JP2807471B2 - DNA and its uses - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a DNA containing DNA encoding mouse endothelin (β), which is a mouse vasoconstrictor peptide;
The present invention relates to a precursor protein (also referred to as a precursor polypeptide) and a mature protein (also referred to as a mature polypeptide) of endothelin (β), and a method for producing the precursor protein and a mature protein (endothelin (β)). In this specification,
The precursor protein is a mature peptide (endothelin (β)
And a protein having a part or all of the amino acid sequence encoded by endothelin (β) DNA on its N-terminal side or C-terminal side, or both.

[Conventional technology]

Along with the endothelium-dependent vasodilator response, endothelium-dependent vasoconstrictor responses to various stimuli have been reported. Contraction due to mechanical load such as blood vessel stretching and internal pressure increase,
A contraction by thrombin, a contraction by a decrease in blood oxygen, and a neuropeptide Y [Proceedings of the National Academy of Science of the USA (Proc. Natl. Acad. Sci., U.
SA), 79 , 5485 (1982); and 81,4577 (1984)], for example, to enhance noradrenaline contraction.

American Journal of Physiology (Am
er. J. Physiol.), 132 , 263 (1987) describes an endothelial cell-derived coronary vasoconstrictor (molecular weight 8,500, 3,000, respectively), but the structure is unknown. Also, journals
Of Pharmacology And Experimental Serra Beauty box (J.Pharmacl.Exp.Ther.) 236, 33
9 (1985) also describes a peptide-like substance derived from endothelial cells, but the structure is unknown.

On the other hand, Vasopressin is known as a peptide having a vasoconstrictive action and its amino acid sequence is also clarified, but there is no report that vasopressin was obtained from mammalian or bird vascular endothelial cells as an origin. In addition, it has been reported that angiotensin having vasoconstriction can be obtained from bovine aortic endothelial cells [Circulation Research (Ci.
rculation Research), 60 , 422 (1987)], but angiotensin is a peptide having a molecular weight of about 1,000.

Some of the present inventors have previously succeeded in isolating porcine endothelin from porcine aortic endothelial cells as a peptide having a similar vasoconstrictive action (Japanese Patent Application No. 62-255381).
And some of the present inventors have also succeeded in isolating human endothelin and cloning complementary DNA (Japanese Patent Application No.
2-313155 and 63-148158).

The present applicant has also filed an application for isolation of rat endothelin and cloning of complementary DNA (Japanese Patent Application No. 63-174935, filed on July 29, 1988).

In addition, endothelin has a molecular weight of 2500 ± 300,
It is a peptide consisting of one and four Cs in the amino acid
ys has a structure in which two sets of SS bonds are formed.

[Problems to be solved by the invention]

As described above, homologous endothelin has been found in various animals, but a novel homologous gene from the same animal species has not been found. Therefore, further searching for novel homologous endothelins, deepening research on the structure and activity of those endothelins, and examining their usefulness,
The current problem is to clone the novel peptide by genetic recombination technology and to open up a way of mass production.

[Means for solving the problem]

The inventors of the present invention will have great effects on future research and treatment if a novel homologous gene of endothelin having the above-mentioned vasoconstriction action can be collected from mice and produced by genetic recombination technology. As a result of repeated studies, the inventors have obtained the following findings and arrived at the present invention.

That is, the present inventors succeeded in cloning a mouse endothelin-encoding DNA for the first time from a mouse liver-derived DNA library using a DNA encoding a human endothelin cDNA that had been previously filed as a probe. did. As a result, it was revealed that there are two types, a type (α type) conventionally identified in humans and pigs, and a novel homologous type gene (β type) having a different amino acid sequence (formula III). And IV). As a result, they succeeded in cultivating a way to produce them in large quantities by genetic recombination technology.

The present invention relates to (1) a DNA containing a DNA encoding mouse endothelin β-form (2) a DNA containing a mouse endothelin polypeptide (β) (3) (1) a DNA containing a DNA encoding mouse endothelin The present invention relates to (4) a method for producing mature mouse endothelin, which includes culturing the transformant (3), accumulating and accumulating protein in the culture, and collecting the transformant.

