of protein C activation by the method described in Reference Example 1, and a strong activity was observed. On the other hand, this activity was not detected in cells transformed only with the plasmid pBPV-1 (9-1) used as a control.

Example 16 (Transformation of C ₁₂₇ I Cells with Plasmid pSV2TMJ2 and Measurement of the Promoting Effect of Thrombin on Protein C Activation of Peptides Produced by the Transformed Cells) Plasmid pSV2TMJ2 prepared in Example 1- (1) To
Was completely digested with Hind III, the cut ends were blunted using DNA polymerase, by the action of T ₄ DNA ligase, the plasmid pSV2TMJ2 Hind III
The plasmid pSV2TMJ2-1 lacking the site was obtained. Next, this plasmid pSV2TMJ2-1 was replaced with Pv
D of about 4100bp was completely digested with u II and Bam HI
An NA fragment was obtained. Hin of this plasmid pUC18
resulting in plasmid pUCTMJ2-1 inserted completely digested vector c II and Bam HI.

On the other hand, plasmid pBR322 (ATCC
37017) to the method of Kobasrubias et al. [L. Covasrubias et al.
l), Gene, 13 , 25, (1981)
Year), plasmid pBR327 was prepared. Bam HI and Hind The resulting plasmid pBR327
The DNA fragment of about 2960bp obtained by digestion with III, Bam HI and Hind plasmid pUCTMJ2-1
The DNA fragment obtained by complete digestion with III was inserted to obtain plasmid pBRTMJ2-1. This plasmid pBRT
MJ2-1 was completely digested with HindIII and plasmid pBPV-1 (9-1) (ATCC37111).
The fragment obtained by complete digestion with Hind III is ligated with T ₄ DNA ligase to obtain plasmid pdBPVTMJ2-1 for C ₁₂₇ cell expression. The above steps are shown in FIG.
As shown in FIG.

Next, C ₁₂₇ I cells (ATCC CRL) were prepared using pdBPVTMJ2-1 according to the method described in Example 7.
1616). 10% FCS and 1 (v / v)% penicillin-streptomycin (Flow Laboratories, USA, catalog number 16-70)
0-49), the cells were cultured for about 3 weeks in Dulbecco's MEM. As a result, six cells forming focus were obtained.
The cells were grown to confluence on a 0 cm tissue cell plate. Thereafter, the medium was replaced with a medium not containing FCS and cultured. After culturing for 1 day in this medium, the pellet of the cultured cells was scraped and used to measure the promoting effect of protein C activation by the method described in Reference Example 1. As a result, a strong activity was observed. On the other hand, this activity was not detected in cells transformed only with the plasmid pBPV-1 (9-1) used as a control.

Example 17 (Protein C by thrombin from the culture solution of the present invention)
Purification of Peptide Having Action to Promote Activation of Plasmid pSV2-n Cultured by the Method described in Example 7
CHO cells transformed with eo and the plasmid pSV2TMD5 were cultured on 25 tissue culture plates having a diameter of 10 cm. The medium was replaced with fresh medium four times every other day. Collect all this culture (about 100 ml),
After adjusting to pH 7.5, it was prepared by column chromatography of DIP-thrombin-agarose.

That is, N.E.S.
L. Esmon) et al. [The Journal of Biological Chemistry (J. Biol. Chem), 25]
7, p. 859, (1982)] DIP-thrombin [diisopropylphosphorothrombin (diisopropylphosphorphor)].
othrombin) can be used as a peak otrecasus (P.
[The Journal of Biological Chemistry (J. Biol. C)]
hem, 245, 359, (1970)].
-Thrombin-agarose was prepared.

Next, DIP-thrombin-agarose was packed in a column having a size of 2.5 cmφ × 10 cm, and
An IP-thrombin-agarose column was prepared and at room temperature 0.1 M NaCl, 0.5 mM CaCl ₂ , 1 mM
Benzamidine hydrochloride, 0.5% (v / v) Lubrol
The column was equilibrated with 0.02 M Tris-HCl buffer (pH 7.5) containing PX (manufactured by Hanoi Chemicals, Japan). Next, the above supernatant was applied to a column. 0.3 column
M NaCl, 0.5 mM CaCl ₂ , 1 mM benzamidine hydrochloride, 0.5% (v / v) Lubrol PX
After washing with 0.02 M Tris-HCl buffer (pH 7.5) containing 1 M NaCl, 0.1 mM EDTA, 1 m
M benzamidine hydrochloride 0.5% (v / v) Lubrol
0.02 M Tris-HCl buffer containing PX (pH 7.
Elution was performed in 5), and 2.0 ml fractions were collected.

For each fraction obtained by elution,
And measuring the promoting effect of protein C activation by the method described above.
did. At the same time, spectrophotometer manufactured by Shimadzu (Japan)
Wavelength of each fraction using a UV-240
Absorbance at 80 nm (A ₂₈₀) Was measured. Active
A certain fraction was collected, and 0.1 M NaCl, 0.5 mM C
aCl_Two, 0.5% (v / v) Lubrol PX (half
0.02M Tris-HCl buffer
The solution (pH 7.5) was dialyzed. The obtained dialysate is used for the second time.
DIP-thrombin-agarose column chromatography
I was offered a fee.

That is, the dialysate was passed through a DIP-thrombin-agarose column having a size of 1.5 cmφ × 10 cm to obtain 0.4 M NaCl, 0.5 mM CaCl ₂ ,
0.02 with 0.1% (v / v) Lubrol PX
After washing with M Tris-HCl buffer (pH 7.5), the solution was further washed with 0.4 M NaCl, 0.1 mM EDTA, 0.1%
(V / v) Wash with 0.02 M Tris buffer (pH 7.5) containing Lubrol PX, then 1 M NaC
1, 0.5 mM EDTA, 0.1% (v / v) Lub
PX with 0.02 M Tris-HCl buffer (pH
Eluted in 7.5). Collect the active fraction and purify the purified product at -80 ° C
And stored frozen. The molecular extinction coefficient of this product is adjusted to 10.0 (E ^1% _{1 cm} 2
80 nm = 10.0), and the amount of the purified product was calculated based on the definition, and it was about 4.7 μg. The purified product was subjected to SDS-polyacrylamide gel electrophoresis using a gradient having a polyacrylamide gel concentration of 5 to 10%, and the band was observed by silver staining. Only a single band was confirmed.

Example 18 Example 17 except that the plasmid pSV2TMD4 was used.
A purified product of the peptide of the present invention is obtained
The absorbance at a length of 280 nm was measured. This refined product
Follow the molecular extinction coefficient of a typical protein
10.0 (E^1% _1cm・ 280nm = 10.0)
Then, the amount of purified product was calculated based on it and found to be about 4.5
μg. In addition, this purified product is polyacrylamide gel
SDS-poly with gradient of 5-10%
Perform acrylamide gel electrophoresis.
Observation of the band revealed only a single band.

Example 19 Example 17 except that the plasmid pSV2TMD2 was used.
A purified product of the peptide of the present invention is obtained
The absorbance at a length of 280 nm was measured. This refined product
Follow the molecular extinction coefficient of a typical protein
10.0 (E^1% _1cm・ 280nm = 10.0)
Then, the amount of purified product was calculated based on it and found to be about 4μg
Met. In addition, this purified product was
SDS-Polyac using a gradient of 5-10%
Perform lilamide gel electrophoresis and band by silver staining.
As a result, only a single band was confirmed.

