JP2000060583A - Dna and its use - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new DNA comprising a DNA containing a DNA encoding a human endothelin-3 having a specific amino acid sequence, being a homologous gene of endothelin having vasoconstrictor action useful for producing the peptide. SOLUTION: This new DNA contains a DNA encoding a human endothelin-3 of an amino acid sequence of the formula, is a new homologous gene of endothelin having vasoconstrictor action and is useful for mass-producing a human endothelin-3 mature peptide. The new DNA is obtained by using a synthetic DNA encoding a part of a human endothelin from a human genome DNA library as a probe, isolating hybridization positive clones, cleaving out one mature body coding region among the clones with a restriction enzyme and subcloning the region.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a DNA containing a DNA encoding endothelin-2 which is a human vasoconstrictor peptide, a precursor protein of endothelin-2 (also referred to as precursor polypeptide) and a mature protein (also referred to as mature polypeptide). Said), a method for producing the precursor protein and mature protein [endothelin-2], and a DNA containing a DNA encoding human endothelin-3, a precursor protein for human endothelin-3 and endothelin-3 Regarding the method. In the present specification, the precursor protein has the amino acid sequence of a mature peptide and has the peptide D at its N-terminal side or C-terminal side or both.
A protein having a part or all of the amino acid sequence encoded by NA.

[0002]

2. Description of the Related Art In addition to endothelium-dependent vasodilation reaction,
Endothelium-dependent vasoconstrictor responses to various stimuli have been reported. Contraction due to mechanical load such as blood vessel expansion and increased internal pressure, contraction due to thrombin, contraction due to decrease in blood oxygen, and neuropeptide Y [Proceedings of the National Academy of Science of Science] The USA (Proc. Natl.
Acad. Sci., USA), 79 , 5485 (1982); ibid, 81, 4577.
(1984)], for example, and enhancement of noradrenaline contraction. American Journal of Physiology (Amer. J. Physiol.), 248, C550 (1985) and Journal of Cell Physio (J. Cell Physio
l.), 132 , 263 (1987) describe a coronary vasoconstrictor derived from endothelial cells (molecular weights are 8,500 and 3,000, respectively), but the structure is unknown. In addition, peptidomimetics derived from endothelial cells are also described in Journal of Pharmacology and Experimental Therapeutics (J. Pharmacl. Exp. Ther.) 236 , 339 (1985).
The structure of this is also unknown. On the other hand, vasopressin (Vasopressin) is known as a peptide having a vasoconstrictor action, and its amino acid sequence has been clarified, but there has been no report that vasopressin was obtained from mammalian or avian vascular endothelial cells as an origin. In addition, it was reported that angiotensin, which has a vasoconstrictor effect, was obtained from bovine aortic endothelial cells [Circulation Research, 60 , 422 (198
7)], angiotensin is a peptide with a molecular weight of about 1,000. Further, some of the present inventors succeeded in previously isolating porcine endothelin from porcine aortic endothelial cells as a peptide having a similar vasoconstrictor action (Japanese Patent Laid-Open No. 1-206997). Have succeeded in isolating human endothelin and cloning porcine endothelin and human endothelin complementary DNA.
(Japanese Patent Application Nos. 62-275613, 62-313155, 63-148158 and 63-274454). The amino acid sequences of the porcine and human endothelin mature polypeptides are identical and are referred to as endothelin-1. In addition, the applicant
An application has also been filed for the isolation of endothelin and cloning of complementary DNA (Japanese Patent Application Nos. 63-174935 and 63-188083), which is called endothelin-3. Furthermore, the present applicant has isolated mouse endothelin, a complementary DNA
We have also applied for the cloning of
-223389), which is called endothelin B. The amino acid sequences of these endothelin-1, B, and -3 are shown in comparison with each other in FIG. In addition, endothelin is a peptide with a molecular weight of 2500 ± 300 and 21 amino acids, and is the first, third, eleventh, and fifteenth counted from the N-terminal of the amino acid sequence. It has a structure in which four Cys located form two sets of SS bonds. The combination of disulfide bonds includes the combination of 1-15 and 3-11 and the combination of 1-11 and 3-15. The former combination has a higher production rate and higher activity. Various names have been used for various endothelins up to now, but this name has been unified this time, and is shown below in comparison with conventional names.

[0003]

As described above, homologous endothelins have been found in various animals, but no novel homologous gene from the same animal species has been found. Therefore, we searched for new homologous endothelins, deepened the research on the structure and activity of these endothelins, examined their usefulness, and cloned the new peptides by gene recombination technology to make them available for mass production. Opening up is the current challenge.