Mouse mature endothelin α, 21 peptides, corresponding to mature endothelin from pigs (endothelin α)
Is represented by [Formula III '], The mouse mature endothelin β of the present invention is represented by [formula IV ′].

The numbers of the amino acids in the above formula are numbered from the first Cys in the amino acid sequence of mouse mature endothelin, and are different from the numbers of the mouse precursor endothelin.

In the above formula, the underlined amino acid is human,
It is different from butane's endothelin.

The position where the SS bond formed by the four Cys in the above formula spans is represented by the number of Cys of the mouse mature endothelin, a combination of 1-15,3-11 and a combination of 3-15,1-11 However, the combination of 1-15 and 3-11 is large, and the vasoconstriction activity is also large.

For DNA sequence, mouse endothelin α,
The β-encoding DNAs are of two types, each containing the base sequence of formula (I) or (II), which are significantly different from those of the respective endothelins of pig, human and rat. . The present invention also relates to a DNA of formula (II) encoding a novel amino acid sequence of mouse endothelin β.

The portion relating to mature mouse endothelin α and β (corresponding to No. 5-25 of Formulas III and IV) was
Unlike the DNA of human / rat endothelin, the DNA of the present invention is novel.

The DNA encoding the mature mouse endothelin peptide (endothelin) of the present invention contains a nucleotide sequence encoding the amino acid sequence (5 to 25 of IV) of the mature mouse endothelin (β) peptide (endothelin). Any DNA may be used as long as it is a DNA containing the nucleotide sequence of formula (II) or a partial DNA thereof.
It is preferred that

The nucleotide sequences of the formulas (I) and (II) are the mouse endothelin DNA sequences obtained in the present invention, and as an example of the nucleotide sequence encoding the mouse mature endothelin amino acid of the formula (III ′) or (IV ′) Is the formula (I) or (I
No. 13-75 of I).

In the method of the present invention, the mouse endothelin has a nucleotide sequence encoding mature mouse endothelin (β)
An expression vector containing DNA can be obtained, for example, by using (i) mouse messenger RNA (mRN
A) and (ii) a single-stranded complementary DNA (cDNA) from the mRNA
Then synthesizing double-stranded DNA, (iii) incorporating the complementary DNA into a phage or plasmid, (iv) transforming a host with the resulting recombinant phage or plasmid,
(V) After culturing the obtained transformant, a suitable method is used from the transformant, for example, hybridization with a DNA probe encoding a part of mouse endothelin (β), or anti-mouse endothelin (β) A phage or plasmid containing the target DNA is isolated by an immunoassay using an antibody, and (vi) the cloned DNA of interest is cut out from the recombinant DNA, and (vi)
i) the cloned DNA or a part thereof is ligated downstream of a promoter in an expression vector.

MRNA encoding mouse endothelin can be obtained from various endothelin-producing cells, such as mouse aortic endothelial cells.

As a method for preparing RNA from mouse endothelin-producing cells, the guanidine thiocyanate method [(J.
J. Chirgwin et al., Bio-chemistry, 18 , 5294 (1979)].

Using the mRNA thus obtained as a template and reverse transcriptase, for example, the method of H. Okayama et al. [Molecular and Cellular Biology (Molecular
and Cellular Biology) 2, 161 ( 1982) and ibid 3, 280
(1983)], and the obtained cDNA is integrated into a plasmid.

Examples of plasmids for incorporating cDNA include E. coli-derived pBR322 [gene, 2, 95 (1977)], pBR32
5 [Gene, 4, 121 (1978)], pUC12 [Gene, 19 ,, 259
(1982)], pUC13 [Gene, 19, 259 (1982)], pUB110 derived from Bacillus subtilis [Biochemical and Biophysical Research Communication (Biochemical and Biophyl).
sical Research Communication), 112, 678 (1983)]
And the like. However, any other substances can be used as long as they are replicated and propagated in the host. As a phage vector incorporating a cDNA, for example, λgt11 [Young and Davis (Young,
R., and Davis, R.,) Proceedings of the National Academy of Sciences of the
USA (Proc. Natl. Acad. Sci., USA), 8
0 , 1194 (1983)], etc., but any other one can be used as long as it can be propagated in the host.