Example 20 Example 17 except that the plasmid pSV2TMD1 was used.
A purified product of the peptide of the present invention is obtained
The absorbance at a length of 280 nm was measured. This refined product
Follow the molecular extinction coefficient of a typical protein
10.0 (E^1% _1cm・ 280nm = 10.0)
Then, based on that, the amount of purified product was calculated to be about 3μg
Met. In addition, this refined product was polyacrylamide concentration 5
SDS-polyacryl with -10% gradient
Perform amide gel electrophoresis and observe bands by silver staining.
As a result, only a single band was confirmed.

Example 21 In the manner described in Example 11, the plasmid pSV2TM
C2 transformed with J2 and plasmid pSV2-neo
HO cells were cultured using 25 tissue culture plates having a diameter of 10 cm. After the culture, the culture supernatant was centrifuged at 800 rpm for 10 minutes to collect the cells. 0.5% (v / v) Triton X-100 was added to the obtained cell pellet.
0.25M sucrose, 1m M Baie Nzuamijin hydrochloric acid, 0.5m
0.02 M Tris-HCl buffer containing M CaCl ₂ (p
H7.5) was suspended in 100 ml and homogenized 5 times at 4 ° C for 5 minutes using a Waring blender to obtain a cell extract.

The obtained extract was centrifuged at 35,000 g and 10 ° C. for 60 minutes, and the supernatant was collected. From the supernatant, a purified product of the peptide of the present invention was obtained in the same manner as in Example 17, and the absorbance at a wavelength of 280 nm was measured. The molecular extinction coefficient of this purified product is determined to be 10.0 (E ^1% _{1 cm} · 280 nm =
10.0), and the amount of purified product was calculated based on the result, and it was about 3 μg. In addition, this purified product was prepared by using S with a gradient of polyacrylamide concentration of 5-10%.
When DS-polyacrylamide gel electrophoresis was performed and the band was observed by silver staining, only a single band was confirmed.

Example 22 (Confirmation of Action to Promote Protein C Activation by Thrombin) The action of promoting the protein C activation of each peptide purified from the culture supernatant of the present invention was evaluated by the following method. That is, 0.1 M NaCl, 3.6 mM CaCl ₂ , 1
To 0.02 M Tris-HCl buffer (pH 7.5) containing 0 mg / ml bovine serum albumin, 50 μg / ml of protein C, 5 nM of thrombin and 5 nM of each purified peptide were added and reacted at 37 ° C. 30 for reactants
The reaction was stopped by adding 0 μg / ml antithrombin III (manufactured by Sigma, USA) and 5 mM EDTA,
The amount of activated protein C produced was measured by the method using the above-mentioned synthetic substrate. The results are shown in FIG. 30 to FIG. 34. In the case where each peptide was not added (B), the generation of activated protein C was not observed (dotted line), but when the peptide was added (A). The amount of activated protein C produced increased with the reaction time (solid line).

Example 23 (Confirmation of anticoagulant action) [0166] It is shown that each peptide purified from the culture supernatant of the present invention inhibits the conversion of fibrinogen to fibrin by thrombin and substantially inhibits blood coagulation.
Coagulometer KC-10 manufactured by Amerung (Germany)
The clotting time was determined by measuring the blood coagulation time using a.
That is, 3.0 μM was added to a 0.05 M Tris-HCl buffer (pH 7.5) containing 5 mM CaCl ₂ and 0.1 M NaCl.
g of fibrinogen (Fraction I, manufactured by Sigma, USA) was added, and 0-50 nM of each purified peptide was added thereto.
Clotting time was measured by adding M thrombin. The results are shown in FIGS. It was confirmed that the blood coagulation time was prolonged as the amount of the purified peptide added increased as compared with thrombin.

Example 24 (Confirmation of Platelet Aggregation Inhibiting Effect) It was confirmed that each peptide purified from the culture supernatant of the present invention substantially inhibited the platelet aggregation effect of thrombin by SIEN.
The evaluation was performed using a platelet aggregometer manufactured by CO (USA). That is, 300,000 cells / μl of a platelet solution (Platelet Rich Plasma,
P. R. P. 1) Addition of 1 unit of thrombin (approximately 0.4 μg) to 250 μl causes aggregation of platelets, but aggregation of platelets does not occur if thrombin is added before addition of thrombin in an amount equal to or higher than that of purified peptide. Was.

[0168]

[Application Examples] Hereinafter, application examples of the peptide purified from the culture supernatant of the present invention will be described with application examples. Application Example 1 Peptide purified from the culture supernatant of the present invention 10 mg Purified gelatin 20 mg Mannitol 100 mg Sodium chloride 7.8 mg Sodium phosphate 15.4 mg The above components were dissolved in 2 ml of distilled water for injection and placed in a sterile vial. Pre-frozen at −35 ° C. for 2 hours, primary dried at −35 ° C. at a vacuum of 0.075 Torr for 35 hours, and then secondary dried at 30 ° C. and a vacuum of 0.03 Torr for 5 hours to obtain vials for injection. Was manufactured. Immediately before administration, the obtained composition was in a physiological saline or glucose injection 500 ml.
Used for intravenous infusion.

Application Example 2 Peptide purified from the culture supernatant of the present invention 2.5 mg Albumin 5 mg Mannitol 25 mg Sodium chloride 1.95 mg Sodium phosphate 3.85 mg The above components are substantially the same as Application Example 1 A vial for injection was produced in the same manner as described above.

[0170]

The culture supernatant of microorganisms or cells containing the novel peptide of the present invention having a function of promoting the activation of protein C by thrombin has an anticoagulant effect, a platelet aggregation inhibitory effect, and a thrombolytic effect. The peptide is useful as an intermediate in the genetic engineering production of the peptide, which is a very useful substance as a therapeutic drug for circulatory diseases and the like with few side effects. Further, the culture supernatant of the present invention may be used in addition to such medical uses, for example, artificial blood vessels, artificial organs,
It can be bound to a medical artificial material such as a catheter and used as an intermediate in the production of the peptide as an agent for preventing thrombus formation. Furthermore, it can be used as a highly reproducible effector or positive standard in large-scale assays such as activated protein C production reaction or blood anticoagulation reaction.

SEQ ID NO: 1 Sequence length: 118 Sequence type: Amino acid Sequence type: Protein sequence Val Glu Pro Val Asp Pro Cys Phe Arg Ala Asn Cys Glu Tyr Gln Cys 1 5 10 15 Gln Pro Leu Asn Gln Thr Ser Tyr Leu Cys Val Cys Ala Glu Gly Phe 20 25 30 Ala Pro Ile Pro His Glu Pro His Arg Cys Gln Met Phe Cys Asn Gln 35 40 45 Thr Ala Cys Pro Ala Asp Cys Asp Pro Asn Thr Gln Ala Ser Cys Glu 50 55 60 Cys Pro Glu Gly Tyr Ile Leu Asp Asp Gly Phe Ile Cys Thr Asp Ile 65 70 75 80 Asp Glu Cys Glu Asn Gly Gly Phe Cys Ser Gly Val Cys His Asn Leu 85 90 95 Pro Gly Thr Phe Glu Cys Ile Cys Gly Pro Asp Ser Ala Leu Val Arg 100 105 110 His Ile Gly Thr Asp Cys 115

SEQ ID NO: 2 Sequence length: 59 Sequence type: amino acid Sequence type: protein sequence Leu Leu Ile Gly Ile Ser Ile Ala Ser Leu Cys Leu Val Val Ala Leu 1 5 10 15 Leu Ala Leu Leu Cys His Leu Arg Lys Lys Gln Gly Ala Ala Arg Ala 20 25 30 Lys Met Glu Tyr Lys Cys Ala Ala Pro Ser Lys Glu Val Val Leu Gln 35 40 45 His Val Arg Thr Glu Arg Thr Pro Gln Arg Leu 50 55