[0004]

[Means for Solving the Problems] If the present inventors can collect a novel homologous gene of endothelin having the above-mentioned vasoconstrictor action and produce it by gene recombination technology, future research and treatment As a result of repeated studies, it is believed that the present invention can achieve a great effect, and the present invention has been achieved by obtaining the following knowledge. That is, the inventors of the present invention used a synthetic DNA encoding a part of human endothelin filed previously as a probe to obtain the above-mentioned endothelin-1 [human endothelin (endothelin A)] from a human genomic DNA library. Succeeded in cloning DNA encoding endothelin having different amino acid sequences. As a result, they succeeded in opening the way to mass-produce these by gene recombination technology. The present inventors have named the human endothelin having this novel amino acid sequence as endothelin-2 (sometimes referred to as human endothelin A-II). Furthermore, the present inventors have found that in the above cloning, the endothelin-3 [rat endothelin (endothelin C)]
Although the amino acid sequences of the matured part and the mature part are the same, the nucleotide sequences encoding them differ, and as a precursor, endothelin having a different amino acid sequence is found, and the DNA is human endothelin-3 DNA, and the precursor is human endothelin. -3 precursor protein.

The present invention provides (1) DNA containing DNA encoding endothelin-2, (2) precursor protein and mature peptide of endothelin-2, and (3) DNA containing DNA encoding endothelin-2. The present invention relates to a method for producing mature endothelin-2, which comprises retaining the transformant, and (4) culturing the transformant of (3) above, accumulating and accumulating protein in the culture, and collecting. ) DNA containing DNA encoding human endothelin-3,
(6) endothelin-3 precursor, (7) a transformant retaining a DNA containing a DNA encoding endothelin-3, and (8) culturing the transformant of (7) above, into a culture. The present invention relates to a method for producing mature endothelin-3, which includes protein production accumulation and collection. The endothelin-2 precursor of the present invention has the amino acid sequence of formula 2. [Formula 2] 1 2 3 4 5 6 7 8 9 10 His Ala Gln Gly Thr His Leu Arg Leu Arg 11 12 13 14 15 16 17 18 19 20 Arg Cys Ser Cys Ser Ser Trp Leu Asp Lys 21 22 23 24 25 26 27 28 29 30 Glu Cys Val Tyr Phe Cys His Leu Asp Ile 31 32 33 34 35 36 37 Ile Trp Val Asn Thr Pro Glu [SEQ ID NO: 4]

Human-derived mature endothelin of the present invention
21 peptides, mature endothelin-2 corresponding to 1, is composed of the 12th to 32nd amino acid sequences of [Formula 2], that is, [Formula 2 ′], 2 '] 1 2 3 4 5 6 Cys Ser Cys Ser Ser T rp 7 8 9 10 11 12 13 14 15 16 Leu Asp Lys Glu Cys Val Tyr Phe Cys His 17 18 19 20 21 Leu Asp Ile Ile Trp MW = 2547 [ SEQ ID NO: 5] The amino acid numbers in the above formula are the numbers assigned from the first Cys with respect to the amino acid sequence of mature endothelin-2, and are different from the numbers of precursor endothelin-2.
In the above formula, the underlined amino acid is different from endothelin A, and instead of 67 Leu-Met of endothelin-1, endothelin-2 has an amino acid of 6 7 Trp-Leu.

Regarding the DNA sequence, the DNA encoding the endothelin-2 of the present invention contains or is a part of the base sequence of [Formula 1], which is a known endothelin-1, -3 is greatly different from the DNA sequence shown in FIG. [Formula 1] Endothelin-2 CAT GCC CAA GGC ACC CAC CTT CGG CTT CGC ↓ 34 CGT TGC TCC TGC AGC TCC TGG CTC GAC AAG GAG TGC GTC TAC TTC TGC CAC TTG GAC ATC 96 ↓ ATC TGG GTG AAC ACT CCT GA [Sequence Number: 6]

The DNA encoding human endothelin-3 of the present invention contains or is a part of the nucleotide sequence of [Formula 3], and this is also a novel one different from known ones. Some things are as shown in FIG. [Formula 3] Endothelin-3 DNA GAG GGG GCC CCT GAG CAC CAC CGA TCC AGG ↓ 34 CGC TGC ACG TGC TTC ACC TAC AAG GAC AAG GAG TGT GTC TAC TAT TGC CAC CTG GAC ATC 96 ↓ ATT TGG ATC AAC ACT CCC GA [ SEQ ID NO: 2]

Part relating to the mature peptide (N in formulas 1 and 3)
o.34-96) is also known DN of endothelin
Unlike A, the DNA of the present invention is novel. The human endothelin-3 precursor of the present invention has the formula (4)
Which is a novel one having an amino acid sequence different from that of rat endothelin-3 precursor as can be seen from FIG. [Formula 4] 1 2 3 4 5 6 7 8 9 10 Glu Gly Ala Pro Glu His His Arg Ser Arg 11 12 13 14 15 16 17 18 19 20 Arg Cys Thr Cys Phe Thr Tyr Lys Asp Lys 21 22 23 24 25 26 27 28 29 30 Glu Cys Val Tyr Tyr Cys His Leu Asp Ile 31 32 33 34 35 36 37 Ile Trp Ile Asn Thr Pro Glu [SEQ ID NO: 3] Encodes the endothelin-2 mature peptide (endothelin-2) of the present invention As DNA, endothelin-2
Any DNA may be used as long as it contains a nucleotide sequence encoding the amino acid sequence of mature peptide (12 to 32 in Formula 2), for example, DNA containing the nucleotide sequence of [Formula 1] or a part thereof. It is preferable that the DNA is The base sequence of [Formula 1] is the endothelin-2 DNA sequence obtained in the present invention, and one example of the base sequence encoding the mature endothelin-2 amino acid of [Formula 2 ′] is No. 34 to [Formula 1]. The one represented by 96 is included.