Examples of the method for incorporation into a plasmid include, for example, T. Maniatis, et al., Molecular Cloning, Cold Spring Harbor Laboratory.
-Boratory), page 239 (1982). As a method for incorporating cDNA into a phage vector, for example, the method of Hyunh. TV et al. [DNA Cloning, A Practical Approac
h) 1 , 49 (1985)].

The plasmid thus obtained can be used in a suitable host such as a bacterium belonging to the genus Escherichia or Bacillus (B.
acillus).

Examples of the genus Escherichia include Escherichia
Escherichia coli K12DH1 [Proceding of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA) 60, 160 (1968)], M103
[Nucleic Acids Research, (Nucleic Acid
Research), 9 , 309 (1981)], JA221 [Journal
Journal of Molec
ular Biology)], 120, 517 (1978)], HB101 [Journal of Molecular Biology 41, 459 (196
9)], C600 [Genetics, 39, 440
(1954)].

Examples of the Bacillus genus include Bacillus subtilis MI114 (Gene, 24, 255 (19
83)], 207-21 [Journal of Biochemistry 95, 87 (1984)].

Methods for transforming a host with a plasmid include, for example, T. Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratory.
Harbor Laboratory), page 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, propagated Escherichia coli using an in vitro packaging method.

A mouse cDNA library containing mouse endothelin (α and β) cDNAs can be obtained by the above-mentioned method or the like, but can also be purchased as a commercial product. For example, a mouse cDNA library is available from Clonetech Laboratories. (Clontech Laboratories, Inc., USA).

Mouse endothelin D from mouse DNA library
As a method for cloning NA, for example, a plaque hybridization method using a phage vector λcharon4A and an oligonucleotide chemically synthesized based on the amino acid sequence of human endothelin as a probe [T. Maniatis et al., Molecular Biochem. Cloning (Molecular Cloning) Cold Spring Harbor Laboratory
-Boratory), (1982)].

The mouse endothelin DNA cloned in this way can be used as a plasmid if necessary, for example, pBR322, pUC12,
Mouse endothelin DNA can be obtained by subcloning into pUC13, pUC18, pUC19, pUC118, pUC119 and the like.

The nucleotide sequence of the DNA thus obtained is used, for example, by the Maxam-Gilbert method [Maxam, A. et al.
M. and Gilbert, w., Proceedings of the National Academy of Sciences of the
USA (Proc. Natl. Acad. Sci., USA), 7
4, 560 (1977)] or the dideoxy method [Messing, J.
Et al., Nucleic Acids Research
s Research) 9, 309 (1981)] and compared with known amino acid sequences to determine mouse endothelin (β)
Check for the presence of DNA.

Thus, DNA encoding mouse endothelin (mouse endothelin DNA) (formula II) is obtained.

Mouse endothelin (α) obtained in Example 1 described later
FIG. 1 shows a restriction enzyme fragment map of DNA containing DNAs encoding β and β). The nucleotide sequence of DNA determined by the dideoxy method and the amino acid sequence determined from
Shown in the figure.

The DNA encoding mouse endothelin cloned as described above can be used as it is depending on the purpose, or digested with a restriction enzyme if desired.

A region to be expressed is excised from the cloned DNA and ligated downstream of a promoter in a vehicle (vector) suitable for expression to obtain an expression type vector.

The DNA has ATG at its 5 'end as a translation initiation codon and TAA, TG at its 3' end as a translation stop codon.
It may have A or TAG. These translation initiation codon and translation termination codon can also be added using an appropriate synthetic DNA adapter. To express the DNA, a promoter is connected upstream thereof.

Examples of the vector include the above-mentioned Escherichia coli-derived plasmids (eg, pBR322, pBR325, pUC12, pUC13), Bacillus subtilis-derived plasmids (eg, pUB110, pTP5, pC194), yeast-derived plasmids (eg, pSH19, pSH15), or λ phage. And animal viruses such as vaccinia virus and retroviruses.

The promoter used in the present invention may be any promoter as long as it is appropriate for the host used for gene expression.