SEQ ID NO: 3 Sequence length: 236 Sequence type: amino acid Sequence type: protein sequence Cys Ser Val Glu Asn Gly Gly Cys Glu His Ala Cys Asn Ala Ile Pro 1 5 10 15 Gly Ala Pro Arg Cys Gln Cys Pro Ala Gly Ala Ala Leu Gln Ala Asp 20 25 30 Gly Arg Ser Cys Thr Ala Ser Ala Thr Gln Ser Cys Asn Asp Leu Cys 35 40 45 Glu His Phe Cys Val Pro Asn Pro Asp Gln Pro Gly Ser Tyr Ser Cys 50 55 60 Met Cys Glu Thr Gly Tyr Arg Leu Ala Ala Asp Gln His Arg Cys Glu 65 70 75 80 Asp Val Asp Asp Cys Ile Leu Glu Pro Ser Pro Cys Pro Gln Arg Cys 85 90 95 Val Asn Thr Gln Gly Gly Phe Glu Cys His Cys Tyr Pro Asn Tyr Asp 100 105 110 Leu Val Asp Gly Glu Cys Val Glu Pro Val Asp Pro Cys Phe Arg Ala 115 120 125 Asn Cys Glu Tyr Gln Cys Gln Pro Leu Asn Gln Thr Ser Tyr Leu Cys 130 135 140 Val Cys Ala Glu Gly Phe Ala Pro Ile Pro His Glu Pro His Arg Cys 145 150 155 160 Gln Met Phe Cys Asn Gln Thr Ala Cys Pro Ala Asp Cys Asp Pro Asn 165 170 175 Thr Gln Ala Ser Cys Glu Cys Pro Glu Gly Tyr I le Leu Asp Asp Gly 180 185 190 Phe Ile Cys Thr Asp Ile Asp Glu Cys Glu Asn Gly Gly Phe Cys Ser 195 200 205 Gly Val Cys His Asn Leu Pro Gly Thr Phe Glu Cys Ile Cys Gly Pro 210 215 220 Asp Ser Ala Leu Val Arg His Ile Gly Thr Asp Cys 225 230 235

SEQ ID NO: 4 Sequence length: 462 Sequence type: amino acid Sequence type: protein sequence Ala Pro Ala Glu Pro Gln Pro Gly Gly Ser Gln Cys Val Glu His Asp 1 5 10 15 Cys Phe Ala Leu Tyr Pro Gly Pro Ala Thr Phe Leu Asn Ala Ser Gln 20 25 30 Ile Cys Asp Gly Leu Arg Gly His Leu Met Thr Val Arg Ser Ser Val 35 40 45 Ala Ala Asp Val Ile Ser Leu Leu Leu Asn Gly Asp Gly Gly Val Gly 50 55 60 Arg Arg Arg Leu Trp Ile Gly Leu Gln Leu Pro Pro Gly Cys Gly Asp 65 70 75 80 Pro Lys Arg Leu Gly Pro Leu Arg Gly Phe Gln Trp Val Thr Gly Asp 85 90 95 Asn Asn Thr Ser Tyr Ser Arg Trp Ala Arg Leu Asp Leu Asn Gly Ala 100 105 110 Pro Leu Cys Gly Pro Leu Cys Val Ala Val Ser Ala Ala Glu Ala Thr 115 120 125 Val Pro Ser Glu Pro Ile Trp Glu Glu Gln Gln Cys Glu Val Lys Ala 130 135 140 Asp Gly Phe Leu Cys Glu Phe His Phe Pro Ala Thr Cys Arg Pro Leu 145 150 155 160 Ala Val Glu Pro Gly Ala Ala Ala Ala Ala Val Ser Ile Thr Tyr Gly 165 170 175 Thr Pro Phe Ala Ala Arg Gly Ala Asp Phe Gln A la Leu Pro Val Gly 180 185 190 Ser Ser Ala Ala Val Ala Pro Leu Gly Leu Gln Leu Met Cys Thr Ala 195 200 205 Pro Pro Gly Ala Val Gln Gly His Trp Ala Arg Glu Ala Pro Gly Ala 210 215 220 Trp Asp Cys Ser Val Glu Asn Gly Gly Cys Glu His Ala Cys Asn Ala 225 230 235 240 Ile Pro Gly Ala Pro Arg Cys Gln Cys Pro Ala Gly Ala Ala Leu Gln 245 250 255 Ala Asp Gly Arg Ser Cys Thr Ala Ser Ala Thr Gln Ser Cys Asn Asp 260 265 270 Leu Cys Glu His Phe Cys Val Pro Asn Pro Asp Gln Pro Gly Ser Tyr 275 280 285 Ser Cys Met Cys Glu Thr Gly Tyr Arg Leu Ala Ala Asp Gln His Arg 290 295 300 Cys Glu Asp Val Asp Asp Cys Ile Leu Glu Pro Ser Pro Cys Pro Gln 305 310 315 320 Arg Cys Val Asn Thr Gln Gly Gly Phe Glu Cys His Cys Tyr Pro Asn 325 330 335 Tyr Asp Leu Val Asp Gly Glu Cys Val Glu Pro Val Asp Pro Cys Phe 340 345 350 Arg Ala Asn Cys Glu Tyr Gln Cys Gln Pro Leu Asn Gln Thr Ser Tyr 355 360 365 Leu Cys Val Cys Ala Glu Gly Phe Ala Pro Ile Pro His Glu Pro His 370 375 380 Arg Cys Gln Met Phe Cys Asn Gln Thr Ala Cys Pro A la Asp Cys Asp 385 390 395 400 400 Pro Asn Thr Gln Ala Ser Cys Glu Cys Pro Glu Gly Tyr Ile Leu Asp 405 410 415 Asp Gly Phe Ile Cys Thr Asp Ile Asp Glu Cys Glu Asn Gly Gly Phe 420 425 430 Cys Ser Gly Val Cys His Asn Leu Pro Gly Thr Phe Glu Cys Ile Cys 435 440 445 Gly Pro Asp Ser Ala Leu Val Arg His Ile Gly Thr Asp Cys 450 455 460

SEQ ID NO: 5 Sequence length: 272 Sequence type: amino acid Sequence type: protein sequence Cys Ser Val Glu Asn Gly Gly Cys Glu His Ala Cys Asn Ala Ile Pro 1 5 10 15 Gly Ala Pro Arg Cys Gln Cys Pro Ala Gly Ala Ala Leu Gln Ala Asp 20 25 30 Gly Arg Ser Cys Thr Ala Ser Ala Thr Gln Ser Cys Asn Asp Leu Cys 35 40 45 Glu His Phe Cys Val Pro Asn Pro Asp Gln Pro Gly Ser Tyr Ser Cys 50 55 60 Met Cys Glu Thr Gly Tyr Arg Leu Ala Ala Asp Gln His Arg Cys Glu 65 70 75 80 Asp Val Asp Asp Cys Ile Leu Glu Pro Ser Pro Cys Pro Gln Arg Cys 85 90 95 Val Asn Thr Gln Gly Gly Phe Glu Cys His Cys Tyr Pro Asn Tyr Asp 100 105 110 Leu Val Asp Gly Glu Cys Val Glu Pro Val Asp Pro Cys Phe Arg Ala 115 120 125 Asn Cys Glu Tyr Gln Cys Gln Pro Leu Asn Gln Thr Ser Tyr Leu Cys 130 135 140 Val Cys Ala Glu Gly Phe Ala Pro Ile Pro His Glu Pro His Arg Cys 145 150 155 160 Gln Met Phe Cys Asn Gln Thr Ala Cys Pro Ala Asp Cys Asp Pro Asn 165 170 175 Thr Gln Ala Ser Cys Glu Cys Pro Glu Gly Tyr I le Leu Asp Asp Gly 180 185 190 Phe Ile Cys Thr Asp Ile Asp Glu Cys Glu Asn Gly Gly Phe Cys Ser 195 200 205 Gly Val Cys His Asn Leu Pro Gly Thr Phe Glu Cys Ile Cys Gly Pro 210 215 220 Asp Ser Ala Leu Val Arg His Ile Gly Thr Asp Cys Asp Ser Gly Lys 225 230 235 240 Val Asp Gly Gly Asp Ser Gly Ser Gly Glu Pro Pro Pro Ser Pro Thr 245 250 255 Pro Gly Ser Thr Leu Thr Pro Pro Ala Val Gly Leu Val His Ser Gly 260 265 270