Matured endothelin-2 in the method of the present invention DN having a nucleotide sequence encoding endothelin-2
Expression vectors containing A include, for example, (i) messenger RNA (mRNA) isolated from endothelin-2 producing cells, and (ii) single-stranded complementary DNA (cDNA) from the mRNA.
And then double-stranded DNA is synthesized, and (iii) the complementary DNA
In a phage or plasmid, (iv) transforming the host with the obtained recombinant phage or plasmid, (v) culturing the obtained transformant, and then transforming the transformant with an appropriate method, for example endothelin-2 By hybridization with a DNA probe encoding a part,
Alternatively, a phage or plasmid containing the target DNA is isolated by an immunoassay method using an anti-endothelin-2 antibody, (vi) the target cloned DNA is cut out from the recombinant DNA, and (vii) the cloned DNA or It can be produced by ligating a part thereof downstream of the promoter in the expression vector. The mRNA encoding endothelin-2 can be obtained from various endothelin-producing cells such as human aortic endothelial cells and human placenta.

Examples of the method for preparing RNA from cells producing endothelin-2 include the guanidine thiocyanate method [(JM. Chirgwin et al., Biochemistry, 18 , 5294 (1979)] and the like. Using the mRNA thus obtained as a template and reverse transcriptase, for example, Okayama (H. Okayama)
Et al method [Molecular - And Cellular - & Baioroji - (Molecular and Cellular Biology) 2 , 161 (1982) and ibid 3, 280 (1983)] according to synthesize cDNA, incorporated resulting cDNA into the plasmid. Examples of plasmids incorporating cDNA include pB derived from E. coli.
R322 [gene, 2 , 95 (1977)], pBR325 [gene, 4 , 121 (1978)], pUC12 [gene, 19 , 259 (19)
82)], pUC13 [Gene, 19 , 259 (1982)], pUB110 derived from Bacillus subtilis [Biochemical and Biophysic Research Communication (Biochemical and Biophysic
al Research Communication), 112 , 678 (1983)] and the like, but any of them may be used as long as they can be replicated and propagated in the host. Examples of phage vectors into which cDNA is incorporated include λgt11 [Young and Davis (Young,
R., and Davis, R.,) Proceedings of the National Academy of Science of the
USA (Proc. Natl. Acad. Sci., USA), 8
0 , 1194 (1983)] and the like, but other ones can be used as long as they can grow in the host.

[0012] Examples of the method for incorporating into a plasmid include, for example, T. Maniatis et al., Molecular Cloning Cold Spring Harbor Laboratory.
or Laboratory), page 239 (1982), and the like. In addition, as a method of incorporating cDNA into a phage vector, for example, the method of Hyunh, TV, et al. [DNA Cloning, A Practical Approach]
1 , 49 (1985)] and the like. The plasmid thus obtained is introduced into an appropriate host such as Escherichia spp. Or Bacillus spp. Examples of the Escherichia bacterium include Escherichia coli K12DH1 [Procedure of the National Academy of
Science (Proc. Natl. Acad. Sci. USA) 60 , 160
(1968)], M103 [Nucleic Acids Research, (Nu
cleic Acids Research), 9 , 309 (1981)], JA221 [Journal of Molecular Biology (Journal)
of Molecular Biology)], 120 , 517 (1978)], HB1
01 [Journal of Molecular Biology 4
1 , 459 (1969)], C600 [Genetics,
39 , 440 (1954)] and the like. Examples of the bacterium of the genus Bacillus include, for example, Bacillus subtilis.
subtilis) MI114 (Gene, 24 , 255 (1983)], 207-
21 (Journal of Biochemistry
f Biochemistry) 95 , 87 (1984)] and the like.

A method for transforming a host with a plasmid is, for example, T. Maniatis.
Et al., Molecular Cloning,
Cold Spring Harbor Laboratory (Cold
Spring Harbor Laboratory), p. 249 (1982), the calcium chloride method or the calcium chloride / rubidium chloride method. When a phage vector is used, it can be introduced into, for example, grown Escherichia coli by the in vitro packaging method. A human cDNA library containing endothelin-2 cDNA can be obtained by the above method or the like, or can be purchased as a commercial product. For example, a human cDNA library can be obtained by Clontech Laboratories, Inc. ., USA).