The trp promoter, lac promoter, recA promoter, λPL promoter, lpp promoter and the like are used when the host used for transformation is Escherichia, and the SPO1 promoter, SPO2 promoter and penP promoter are used when the host is Bacillus. When the host is yeast, for example, the PHO5 promoter, PGK promoter, GAP promoter, ADH promoter and the like are preferable. Especially it is preferred host promoters in Escherichia is trp promoter or .lambda.P _L promoter.

When the host is an animal cell, an SV40-derived promoter, a retrovirus promoter, a metallothionein promoter, a heat shock promoter, or the like can be used.

 The use of enhancers for expression is also effective.

Using the vector containing the DNA encoding the mature peptide of mouse endothelin (endothelin) thus constructed, a transformant is produced.

Examples of the host include Escherichia, Bacillus, yeast, animal cells and the like.

Specific examples of the genus Escherichia and Bacillus include those similar to those described above.

Examples of the yeast include Saccharomyces cerevisiae AH22, AH22R ⁻ , and NA87-1.
1A, DKD-5D and the like.

Examples of animal cells include monkey cells COS-7, Vero, Chinese hamster cells CHO, mouse L cells, human FL cells, and the like.

In order to transform the above Escherichia, for example, proceeding of the National Academy
Science of Proc. Natl. Acad. Sci. USA, 69 , 211
0 (1972) and Gene, 17 , 107 (1982).

To transform Bacillus sp., For example, Molecular and General Genetics (Molecu
lar & General Gentetics), 168 , 111 (1979).

To transform yeast, for example,
Acad. Sci. USA, Proc. Natl. Acad. Sci. USA, 75 1929 (1978).

Transformation of animal cells is performed, for example, according to the method described in Virology 52 , 456 (1973).

Thus, a transformant transformed with the expression vector containing the DNA encoding the mouse endothelin mature peptide [endothelin (β)] is obtained.

When culturing a transformant whose host is a bacterium belonging to the genus Escherichia or Bacillus, a liquid medium is suitable as a medium used for the culturing, and a carbon source necessary for growth of the transformant is used therein. Nitrogen sources, inorganic substances, etc. are contained. Examples of carbon sources include glucose, dextrin, plastic starch, and sucrose. Examples of nitrogen sources include ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extracts, soybean meal, and potato extract. Alternatively, examples of the organic substance and the inorganic substance include calcium chloride, sodium dihydrogen phosphate, and magnesium chloride. In addition, yeast, vitamins, growth promoting factors and the like may be added.

 The pH of the medium is preferably about 5 to 8.

As a medium for culturing Escherichia, for example, M9 medium containing glucose and casamino acid [Mill (Mill
er), Journal of Experimens in Molecular Genetics (Journal of Experimen)
ts in Molecular Genetics), 431-433, Cold Spring Ha
rbor Laboratory, New York 1972]. If necessary, an agent such as 3β-indolyl acrylic acid can be added in order to make the promoter work efficiently.

When the host is Escherichia, the culture is usually about 15 to 43.
C. for about 3 to 24 hours, and if necessary, aeration and stirring can be added.

When the host is a bacterium belonging to the genus Bacillus, the cultivation is usually carried out at about 30 to 40 ° C. for about 6 to 24 hours, and if necessary, aeration and stirring can be applied.

When culturing a transformant whose host is yeast, for example, Burkholder's minimal medium [Bostian, KL et al., “Brothing of the National Academy of Sciences (Proc. .Ac
ad.Sci.USA) 77 , 4505 (1980)]. Medium p
Preferably, H is adjusted to about 5-8. Culture is usually about 20
C. to 35.degree. C. for about 24 to 72 hours, with aeration and agitation as needed.

When culturing a transformant in which the host is an animal cell, the medium may be, for example, MEM containing about 5 to 20% of fetal bovine blood.
Medium [Science 122 , 501 (1952)], DEME
Medium [Viroroji (Virology), 8 396 (1959 ) ], RP
MI1640 medium [The Journal of the American Medical Association (The Jounal of the Americ
an Medical Association) 199, 519 ( 1967) ], 199 medium [Proceedings Managing Of The Society FOR
The Biological Medicine (Pro-ceeding of th
e Society for Biological Medicine) 73 , 1 (1950)]
And the like. Preferably, the pH is between about 6 and 8.
The cultivation is usually performed at about 30 to 40 ° C. for about 15 to 60 hours, and if necessary, aeration or stirring is added.