SEQ ID NO: 6 Sequence length: 498 Sequence type: amino acid Sequence type: protein sequence Ala Pro Ala Glu Pro Gln Pro Gly Gly Ser Gln Cys Val Glu His Asp 1 5 10 15 Cys Phe Ala Leu Tyr Pro Gly Pro Ala Thr Phe Leu Asn Ala Ser Gln 20 25 30 Ile Cys Asp Gly Leu Arg Gly His Leu Met Thr Val Arg Ser Ser Val 35 40 45 Ala Ala Asp Val Ile Ser Leu Leu Leu Asn Gly Asp Gly Gly Val Gly 50 55 60 Arg Arg Arg Leu Trp Ile Gly Leu Gln Leu Pro Pro Gly Cys Gly Asp 65 70 75 80 Pro Lys Arg Leu Gly Pro Leu Arg Gly Phe Gln Trp Val Thr Gly Asp 85 90 95 Asn Asn Thr Ser Tyr Ser Arg Trp Ala Arg Leu Asp Leu Asn Gly Ala 100 105 110 Pro Leu Cys Gly Pro Leu Cys Val Ala Val Ser Ala Ala Glu Ala Thr 115 120 125 Val Pro Ser Glu Pro Ile Trp Glu Glu Gln Gln Cys Glu Val Lys Ala 130 135 140 Asp Gly Phe Leu Cys Glu Phe His Phe Pro Ala Thr Cys Arg Pro Leu 145 150 155 160 Ala Val Glu Pro Gly Ala Ala Ala Ala Ala Val Ser Ile Thr Tyr Gly 165 170 175 Thr Pro Phe Ala Ala Arg Gly Ala Asp Phe Gln Al a Leu Pro Val Gly 180 185 190 Ser Ser Ala Ala Val Ala Pro Leu Gly Leu Gln Leu Met Cys Thr Ala 195 200 205 Pro Pro Gly Ala Val Gln Gly His Trp Ala Arg Glu Ala Pro Gly Ala 210 215 220 Trp Asp Cys Ser Val Glu Asn Gly Gly Cys Glu His Ala Cys Asn Ala 225 230 235 240 Ile Pro Gly Ala Pro Arg Cys Gln Cys Pro Ala Gly Ala Ala Leu Gln 245 250 255 Ala Asp Gly Arg Ser Cys Thr Ala Ser Ala Thr Gln Ser Cys Asn Asp 260 265 270 Leu Cys Glu His Phe Cys Val Pro Asn Pro Asp Gln Pro Gly Ser Tyr 275 280 285 Ser Cys Met Cys Glu Thr Gly Tyr Arg Leu Ala Ala Asp Gln His Arg 290 295 300 Cys Glu Asp Val Asp Asp Cys Ile Leu Glu Pro Ser Pro Cys Pro Gln 305 310 315 320 Arg Cys Val Asn Thr Gln Gly Gly Phe Glu Cys His Cys Tyr Pro Asn 325 330 335 Tyr Asp Leu Val Asp Gly Glu Cys Val Glu Pro Val Asp Pro Cys Phe 340 345 350 Arg Ala Asn Cys Glu Tyr Gln Cys Gln Pro Leu Asn Gln Thr Ser Tyr 355 360 365 Leu Cys Val Cys Ala Glu Gly Phe Ala Pro Ile Pro His Glu Pro His 370 375 380 Arg Cys Gln Met Phe Cys Asn Gln Thr Ala Cys Pro A la Asp Cys Asp 385 390 395 400 400 Pro Asn Thr Gln Ala Ser Cys Glu Cys Pro Glu Gly Tyr Ile Leu Asp 405 410 415 Asp Gly Phe Ile Cys Thr Asp Ile Asp Glu Cys Glu Asn Gly Gly Phe 420 425 430 Cys Ser Gly Val Cys His Asn Leu Pro Gly Thr Phe Glu Cys Ile Cys 435 440 445 Gly Pro Asp Ser Ala Leu Val Arg His Ile Gly Thr Asp Cys Asp Ser 450 455 460 Gly Lys Val Asp Gly Gly Asp Ser Gly Ser Gly Glu Pro Pro Pro Ser 465 470 475 480 Pro Thr Pro Gly Ser Thr Leu Thr Pro Pro Ala Val Gly Leu Val His 485 490 495 Ser Gly