As a method for cloning endothelin-2 DNA from a human DNA library, for example, a plaque hybridization method using oligonucleotides chemically synthesized based on the amino acid sequences of phage vector λcharon4A and endothelin-2 as a probe [tee・ T.Maniatis et al., Molecular Cloning Cold Spring Harbor Laboratory (Cold Spring)
Harbor Laboratory), (1982)] and the like. Endothelin-2 DNA cloned in this way
Is a plasmid if necessary, eg pBR322, pUC12, pUC
Endothelin-2 DNA can be obtained by subcloning into 13, pUC18, pUC19, pUC118, pUC119 and the like. The nucleotide sequence of the DNA thus obtained is determined, for example, by the Maxam-Gilbert method [Maxa
m, AM and Gilbert, w., Proceedings of the National Academy of Science of the USA (Proc. Natl. Acad. Sci.,
USA), 74 , 560 (1977)] or dideoxy method [Mes
sing, J. et al., Nucleic Acids Research (Nuc
leic Acids Research) 9 , 309 (1981)], and compared with a known amino acid sequence, endothelin-2
Confirm the presence of DNA.

As described above, the DNA encoding endothelin-2 (endothelin-2 DNA) [Formula 1] is obtained. Endothelin-2 obtained in Example 2 described later
And DNA containing DNA encoding
A restriction enzyme fragment map of DNA containing 3 DNA is shown in FIG. The nucleotide sequence of DNA determined by the dideoxy method is shown in FIG. 2, and the amino acid sequence found from the nucleotide sequence is shown in FIG. The DNA encoding the endothelin-2 cloned as described above can be used as it is, or if desired after digestion with a restriction enzyme, depending on the purpose.
The region to be expressed can be excised from the cloned DNA and ligated downstream of the promoter in a vehicle (vector) suitable for expression to obtain an expression type vector. The DNA may have ATG as a translation initiation codon at its 5 ′ end and TAA, TGA or TAG as a translation stop codon at its 3 ′ end. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adaptor. Furthermore, in order to express the DNA, a promoter is connected upstream thereof. As the vector, a plasmid derived from the above E. coli (eg, pBR322, pBR325, pUC1
2, pUC13), a Bacillus subtilis-derived plasmid (eg, pUB
110, pTP5, pC194), yeast-derived plasmids (eg, pSH19, pSH15), bacteriophages such as λ phage and retroviruses, and animal viruses such as vaccinia virus.

The promoter used in the present invention may be any promoter as long as it is suitable for the host used for gene expression. When the host for transformation is a genus Escherichia, trp promoter, lac promoter, recA promoter, λPL promoter, lpp promoter, etc.
When the host is a genus Bacillus, SPO1 promoter, SPO2 promoter, penP promoter and the like, when the host is yeast, PHO5 promoter,
PGK promoter, GAP promoter, ADH promoter and the like are preferable. Particularly, the host is Escherichia and the promoter is the trp promoter or λPL.
It is preferably a promoter. When the host is an animal cell, SV40-derived promoter, retrovirus promoter, metallothionein promoter, heat shock promoter and the like can be used. The use of enhancers for expression is also effective. A transformant is produced using the vector containing the DNA encoding the mature peptide of endothelin-2 thus constructed (endothelin-2).

Examples of the host include Escherichia, Bacillus, yeast, animal cells and the like.
Specific examples of the Escherichia genus and Bacillus genus include the same ones as described above. Examples of the yeast include Saccharomyces cerevisiae (Sacc
^{haromyces cerevisiae) AH22, AH22R -,} NA8
7-11A, DKD-5D and the like. Animal cells include, for example, monkey cell COS-7, Vero, Chinese hamster cell CHO, mouse L cell, human F
L cells and the like can be mentioned. For transforming the above Escherichia bacterium, for example, the Proceeding of the National Academy of Science (Proc.Natl
.Acad.Sci.USA), 6 9 , 2110 (1972) and Jean, 17 , 107
(1982) and the like. Transformation of Bacillus can be accomplished, for example, by Molecular & Gene Genetics.
ral Genetics), 168 , 111 (1979) and the like. To transform yeast, for example, the Procedure of the National Academy
Of Science (Proc.Natl.Acad.Sci.USA), 75 , 19
29 (1978). For transforming animal cells, for example, Virology
52 , 456 (1973). Thus, a transformant transformed with the expression vector containing the DNA encoding the endothelin-2 mature peptide (endothelin-2) is obtained. When the host is culturing a transformant that is an Escherichia genus, a Bacillus genus, a liquid medium is suitable as a medium used for the culture, in which a carbon source necessary for the growth of the transformant,
A nitrogen source, an inorganic substance, etc. are contained. Examples of the carbon source include glucose, dextrin, soluble starch, sucrose, etc., and examples of the nitrogen source include ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean meal, potato extract, and other inorganic substances. Examples of organic substances and inorganic substances include calcium chloride, sodium dihydrogen phosphate, magnesium chloride and the like. In addition, yeast, vitamins, growth promoting factors and the like may be added.