Separation and purification of the mature mouse endothelin (β) peptide (endothelin) from the above culture can be performed, for example, by the following method.

When extracting mature mouse endothelin peptide (β) (endothelin) from cultured cells or cells, the cells or cells are collected by a known method after culture, suspended in an appropriate buffer, and sonicated. After disrupting cells or cells by lysozyme and / or freeze-thawing, a method of obtaining a crude extract of a mature rat endothelin peptide by centrifugation or filtration may be used as appropriate. The buffer may contain a protein denaturing agent such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100.

If the mouse endothelin precursor protein or mature peptide (β) is secreted into the culture,
The supernatant is separated from the cells or cells by a method known per se, and the supernatant is collected. The culture supernatant thus obtained,
Alternatively, the rat endothelin precursor protein or mature peptide contained in the extract can be obtained by appropriately combining known separation and purification methods. As these known separation and purification methods, methods utilizing solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis, mainly molecular weight such as Using differences in charge, methods using charge differences such as ion exchange chromatography, methods using specific affinity such as affinity chromatography, and using differences in hydrophobicity such as reversed-phase high-performance liquid chromatography And methods utilizing the difference in isoelectric points, such as isoelectric focusing.

The thus produced mouse endothelin (α and β) precursor protein and mature peptide can be measured by an enzyme immunoassay using a specific antibody or the like.
When the product has vasoconstriction activity, the activity can be measured using the activity as an index.

[Action / Effect]

In cells and cells infected or transformed with the DNA of the present invention, a large amount of mouse endothelin mature peptide (β)
Can be produced, and the production of mature mouse endothelin peptide can be advantageously induced.

The mouse endothelin mature peptide produced here not only provides a clue to the analysis of the mechanism of vasoconstriction in living organisms, especially humans, and elucidation of vasoconstrictor antagonists, but also as a therapeutic agent for hypotension and a local vasoconstrictor. Can be used.

The DNA encoding mouse endothelin (β)
Cloning, production of a mouse endothelin mature peptide expression vector, production of a transformant using them, and mouse endothelin (β) using the transformant
The production of the mature peptide and its usefulness have been described in detail.

In the specification and the drawings of the present invention, when bases, amino acids and the like are indicated by abbreviations, IUPAC-IUB Commision on Bi
This is based on an abbreviation by ochemical Nomenclature or an abbreviation commonly used in the art, and examples thereof are described below. When amino acids may have optical isomers,
Unless otherwise specified, L-form is shown.

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: Aspartic 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: P Phosphorus Asn or N: Asparagine Gln or Q: Glutamine In the mature mouse endothelin peptide of the present invention, a part of its amino acid sequence may be modified (addition, removal, substitution with another amino acid, etc.) .

Examples 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 obtained in Example 1 was obtained from Escherichia
coli HB101 / pmET5 and Escherichia coli HB101 / pmE
T10 has been assigned to the Microorganisms and Industrial Technology Research Institute (FRI) of the Ministry of International Trade and Industry of the Ministry of International Trade and Industry on September 6,
Deposited and stored as -2032 and FERM BP-2033.

Reference example (1) Assay method for vascular smooth muscle contraction effect A swine right coronary artery spiral specimen (0.5 × 20 mm) from which endothelium was removed by abrasion with a syringe needle was mixed with a mixed gas of carbon dioxide and oxygen (5:95, V / V). Suspension in Krebs-Ringer solution (3 ml) at 37 ° C. saturated with). Basal tensio
After setting n) to 2 g, measure the isometric tension with a tension transducer.