SEQ ID NO: 7 Sequence length: 557 Sequence type: amino acid Sequence type: protein sequence Ala Pro Ala Glu Pro Gln Pro Gly Gly Ser Gln Cys Val Glu His Asp 1 5 10 15 Cys Phe Ala Leu Tyr Pro Gly Pro Ala Thr Phe Leu Asn Ala Ser Gln 20 25 30 Ile Cys Asp Gly Leu Arg Gly His Leu Met Thr Val Arg Ser Ser Val 35 40 45 Ala Ala Asp Val Ile Ser Leu Leu Leu Asn Gly Asp Gly Gly Val Gly 50 55 60 Arg Arg Arg Leu Trp Ile Gly Leu Gln Leu Pro Pro Gly Cys Gly Asp 65 70 75 80 Pro Lys Arg Leu Gly Pro Leu Arg Gly Phe Gln Trp Val Thr Gly Asp 85 90 95 Asn Asn Thr Ser Tyr Ser Arg Trp Ala Arg Leu Asp Leu Asn Gly Ala 100 105 110 Pro Leu Cys Gly Pro Leu Cys Val Ala Val Ser Ala Ala Glu Ala Thr 115 120 125 Val Pro Ser Glu Pro Ile Trp Glu Glu Gln Gln Cys Glu Val Lys Ala 130 135 140 Asp Gly Phe Leu Cys Glu Phe His Phe Pro Ala Thr Cys Arg Pro Leu 145 150 155 160 Ala Val Glu Pro Gly Ala Ala Ala Ala Ala Val Ser Ile Thr Tyr Gly 165 170 175 Thr Pro Phe Ala Ala Arg Gly Ala Asp Phe Gln A la Leu Pro Val Gly 180 185 190 Ser Ser Ala Ala Val Ala Pro Leu Gly Leu Gln Leu Met Cys Thr Ala 195 200 205 Pro Pro Gly Ala Val Gln Gly His Trp Ala Arg Glu Ala Pro Gly Ala 210 215 220 Trp Asp Cys Ser Val Glu Asn Gly Gly Cys Glu His Ala Cys Asn Ala 225 230 235 240 Ile Pro Gly Ala Pro Arg Cys Gln Cys Pro Ala Gly Ala Ala Leu Gln 245 250 255 Ala Asp Gly Arg Ser Cys Thr Ala Ser Ala Thr Gln Ser Cys Asn Asp 260 265 270 Leu Cys Glu His Phe Cys Val Pro Asn Pro Asp Gln Pro Gly Ser Tyr 275 280 285 Ser Cys Met Cys Glu Thr Gly Tyr Arg Leu Ala Ala Asp Gln His Arg 290 295 300 Cys Glu Asp Val Asp Asp Cys Ile Leu Glu Pro Ser Pro Cys Pro Gln 305 310 315 320 Arg Cys Val Asn Thr Gln Gly Gly Phe Glu Cys His Cys Tyr Pro Asn 325 330 335 Tyr Asp Leu Val Asp Gly Glu Cys Val Glu Pro Val Asp Pro Cys Phe 340 345 350 Arg Ala Asn Cys Glu Tyr Gln Cys Gln Pro Leu Asn Gln Thr Ser Tyr 355 360 365 Leu Cys Val Cys Ala Glu Gly Phe Ala Pro Ile Pro His Glu Pro His 370 375 380 Arg Cys Gln Met Phe Cys Asn Gln Thr Ala Cys Pro A la Asp Cys Asp 385 390 395 400 400 Pro Asn Thr Gln Ala Ser Cys Glu Cys Pro Glu Gly Tyr Ile Leu Asp 405 410 415 Asp Gly Phe Ile Cys Thr Asp Ile Asp Glu Cys Glu Asn Gly Gly Phe 420 425 430 Cys Ser Gly Val Cys His Asn Leu Pro Gly Thr Phe Glu Cys Ile Cys 435 440 445 Gly Pro Asp Ser Ala Leu Val Arg His Ile Gly Thr Asp Cys Asp Ser 450 455 460 Gly Lys Val Asp Gly Gly Asp Ser Gly Ser Gly Glu Pro Pro Pro Ser 465 470 475 480 Pro Thr Pro Gly Ser Thr Leu Thr Pro Pro Ala Val Gly Leu Val His 485 490 495 Ser Gly Leu Leu Ile Gly Ile Ser Ile Ala Ser Leu Cys Leu Val Val 500 505 510 Ala Leu Leu Ala Leu Leu Cys His Leu Arg Lys Lys Gln Gly Ala Ala 515 520 525 Arg Ala Lys Met Glu Tyr Lys Cys Ala Ala Pro Ser Lys Glu Val Val 530 535 540 Leu Gln His Val Arg Thr Glu Arg Thr Pro Gln Arg Leu 545 550 555

SEQ ID NO: 8 Sequence length: 354 Sequence type: base sequence Sequence type: DNA sequence GTG GAG CCC GTG GAC CCG TGC TTC AGA GCC AAC TGC GAG TAC CAG TGC 48 CAG CCC CTG AAC CAA ACT AGC TAC CTC TGC GTC TGC GCC GAG GGC TTC 96 GCG CCC ATT CCC CAC GAG CCG CAC AGG TGC CAG ATG TTT TGC AAC CAG 144 ACT GCC TGT CCA GCC GAC TGC GAC CCC AAC ACC CAG GCT AGC TGT GAG 192 TGC CCT GAA GGC TAC ATC C GAC GAC GGT TTC ATC TGC ACG GAC ATC 240 GAC GAG TGC GAA AAC GGC GGC TTC TGC TCC GGG GTG TGC CAC AAC CTC 288 CCC GGT ACC TTC GAG TGC ATC TGC GGG CCC GAC TCG GCC CTT GTC CGC 336 CAC ATT GGC ACC GAC 354

SEQ ID NO: 9 Sequence length: 708 Sequence type: base sequence Sequence type: DNA sequence TGC AGC GTG GAG AAC GGC GGC TGC GAG CAC GCG TGC AAT GCG ATC CCT 48 GGG GCT CCC CGC TGC CAG TGC CCA GCC GGC GCC GCC CTG CAG GCA GAC 96 GGG CGC TCC TGC ACC GCA TCC GCG ACG CAG TCC TGC AAC GAC CTC TGC 144 GAG CAC TTC TGC GTT CCC AAC CCC GAC CAG CCG GGC TCC TAC TCG TGC 192 ATG TGC GAG ACC GGC TAC CGG CTG GCG GCC GAC CAA CAC CGG TGC GAG 240 GAC GTG GAT GAC TGC ATA CTG GAG CCC AGT CCG TGT CCG CAG CGC TGT 288 GTC AAC ACA CAG GGT GGC TTC GAG TGC CAC TGC TAC CCT AAC TAC GAC 336 CTG GTG GAC GGC TGC GTG GAG CCC GTG GAC CCG TGC TTC AGA GCC 384 AAC TGC GAG TAC CAG TGC CAG CCC CTG AAC CAA ACT AGC TAC CTC TGC 432 GTC TGC GCC GAG GGC TTC GCG CCC ATT CCC CAC GAG CCG CAC AGG TGC 480 CAG ATG TTT CAG ACT GCC TGT CCA GCC GAC TGC GAC CCC AAC 528 ACC CAG GCT AGC TGT GAG TGC CCT GAA GGC TAC ATC CTG GAC GAC GGT 576 TTC ATC TGC ACG GAC ATC GAC GAG TGC GAA AAC GGC GGC TTC TG C TCC 624 GGG GTG TGC CAC AAC CTC CCC GGT ACC TTC GAG TGC ATC TGC GGG CCC 672 GAC TCG GCC CTT GTC CGC CAC ATT GGC ACC GAC TGT 708

SEQ ID NO: 10 Sequence length: 1386 Sequence type: base sequence Sequence type: DNA sequence GCA CCC GCA GAG CCG CAG CCG GGT GGC AGC CAG TGC GTC GAG CAC GAC 48 TGC TTC GCG CTC TAC CCG GGC CCC GCG ACC TTC CTC AAT GCC AGT CAG 96 ATC TGC GAC GGA CTG CGG GGC CAC CTA ATG ACA GTG CGC TCC TCG GTG 144 GCT GCC GAT GTC ATT TCC TTG CTA CTG AAC GGC GAC GGC GGC GTT GGC 192 CGC CGG CGC CTC TGG AGC CTG CAG CTG CCA CCC GGC TGC GGC GAC 240 CCC AAG CGC CTC GGG CCC CTG CGC GGC TTC CAG TGG GTT ACG GGA GAC 288 AAC AAC ACC AGC TAT AGC AGG TGG GCA CGG CTC GAC CTC AAT GGG GCT 336 CCC CTC TGC GGC CC TGC GTC GCT GTC TCC GCT GCT GAG GCC ACT 384 GTG CCC AGC GAG CCG ATC TGG GAG GAG CAG CAG TGC GAA GTG AAG GCC 432 GAT GGC TTC CTC TGC GAG TTC CAC TTC CCA GCC ACC TGC AGG CCA CTG 480 GCT GTG GAG CCC GCC GCG GCT GCC GCC GTC TCG ATC ACC TAC GGC 528 ACC CCG TTC GCG GCC CGC GGA GCG GAC TTC CAG GCG CTG CCG GTG GGC 576 AGC TCC GCC GCG GTG GCT CCC CTC GGC TTA CAG CTA ATG TGC ACC GCG 624 CCG CCC GGA GCG GTC CAG GGG CAC TGG GCC AGG GAG GCG CCG GGC GCT 672 TGG GAC TGC AGC GTG GAG AAC GGC GGC TGC GAG CAC GCG TGC AAT GCG 720 ATC CCT GGG GCT CCC CGC TGC CAG TGC CCA GCC GCC GCC CTG CAG 768 GCA GAC GGG CGC TCC TGC ACC GCA TCC GCG ACG CAG TCC TGC AAC GAC 816 CTC TGC GAG CAC TTC TGC GTT CCC AAC CCC GAC CAG CCG GGC TCC TAC 864 TCG TGC ATG TGC GAG ACC GGC TAC CTG CTG GCC GAC CAA CAC CGG 912 TGC GAG GAC GTG GAT GAC TGC ATA CTG GAG CCC AGT CCG TGT CCG CAG 960 CGC TGT GTC AAC ACA CAG GGT GGC TTC GAG TGC CAC TGC TAC CCT AAC 1008 TAC GAC CTG GTG GAC GGC GAG TGT CCC GTG GAC CCG TGC TTC 1056 AGA GCC AAC TGC GAG TAC CAG TGC CAG CCC CTG AAC CAA ACT AGC TAC 1104 CTC TGC GTC TGC GCC GAG GGC TTC GCG CCC ATT CCC CAC GAG CCG CAC 1152 AGG TGC CAG ATG TTT TGC AAC GCC TGT CCA GCC GAC TGC GAC 1200 CCC AAC ACC CAG GCT AGC TGT GAG TGC CCT GAA GGC TAC ATC CTG GAC 1248 GAC GGT TTC ATC TGC ACG GAC ATC GAC GAG TGC GAA AAC GGC GGC TTC 1296 TGC TCC GGG GTG TGC CAC A AC CTC CCC GGT ACC TTC GAG TGC ATC TGC 1344 GGG CCC GAC TCG GCC CTT GTC CGC CAC ATT GGC ACC GAC TGT 1386