The pH of the medium is preferably about 5-8. As a medium for culturing Escherichia, for example, M9 medium containing glucose and casamino acid [Miller, Journal of Experiments in Molecular.
Genetics (Journal of Experiments in Molecul
ar Genetics), 431-433, Cold Spring Harbor Laborat
ory, New York 1972] is preferred. If necessary, in order to make the promoter work efficiently, for example, 3β-
Indolyl A drug such as acrylic acid can be added. When the host is Escherichia, the culture is usually about 15
It is carried out at about 43 ° C for about 3 to 24 hours, and if necessary, aeration and stirring can be added. When the host is a bacterium of the genus Bacillus, the culture is usually carried out at about 30 to 40 ° C. for about 6 to 24 hours, and aeration and stirring can be added if necessary. When culturing a transformant whose host is yeast, examples of the medium include Burkholder's minimum medium [Bostian, KL
, "Proceding of the National Academy of Science (Proc.Natl.Acad.Sci.USA) 77 , 4505.
(1980)]. The pH of the medium is preferably adjusted to about 5-8. Culturing is usually performed at about 20 ° C to 35 ° C for about 24 hours.
Perform for ~ 72 hours, add aeration and agitation as needed. When culturing a transformant whose host is an animal cell, the medium is, for example, MEM containing about 5 to 20% fetal bovine serum.
Medium [Science 122 , 501 (1952)], DM
EM medium [Virology, 8 , 396 (195
9)], RPMI 1640 medium [Journal of the
American Medical Association (The Jour
nal of the American Medical Association) 199 , 519
(1967)], 199 Medium [Procedure of the Society for the Biological Medicine (Pr.
oceeding of the Society for the Biological Medicin
e) 73, 1 (1950)] and the like. pH is about 6-8
Is preferred. Culturing is usually at about 30-40 ° C for about 15-6
Perform for 0 hours and add aeration and agitation as needed.

The mature peptide of endothelin-2 (endothelin-2) can be separated and purified from the above culture, for example, by the following method. When the mature peptide of endothelin-2 is extracted from the cultured bacterial cells or cells, after the culture, the bacterial cells or cells are collected by a known method, suspended in an appropriate buffer solution, and then ultrasonicated, lysozyme and / or frozen. A method of obtaining a crude extract of a mature peptide of human endothelin A-II by disrupting cells or cells by thawing or the like and then centrifuging or filtering can be appropriately used. The buffer solution may contain a protein denaturing agent such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100. When the endothelin-2 precursor protein or mature peptide is secreted into the culture medium, after the completion of the culture, the cells or cells are separated from the supernatant by a method known per se, and the supernatant is collected. The endothelin-2 precursor protein or mature peptide contained in the culture supernatant or the extract thus obtained can be subjected to an appropriate combination of separation / purification methods known per se. As the known separation and purification methods thereof, a method utilizing solubility such as salting out or solvent precipitation method, a dialysis method, an ultrafiltration method, a gel filtration method, and an SDS-polyacrylamide gel electrophoresis method are mainly used. The difference in charge, the difference in charge such as ion exchange chromatography, the method using specific affinity such as affinity chromatography, and the difference in hydrophobicity such as reverse phase high performance liquid chromatography Examples of the method include a method utilizing a difference in isoelectric points such as an isoelectric focusing method. The endothelin-2 precursor protein or mature peptide thus produced can be measured by an enzyme immunoassay using a specific antibody. When the product has vasoconstrictor activity, it can be measured using the activity as an index. Although endothelin-2 has been described in detail above, the same can be said for endothelin-3.

[Action / Effect] A large amount of endothelin-2 or endothelin-3 mature peptide can be produced in the cells or cells infected with DNA or transformed with the DNA of the present invention, and these peptides can be produced advantageously. I can guide you. Like other endothelins, the endothelin-2 mature peptide and endothelin-3 produced here can be used not only as a hypotensive therapeutic agent or a local vasoconstrictor, but also for the analysis of the mechanism of vasoconstriction reaction in the living body and It provides a clue to the elucidation of vasoconstrictor antagonists. Regarding vasoconstriction, the activity of endothelin-2 is high, for example, 2 of conventional endothelin.
It has twice the activity. Further, these endothelins have an effect as a vasoconstrictor to prevent various bleeding, for example, gastric or esophageal bleeding. It also has the effect of relieving various shock symptoms. The peptide can be administered orally, topically, intravenously or parenterally, with topical or intravenous administration being preferred. The dosage is
0.001μg-100μg / kg, preferably 0.01μg-10μg / kg
It is used by dissolving it in a physiological saline solution of 1 to 10 ml at a dose corresponding to the body weight. The peptide of the present invention is an emulsion, a wettable powder, a tablet, a water solution, a powder containing the peptide and subcomponents,
It can be used as a formulation in various forms such as granules, capsules and pills. As the sub-components, pharmacologically acceptable excipients, disintegrants, lubricants, binders, dispersants, plasticizers, fillers, carriers and the like are used. Examples of these subcomponents are lactose, glucose, sucrose and the like as excipients, and starch, sodium alginate, agar powder, carboxymethylcellulose calcium and the like as disintegrants.
Magnesium stearate, talc, liquid paraffin, etc. as lubricants, simple syrup, gelatin solution, ethanol, polyvinyl alcohol etc. as binders,
As the dispersant, methyl cellulose, ethyl cellulose,
Examples of the plasticizer include shellac, and glycerin, starch, and the like.

In the specification and drawings of the present invention, when an abbreviation is used for a base or amino acid, IUPAC-IU
It is based on the abbreviations by the B Commission on Biochemical Nomenclature or the abbreviations commonly used in this field, and examples are given below. When amino acids may have optical isomers, the L-form is shown unless otherwise specified. DNA: deoxyribonucleic acid cDNA: complementary deoxyribonucleic acid A: adenine T: thymine G: guanine C: cytosine RNA: ribonucleic acid mRNA: messenger ribonucleic acid dATP: deoxyadenosine triphosphate dTTP: deoxythymidine triphosphate dGTP: deoxyguanosine tri Phosphate dCTP: Deoxycytidine triphosphate ATP: Adenosine triphosphate EDTA: Ethylenediaminetetraacetic acid SDS: Sodium dodecyl sulfate Gly or G: Glycine Ala or A: Alanine Val or V: Valine Leu or L: Leucine Ile or I: Isoleucine Ser or S: Serine Thr or T: Threonine Cys or C: Cysteine Met or M: Methionine Glu or E: Glutamic acid Asp or D: Asparagi Acid Lys or K: Lysine Arg or R: Arginine His or H: Histidine Phe or F: Phenylalanine Tyr or Y: Tyrosine Trp or W: Tryptophan Pro or P: Proline Asn or N: Asparagine Gln or Q: Glutamine In the endothelin-2 mature peptide and endothelin-3 of the present invention, a part of the amino acid sequence thereof may be modified (addition, removal, substitution with other amino acids, etc.).

[0022]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in more detail with reference to the following Reference Examples and Examples, but the present invention is not limited thereto. The transformant Escherichia obtained in Example 2 was used.
coli XL-1 / pghET20SG1 and Esc obtained in Example 3
herichia coli XL-1 / pghET3E1 has been available from the Ministry of International Trade and Industry, Institute of Industrial Technology, Microbial Research Institute (FR) since October 24, 1988.
I) [Presently, Institute of Biotechnology, Industrial Technology Institute (NI)
Has been renamed to BH)], and the accession number FERM
Deposited and stored as BP-2118 and FERM BP-2119.

Reference Example (1) Assay Method for Contraction of Vascular Smooth Muscle A pig right coronary artery spiral specimen (0.5 × 20 mm) from which the endothelium was removed by scraping with an injection needle was mixed with carbon dioxide and oxygen (5:95). , K / V) saturated with Krebs-Ringer solution (3 ml) at 37 ° C. Pre-stimulation tension (basal t
tension) is set to 2 g, and isometric tension is measured with a tension transducer. (2) Assay for cardiotonic action In place of the pig right coronary artery spiral specimen used in the assay method of (1) above, a suspended specimen of the right atrium of a guinea pig was used, and the same operation as in (1) was performed to determine tension and Measure the heart rate per minute.

Example 1 D encoding a part of endothelin-1 [porcine endothelin (human endothelin I)]
Preparation of NA probe 7 to 20 of endothelin-1 (human endothelin I)
Amino acid sequence of residue Met Asp Lys Glu Cys Val Tyr Phe Cys His Leu Asp Il
deducing the expected sequence of messenger RNA from e Ile [SEQ ID NO: 7],
A DNA probe having the following sequence was chemically synthesized. 5 ′ ATG GAC AAG GAG TGT GTC TAC TTC TGC CAT CTG GAC ATC ATC 3 ′ [SEQ ID NO: 8] The 5 ′ end of this DNA probe was [γ- ³² P] ATP-phosphorylated using T4 polynucleotide kinase. , Used for screening of genomic DNA library.