SEQ ID NO: 11 Sequence length: 816 Sequence type: base sequence Sequence type: DNA sequence TGC AGC GTG GAG AAC GGC GGC TGC GAG CAC GCG TGC AAT GCG ATC CCT 48 GGG GCT CCC CGC TGC CAG TGC CCA GCC GGC GCC GCC CTG CAG GCA GAC 96 GGG CGC TCC TGC ACC GCA TCC GCG ACG CAG TCC TGC AAC GAC CTC TGC 144 GAG CAC TTC TGC GTT CCC AAC CCC GAC CAG CCG GGC TCC TAC TCG TGC 192 ATG TGC GAG ACC GGC TAC CGG CTG GCG GCC GAC CAA CAC CGG TGC GAG 240 GAC GTG GAT GAC TGC ATA CTG GAG CCC AGT CCG TGT CCG CAG CGC TGT 288 GTC AAC ACA CAG GGT GGC TTC GAG TGC CAC TGC TAC CCT AAC TAC GAC 336 CTG GTG GAC GGC TGC GTG GAG CCC GTG GAC CCG TGC TTC AGA GCC 384 AAC TGC GAG TAC CAG TGC CAG CCC CTG AAC CAA ACT AGC TAC CTC TGC 432 GTC TGC GCC GAG GGC TTC GCG CCC ATT CCC CAC GAG CCG CAC AGG TGC 480 CAG ATG TTT CAG ACT GCC TGT CCA GCC GAC TGC GAC CCC AAC 528 ACC CAG GCT AGC TGT GAG TGC CCT GAA GGC TAC ATC CTG GAC GAC GGT 576 TTC ATC TGC ACG GAC ATC GAC GAG TGC GAA AAC GGC GGC TTC TGC TCC 624 GGG GTG TGC CAC AAC CTC CCC GGT ACC TTC GAG TGC ATC TGC GGG CCC 672 GAC TCG GCC CTT GTC CGC CAC ATT GGC ACC GAC TGT GAC TCC GGC AAG 720 GTG GAC GGT GGC GAC AGC GGC TCT GGC GAG CCC CC AGC CCG ACG 768 CCC GGC TCC ACC TTG ACT CCT CCG GCC GTG GGG CTC GTG CAT TCG GGC 816

SEQ ID NO: 12 Sequence length: 1494 Sequence type: base sequence Sequence type: DNA sequence GCA CCC GCA GAG CCG CAG CCG GGT GGC AGC CAG TGC GTC GAG CAC GAC 48 TGC TTC GCG CTC TAC CCG GGC CCC GCG ACC TTC CTC AAT GCC AGT CAG 96 ATC TGC GAC GGA CTG CGG GGC CAC CTA ATG ACA GTG CGC TCC TCG GTG 144 GCT GCC GAT GTC ATT TCC TTG CTA CTG AAC GGC GAC GGC GGC GTT GGC 192 CGC CGG CGC CTC TGG AGC CTG CAG CTG CCA CCC GGC TGC GGC GAC 240 CCC AAG CGC CTC GGG CCC CTG CGC GGC TTC CAG TGG GTT ACG GGA GAC 288 AAC AAC ACC AGC TAT AGC AGG TGG GCA CGG CTC GAC CTC AAT GGG GCT 336 CCC CTC TGC GGC CC TGC GTC GCT GTC TCC GCT GCT GAG GCC ACT 384 GTG CCC AGC GAG CCG ATC TGG GAG GAG CAG CAG TGC GAA GTG AAG GCC 432 GAT GGC TTC CTC TGC GAG TTC CAC TTC CCA GCC ACC TGC AGG CCA CTG 480 GCT GTG GAG CCC GCC GCG GCT GCC GCC GTC TCG ATC ACC TAC GGC 528 ACC CCG TTC GCG GCC CGC GGA GCG GAC TTC CAG GCG CTG CCG GTG GGC 576 AGC TCC GCC GCG GTG GCT CCC CTC GGC TTA CAG CTA ATG TGC ACC GCG 624 CCG CCC GGA GCG GTC CAG GGG CAC TGG GCC AGG GAG GCG CCG GGC GCT 672 TGG GAC TGC AGC GTG GAG AAC GGC GGC TGC GAG CAC GCG TGC AAT GCG 720 ATC CCT GGG GCT CCC CGC TGC CAG TGC CCA GCC GCC GCC CTG CAG 768 GCA GAC GGG CGC TCC TGC ACC GCA TCC GCG ACG CAG TCC TGC AAC GAC 816 CTC TGC GAG CAC TTC TGC GTT CCC AAC CCC GAC CAG CCG GGC TCC TAC 864 TCG TGC ATG TGC GAG ACC GGC TAC CTG CTG GCC GAC CAA CAC CGG 912 TGC GAG GAC GTG GAT GAC TGC ATA CTG GAG CCC AGT CCG TGT CCG CAG 960 CGC TGT GTC AAC ACA CAG GGT GGC TTC GAG TGC CAC TGC TAC CCT AAC 1008 TAC GAC CTG GTG GAC GGC GAG TGT CCC GTG GAC CCG TGC TTC 1056 AGA GCC AAC TGC GAG TAC CAG TGC CAG CCC CTG AAC CAA ACT AGC TAC 1104 CTC TGC GTC TGC GCC GAG GGC TTC GCG CCC ATT CCC CAC GAG CCG CAC 1152 AGG TGC CAG ATG TTT TGC AAC GCC TGT CCA GCC GAC TGC GAC 1200 CCC AAC ACC CAG GCT AGC TGT GAG TGC CCT GAA GGC TAC ATC CTG GAC 1248 GAC GGT TTC ATC TGC ACG GAC ATC GAC GAG TGC GAA AAC GGC GGC TTC 1296 TGC TCC GGG GTG TGC CAC A AC CTC CCC GGT ACC TTC GAG TGC ATC TGC 1344 GGG CCC GAC TCG GCC CTT GTC CGC CAC ATT GGC ACC GAC TGT GAC TCC 1392 GGC AAG GTG GAC GGT GGC GAC AGC GGC TCT GGC GAG CCC CCG CCC AGC 1440 CCG ACG CCC GGC ACC TTG ACT CCT CCG GCC GTG GGG CTC GTG CAT 1488 TCG GGC 1494

SEQ ID NO: 13 Sequence length: 1671 Sequence type: base sequence Sequence type: DNA sequence GCA CCC GCA GAG CCG CAG CCG GGT GGC AGC CAG TGC GTC GAG CAC GAC 48 TGC TTC GCG CTC TAC CCG GGC CCC GCG ACC TTC CTC AAT GCC AGT CAG 96 ATC TGC GAC GGA CTG CGG GGC CAC CTA ATG ACA GTG CGC TCC TCG GTG 144 GCT GCC GAT GTC ATT TCC TTG CTA CTG AAC GGC GAC GGC GGC GTT GGC 192 CGC CGG CGC CTC TGG AGC CTG CAG CTG CCA CCC GGC TGC GGC GAC 240 CCC AAG CGC CTC GGG CCC CTG CGC GGC TTC CAG TGG GTT ACG GGA GAC 288 AAC AAC ACC AGC TAT AGC AGG TGG GCA CGG CTC GAC CTC AAT GGG GCT 336 CCC CTC TGC GGC CC TGC GTC GCT GTC TCC GCT GCT GAG GCC ACT 384 GTG CCC AGC GAG CCG ATC TGG GAG GAG CAG CAG TGC GAA GTG AAG GCC 432 GAT GGC TTC CTC TGC GAG TTC CAC TTC CCA GCC ACC TGC AGG CCA CTG 480 GCT GTG GAG CCC GCC GCG GCT GCC GCC GTC TCG ATC ACC TAC GGC 528 ACC CCG TTC GCG GCC CGC GGA GCG GAC TTC CAG GCG CTG CCG GTG GGC 576 AGC TCC GCC GCG GTG GCT CCC CTC GGC TTA CAG CTA ATG TGC ACC GCG 624 CCG CCC GGA GCG GTC CAG GGG CAC TGG GCC AGG GAG GCG CCG GGC GCT 672 TGG GAC TGC AGC GTG GAG AAC GGC GGC TGC GAG CAC GCG TGC AAT GCG 720 ATC CCT GGG GCT CCC CGC TGC CAG TGC CCA GCC GCC GCC CTG CAG 768 GCA GAC GGG CGC TCC TGC ACC GCA TCC GCG ACG CAG TCC TGC AAC GAC 816 CTC TGC GAG CAC TTC TGC GTT CCC AAC CCC GAC CAG CCG GGC TCC TAC 864 TCG TGC ATG TGC GAG ACC GGC TAC CTG CTG GCC GAC CAA CAC CGG 912 TGC GAG GAC GTG GAT GAC TGC ATA CTG GAG CCC AGT CCG TGT CCG CAG 960 CGC TGT GTC AAC ACA CAG GGT GGC TTC GAG TGC CAC TGC TAC CCT AAC 1008 TAC GAC CTG GTG GAC GGC GAG TGT CCC GTG GAC CCG TGC TTC 1056 AGA GCC AAC TGC GAG TAC CAG TGC CAG CCC CTG AAC CAA ACT AGC TAC 1104 CTC TGC GTC TGC GCC GAG GGC TTC GCG CCC ATT CCC CAC GAG CCG CAC 1152 AGG TGC CAG ATG TTT TGC AAC GCC TGT CCA GCC GAC TGC GAC 1200 CCC AAC ACC CAG GCT AGC TGT GAG TGC CCT GAA GGC TAC ATC CTG GAC 1248 GAC GGT TTC ATC TGC ACG GAC ATC GAC GAG TGC GAA AAC GGC GGC TTC 1296 TGC TCC GGG GTG TGC CAC A AC CTC CCC GGT ACC TTC GAG TGC ATC TGC 1344 GGG CCC GAC TCG GCC CTT GTC CGC CAC ATT GGC ACC GAC TGT GAC TCC 1392 GGC AAG GTG GAC GGT GGC GAC AGC GGC TCT GGC GAG CCC CCG CCC AGC 1440 CCG ACG CCC GGC ACC TTG ACT CCT CCG GCC GTG GGG CTC GTG CAT 1488 TCG GGC TTG CTC ATA GGC ATC TCC ATC GCG AGC CTG TGC CTG GTG GTG 1536 GCG CTT TTG GCG CTC CTC TGC CAC CTG CGC AAG AAG CAG GGC GCC GCC A584 AGG AGCAG GAG TAC AAG TGC GCG GCC CCT TCC AAG GAG GTA GTG 1632 CTG CAG CAC GTG CGG ACC GAG CGG ACG CCG CAG AGA CTC 1671

[Brief description of the drawings]

FIG. 1 shows the results of subjecting a peptide obtained by purifying from human lung, which has the effect of promoting protein C activation by thrombin, to a fourth DIP-thrombin-agarose column chromatography in Reference Example 2. It is a graph.

FIG. 2 shows the nucleotide sequence of DNA fragment TM13 obtained in Reference Example 3- (2) of the present invention.

FIG. 3 shows the nucleotide sequence of DNA fragment TM13 following FIG. 2.

FIG. 4 shows the nucleotide sequence of DNA fragment TM137 obtained in Reference Example 3- (5) of the present invention.

FIG. 5 shows the nucleotide sequence of DNA fragment TM137 following FIG. 4;

FIG. 6 shows the nucleotide sequence of DNA fragment TM137 following FIG. 5;

FIG. 7 shows the nucleotide sequence of DNA fragment TM137 following FIG. 6;

FIG. 8 shows the nucleotide sequence of DNA fragment TMP5 obtained in Reference Example 3- (7) of the present invention.

FIG. 9 shows the nucleotide sequence of DNA fragment TMP5 following FIG.

FIG. 10 shows the DN obtained in Reference Example 3- (10) of the present invention.
1 shows the nucleotide sequence of A fragment TMP26.

FIG. 11 shows the above DNA fragments TMP13, TM137,
TMP5 and TMP26 and DNA fragments TMJ1 and TJ1 obtained in Reference Examples 3- (13-1) and 3- (13-2)
It shows the correspondence between each restriction map of MJ2 and the base sequences of these DNA fragments, and shows that the overlapping portions of the DNA fragments in the vertical direction have a common base sequence. In the figure, the portion of the restriction fragment map of the DNA fragment TMJ2 containing the hatched portion and the oblique line is a possible open reading frame, and the base sequence of the present invention is present in the oblique line portion.

FIG. 12 is a flow chart showing the construction of plasmid pUC18TMJ1 containing TMJ1 and TMP5 bound thereto.

FIG. 13 is a flow chart showing construction of plasmid pUC18TMJ2 containing TMJ1 and TMP26 bound thereto.

FIG. 14 shows that TMJ2 was used as an expression vector for animal cell hosts.
Plasmid pSV2TMJ was inserted by inserting MJ2.
3 is a flowchart showing the construction of the second embodiment.

FIG. 15 shows that the releaser TMD hybridized complementarily to the recombinant plasmid M-13mp19TMJ3 obtained in Example 1- (2)-(a), in which the releaser hybridized to the plasmid. This shows the base sequence around the portion and the amino acid sequence encoded thereby.