Example 2 Isolation of Endothelin-2 Precursor Genomic DNA and Determination of Its Nucleotide Sequence Escherichia coli Le 392 was infected with the above-mentioned human genomic DNA library (manufactured by Clontech Laboratories, Inc.) and plated to obtain a phage plaque. Has appeared.
According to the report by Benton, W., Davis, R. [Science 196 , 180-182 (1977)], a part of the plaque DNA was transferred to a nylon membrane,
Plaque hybridization was performed with the ³² P-labeled DNA probe. Hybridization was carried out at 42 ° C in the presence of 20% formamide and the membrane was adjusted to 0.
It was washed at 20 ° C. in 2 × SSC, 0.1% SDS. Hybridization-positive clones were isolated, and one of them, the mature body coding region of λghET20, was cut out with SacI and subcloned into the plasmid pUC 118. Escherichia coli XL-1 was transformed with this plasmid to obtain a transformant Escherichia coli XL-1 / pghET20SG1.
A simple restriction map of the human genomic DNA fragment contained in this plasmid is shown in FIG. Areas in the figure show the following. ■: Endothelin-2 mature coding region The nucleotide sequence of this mature coding region and its surrounding region is determined by the Sanger method [Proc. Natl. Acad. S.
ci. USA) 74 , 5463-5467 (1977)]. This base sequence and the amino acid sequence deduced therefrom are shown in FIG. 2 (the boxed portion is the mature peptide portion). In addition, FIG. 3 shows the amino acid sequence of the mature peptide of endothelin-1 and -3 previously found together with the amino acid sequence of the mature peptide of human endothelin A-II for comparison.

Example 3 Isolation of Endothelin-3 Mature Body Coding Region and Determination of Its Nucleotide Sequence Hybridization-positive clones were isolated in the same manner as in Example 2, and one of them, the mature body coding region of λghET3. Was excised with EcoRI to obtain plasmid p
Subcloned into UC 118. Escherichia coli XL-1 was transformed with this plasmid, and the transformant Escherichia
E. coli XL-1 / pghET3E1 was obtained. A simple restriction map of the human genomic DNA fragment contained in this plasmid is shown in FIG. Areas in the figure show the following. ■: Endothelin-3 mature coding region The nucleotide sequence of this mature coding region and its surrounding region is determined by the Sanger method [Proc. Natl. Acad. S.
ci. USA) 74 , 5463-5467 (1977)]. This base sequence and the amino acid sequence deduced therefrom are shown in FIG. The area surrounded by a frame is endothelin-
3 mature peptide part [SEQ ID NO: 1].

Example 4 (i) Synthesis of endothelin-2 A commercially available Boc-Trp (CHO) -PAM resin (manufactured by Applied Biosystems) 0.7 g (0.5 m mole) was used and a peptide synthesizer (Applied Biosystems) was used. A model 430A manufactured by Systems, Inc. was used and synthesized by a usual method. The condensation method was carried out by treating the Boc group on the resin with 50% trifluoroacetic acid in methylene chloride to liberate the terminal amino group, and to bind the free amino group to Boc-Ile, Boc-Asp (OB
zl), Boc-Leu, Boc-His (Tos),
Boc-Cys (Acm), Boc-Tyr (Br-
z), Boc-Val, Boc-Phe, Boc-Gl
u (OBzl), Boc-Lys (Cl-Z), Boc
-Trp (CHO), Boc-Ser (Bzl) from the C-terminal side according to the amino acid sequence of endothelin-2, DC
The reaction of condensing in the presence of C was repeated. 890 out of 2.53 g of the protected endothelin-2 resin thus obtained
mg to 1 ml of anisole and 1 of 1,2-ethanedithiol
After swelling with ml and treating with 10 ml of hydrogen fluoride at 0 ° C. for 60 minutes, excess hydrogen fluoride was distilled off under reduced pressure. The residue was washed with 5 ml of diethyl ether, extracted with trifluoroacetic acid, and the resin was filtered off. After trifluoroacetic acid was distilled off under reduced pressure, it was dissolved in 50% acetic acid water and applied to a dextran gel (Sephadex G-50) column (2 x 90 cm). The main fraction eluted with the same solvent was collected and freeze-dried. And 180m
g of white powder was obtained. 31 mg of this is 50% -acetic acid water 4 m
dissolved in 1, and added 19 mg of mercuric trifluoroacetate,
After stirring at room temperature for 16 hours, n-butanol 100m
1, 50 ml of methanol and 50 ml of water were added for dilution, and hydrogen sulfide gas was passed through. Add 5% -NH ₄ OH to this and p
After adjusting to H8, it was subjected to air oxidation for 6 hours, adjusted to pH 3 by adding acetic acid, and then freeze-dried. This was packed on a column of Sephadex G-50 packed with 50% acetic acid (2 x 90
cm), and the main fractions were collected and further subjected to HPLC (column: YMC, solvent: 0.1% -trifluoroacetic acid water and 0.1%).
Fractionation was performed by elution with a linear concentration gradient of acetonitrile containing% -trifluoroacetic acid) to obtain 1.0 mg of the desired product. Synthetic endothelin-2 was eluted at 41.4 minutes by HPLC with respect to endothelin-1 40.2 minutes. Column conditions Wakosil 5C18 (4.6 × 250 mm) Eluent: Solution A (0.1% -trifluoroacetic acid water) Solution B (0.1% -trifluoroacetic acid-containing acetonitrile)
Linear concentration gradient elution from solution A to solution B (50 minutes) Flow rate: 1.0 ml / min Amino acid analysis value: Analysis value (number in synthetic product) Asx 1.92 (2) Ser 2.31 (3) Glx 1.04 ( 1) Cys 1.30 (1) Val 1.16 (1) Ile 1.35 (1) Leu 1.85 (2) Tyr 0.89 (1) Phe 1.01 (1) His 1.10 (1) Lys 1.03 (1) Trp (*) (2) * No data available due to acid hydrolysis. The SS bond position here is Cy of mature endothelin-2.
It was a combination of 1-15 and 3-11 indicated by the number of s. (Ii) Assay The activity of endothelin-2 obtained in (i) above was measured by the method of Reference Example (1). The ED ₅₀ according to assay method (1) (assay with porcine coronary artery) was 8 to 10 × 10 ^-10 mol / l. (Iii) Preparation of injectable preparation 12 μg of endothelin-2 obtained in (i) was dissolved in physiological saline, filtered with a Millipore filter, and then freeze-dried. When producing an intravenous injection at the time of use, the above freeze-dried product was dissolved in physiological saline to make a total volume of 5 ml to give an injection.