FIG. 16 shows that the recombinant plasmid pSV2TMD1 obtained in Example 1- (2)-(b) was replaced with the releaser TMd _2.
Shows the base sequence hybridizing complementarily, and the nucleotide sequence around the portion where the releaser hybridizes to the plasmid and the amino acid sequence encoded thereby.

[17] Example 1- (2) - deleter over TMd ₃ in the recombinant plasmid M-13mp19TMJ3 obtained in (a) is intended to indicate a place where hybridized complementary, hybridized deleter over within plasmid This shows the base sequence around the part shown in the figure and the amino acid sequence encoded thereby.

FIG. 18 shows that the recombinant plasmid M13-TMD3 obtained in Example 1- (4)-(a) has a deleter TMd ₂
Shows the base sequence hybridizing complementarily, and the nucleotide sequence around the portion where the releaser hybridizes to the plasmid and the amino acid sequence encoded thereby.

FIG. 19 shows that the recombinant plasmid M13-TMD3 obtained in Example 1- (4)-(a) has a deleter TMd ₄
Shows the base sequence hybridizing complementarily, and the nucleotide sequence around the portion where the releaser hybridizes to the plasmid and the amino acid sequence encoded thereby.

FIG. 20 is a flowchart showing the construction of plasmid pdBPVTMD5-1, which is a recombinant DNA capable of replication.

FIG. 21 is a flowchart showing the construction of plasmid pdBPVTMD5-1, which is a replicable recombinant DNA, following FIG. 20.

FIG. 22 is a flowchart showing the construction of a plasmid pdBPVTMD4-1 which is a replicable recombinant DNA.

FIG. 23 is a flowchart showing the construction of plasmid pdBPVTMD4-1, which is a replicable recombinant DNA, following FIG. 22.

FIG. 24 is a flow chart showing the construction of a plasmid pdBPVTMD2-1 which is a replicable recombinant DNA.

FIG. 25 is a flow chart showing the construction of plasmid pdBPVTMD2-1 which is a replicable recombinant DNA following FIG. 24.

FIG. 26 is a flowchart showing the construction of a plasmid pdBPVTMD1-1 which is a replicable recombinant DNA.

FIG. 27 is a flowchart showing the construction of plasmid pdBPVTMD1-1, which is a replicable recombinant DNA, following FIG. 26.

FIG. 28 is a flow chart showing the construction of plasmid pdBPVTMJ2-1, which is a replicable recombinant DNA.

FIG. 29 is a flowchart showing the construction of plasmid pdBPVTMJ2-1, which is a replicable recombinant DNA, following FIG. 28.

FIG. 30 shows that the cells transformed with plasmid pSV2TMD5 were cultured and purified, and the amount and reaction time of activated protein C generated by the reaction of protein C with thrombin in the presence and absence of the obtained peptide. 6 is a graph showing the relationship of.

FIG. 31 shows the results of culturing and purifying cells transformed with the plasmid pSV2TMD4, the amount of activated protein C generated by the reaction of protein C with thrombin, and the reaction time in the presence and absence of the obtained peptide. 6 is a graph showing the relationship of.

FIG. 32 shows the results of culturing and purifying cells transformed with the plasmid pSV2TMD2, the amount of activated protein C produced by the reaction of protein C with thrombin, and the reaction time in the presence and absence of the obtained peptide. 6 is a graph showing the relationship of.

FIG. 33 shows the results of culturing and purifying cells transformed with plasmid pSV2TMD1, the amount of activated protein C produced by the reaction of protein C with thrombin, and the reaction time in the presence and absence of the obtained peptide. 6 is a graph showing the relationship of.

FIG. 34 shows that the cells transformed with plasmid pSV2TMJ2 were cultured and purified, and the amount and reaction time of activated protein C generated by the reaction of protein C with thrombin in the presence and absence of the obtained peptide. 6 is a graph showing the relationship of.

FIG. 35 is a graph showing the relationship between the clotting time of blood obtained by culturing and purifying cells transformed with plasmid pSV2TMD5 and the obtained peptide and the amount of the purified peptide of the present invention added.

FIG. 36 is a graph showing the relationship between the coagulation time of blood obtained by culturing and purifying cells transformed with the plasmid pSV2TMD4 and adding the obtained peptide, and the amount of the purified peptide of the present invention added.

FIG. 37 is a graph showing the relationship between the clotting time of blood obtained by culturing and purifying cells transformed with plasmid pSV2TMD2 and the obtained peptide and the amount of the purified peptide of the present invention added.

FIG. 38 is a graph showing the relationship between the clotting time of blood obtained by culturing and purifying cells transformed with the plasmid pSV2TMD1 and adding the obtained peptide and the amount of the purified peptide of the present invention added.

FIG. 39 is a graph showing the relationship between the clotting time of blood obtained by culturing and purifying cells transformed with plasmid pSV2TMJ2 and the obtained peptide and the amount of the purified peptide of the present invention added.

────────────────────────────────────────────────── (5) Continuation of the front page (51) Int.Cl. ⁶ Identification code FI C12R 1:91) (56) References JP-A-62-169728 (JP, A) Reference table Hei 3-5033757 (JP, U) J. Clin. Invest. , 76. (1985) p. 2178-2181 Embo J .; , 6, (1987. July) p. 1891-1897 Biochemistry, 26, (1987) p. 4350-4357 Proc. Natl. Acad. Sc i. USA, 84, (1987) p. 6425-6429 J.P. Biol. Chem, 259, (1984) p. 12246-12251 Proc. Natl. Acad. Sc i. USA, 83, (1986) p. 8834-8838 (58) Fields investigated (Int. Cl. ⁶ , DB name) C12N 15/09 ZNA BIOSIS (DIALOG) GenBank / EMBL / DDBJ (GENETYX)

Claims (1)

(57) [Claims] 1. A culture supernatant of a microorganism or a cell, wherein (a) at least as an amino acid sequence, (i) the following formula (I): Val Glu Pro Val Asp Pro Cys Phe Arg Ala Asn Cys Glu Tyr Gln Cys Gln Pro Leu Asn Gln Thr Ser Tyr Leu Cys Val Cys Ala Glu Gly Phe Ala Pro Ile Pro His Glu Pro His Arg Cys Gln Met Phe Cys Asn Gln Thr Ala Cys Pro Ala Asp Cys Asp Pro Asn Thr Gln Ala Ser Cys Glu Cys Pro Glu Gly Tyr Ile Leu Asp Asp Gly Phe Ile Cys Thr Asp Ile Asp Glu Cys Glu A n Gly Gly Phe Cys Ser Gly Val Cys His Asn Leu Pro Gly Thr Phe Glu Cys Ile Cys Gly Pro Asp Ser Ala Leu Val Arg His Ile Gly Thr Asp Cys amino acid sequence represented by or, (ii) formula (I )of
One or several amino acids are missing in the amino acid sequence
A DNA containing a lost, substituted or added amino acid sequence and a soluble DNA encoding a peptide encoding a peptide having an effect of promoting the activation of protein C by thrombin. Binding to a possible expression vector to obtain a replicable recombinant DNA containing said DNA and said replicable expression vector; and (b) transforming a microorganism or cell with said replicable recombinant DNA. (C) selecting the transformant from the parent cell of the microorganism or cell; and (d) culturing the transformant and adding the DN to the transformant.
A culture supernatant of a microorganism or a cell containing the peptide, wherein A is expressed and a culture supernatant is collected from the culture.