[0028]

[Sequence list] [Sequence Listing] <110> Takeda Chemical Industries, Ltd. <120> DNA and use thereof <130> A99190 <160> 8 <210> 1 <211> 21 <212> PRT <213> Human <400> 1 Cys Thr Cys Phe Thr Tyr Lys Asp Lys Glu Cys Val Tyr Tyr Cys His   1 5 10 15 Leu Asp Ile Ile Trp              20 <210> 2 <211> 110 <212> DNA <213> Human <400> 2 GAGGGGGCCC CTGAGCACCA CCGATCCAGG CGCTGCACGT GCTTCACCTA CAAGGACAAG 60 GAGTGTGTCT ACTATTGCCA CCTGGACATC ATTTGGATCA ACACTCCCGA 110 <210> 3 <211> 37 <212> PRT <213> Human <400> 3 Glu Gly Ala Pro Glu His His Arg Ser Arg Arg Cys Thr Cys Phe Thr   1 5 10 15 Tyr Lys Asp Lys Glu Cys Val Tyr Tyr Cys His Leu Asp Ile Ile Trp              20 25 30 Ile Asn Thr Pro Glu          35 <210> 4 <211> 37 <212> PRT <213> Human <400> 4 His Ala Gln Gly Thr His Leu Arg Leu Arg Arg Cys Ser Cys Ser Ser   1 5 10 15 Trp Leu Asp Lys Glu Cys Val Tyr Phe Cys His Leu Asp Ile Ile Trp              20 25 30 Val Asn Thr Pro Glu          35 <210> 5 <211> 21 <212> PRT <213> Human <400> 5 Cys Ser Cys Ser Ser Trp Leu Asp Lys Glu Cys Val Tyr Phe Cys His   1 5 10 15 Leu Asp Ile Ile Trp              20 <210> 6 <211> 110 <212> DNA <213> Human <400> 6 CATGCCCAAG GCACCCACCT TCGGCTTCGC CGTTGCTCCT GCAGCTCCTG GCTCGACAAG 60 GAGTGCGTCT ACTTCTGCCA CTTGGACATC ATCTGGGTGA ACACTCCTGA 110 <210> 7 <211> 14 <212> PRT <213> Human <400> 7 Met Asp Lys Glu Cys Val Tyr Phe Cys His Leu Asp Ile Ile 14 <210> 8 <211> 42 <212> DNA <213> Artificial Sequence <400> 8 ATGGACAAGG AGTGTGTCTA CTTCTGCCAT CTGGACATCA TC 42

[0029]

[Brief description of drawings]

FIG. 1 Endothelin-2 precursor and mature peptide DNA
Containing DNA and D containing endothelin-3 DNA
It is a simple restriction map of NA.

FIG. 2 Endothelin-2 precursor and mature peptide DNA
And the nucleotide sequence of endothelin-3 DNA as a comparative example, and the amino acid sequences deduced therefrom are also shown.

FIG. 3 is an endothelin-presumed from the nucleotide sequence of FIG.
2 Amino acid sequence of mature peptide, endothelin-1,-
3 shows the amino acid sequence of 3.

Front page continuation (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12N 5/10 C12P 21/02 C C12P 21/02 C12N 5/00 A

Claims (7)

[Claims] 1. A DN encoding human endothelin-3.
DNA containing A. 2. The DN according to claim 1, which encodes human endothelin-3 represented by the amino acid sequence of SEQ ID NO: 1.
A. 3. DN encoding human endothelin-3
The DNA according to claim 1, wherein A contains the base sequence of SEQ ID NO: 2 or is represented by a part thereof. 4. A precursor protein of human endothelin-3. 5. The precursor protein according to claim 4, which is represented by the amino acid sequence of SEQ ID NO: 3. 6. A DN encoding human endothelin-3.
A transformant carrying a DNA containing A. 7. A method for producing a mature endothelin-3 protein, which comprises culturing the transformant according to claim 6, allowing mature endothelin-3 to be produced and accumulated in the culture, and collecting the mature endothelin-3 protein.