Example 1 Isolation of mouse endothelin DNA and determination of its nucleotide sequence A mouse DNA library (derived from mouse liver) was used in E. coli LE392.
DNA partially digested with Sau3A and bound to Caron 28)
Was infected and plated to reveal phage plaques. Benton, W., Davis,
R.) [Science 196 , 180-182 (197
Utsushitori part of plaque DNA to a nitrocellulose membrane according to 7)], ³² was subjected to DNA probe and plaque hybridization of the labeled preceding by P. Hybridization was performed at 65 ° C. in the absence of formamide. 23 clones positive for hybridization were isolated and one of them was EcoR I Xba I DN of mET5.
The A part and the BamHI DNA part of mET10 were cut out and recloned into the corresponding sites of the plasmid pUC18.
T5 and pmET10 were prepared. Escherichia coli HB101
And transformant Escherichia coli HB101 / pmE
T5 and E. coli HB101 / pmET10 were obtained. The DNA portions contained in this plasmid are 1.4 Kbp and 1.8 Kpb, and a simple restriction map of the DNA containing this DNA fragment is shown in FIG. Areas in the figure indicate the following.

The method of the nucleotide sequence of the cDNA portion Sanger (Sanger) [Pro sheathing of The National Academy of Sciences (Proc.Nat.Acad.Sci.USA) 74, 5463
-5467 (1977)]. This base sequence and mouse endoseri (α and β) deduced from it
The amino acid sequence of is shown in FIG.

The region surrounded by is the mature peptide of mouse endothelin (α and β).

Example 2 i) Mouse endothelin β synthesis Using a commercially available Boc-Trp (CHO) -PAM resin, 0.7 g (0.5 mmole) (manufactured by Applied Biosystems), a peptide synthesizer (manufactured by Applied Biosystems) -Using the model 430A), it was synthesized by a usual method. In the condensation method, the Boc group on the resin is treated with 50% trifluoroacetic acid in methylene chloride to release a terminal amino group, and the free amino group is added to Boc-Ile, Boc-Asp (OBzl), Boc-Leu, Boc−
His (Tos), Boc-Cys (Acm), Boc-Tyr (Br-z), Boc
-Val, Boc-Phe, Boc-Glu (OBzl), Boc-Lys (Cl-
Z) The reaction of condensing Boc-Trp (CHO) and Boc-Ser (Bzl) from the C-terminal side according to the amino acid sequence of mouse endothelin in the presence of DCC was repeated.

The protected mouse endothelin resin thus obtained 2.
890 mg of 53 g was expanded with 1 ml of anisole and 1 ml of 1,2-ethanedithiol, treated with 10 ml of hydrogen fluoride at 0 ° C. for 60 minutes, and then 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 removed by filtration. After trifluoroacetic acid was distilled off under reduced pressure, the residue was dissolved in 50% aqueous acetic acid, applied to a dextran gel (Sephadex G-50) column (2 × 90 cm), and the main fraction eluted with the same solvent was collected and freeze-dried. Thus, 180 mg of a white powder was obtained. 31 mg of this was dissolved in 4 ml of 50% aqueous acetic acid, 19 mg of mercuric trifluoroacetate was added, and the mixture was stirred at room temperature for 16 hours.
, And the mixture was diluted with hydrogen sulfide gas. 5% -NH
After adjusting the pH to 8 by adding ₄ OH, the mixture was subjected to air oxidation for 6 hours, acetic acid was added to adjust the pH to 3, and then freeze-dried. This is 5
The mixture was applied to a Sephadex G-50 column (2 × 90 cm) packed with 0% acetic acid, and the main fractions were collected. (Acetonitrile containing fluoroacetic acid was eluted with a linear gradient) to obtain 1.0 mg of the desired product.

Synthetic mouse endothelin β was eluted by HPLC at 23.84 minutes compared to 23.33 minutes for endothelin α.

Column conditions YMC-ODS (4.6 x 150 mm) Eluent: Solution A (0.1% -trifluoroacetic acid aqueous solution) Using solution B (0.1% -trifluoroacetic acid-containing acetonitrile), linear concentration gradient elution from solution A to solution B ( Flow rate: 1.0 ml / min Amino acid analysis value: Asp3.33 (3), Ser2.00 (2), Glu1.32 (1), Cys3.5
2 (4), Val0.92 (1), Ile1.73 (2), Leu2.19 (2),
Try0.93 (1), Phe1.0 (1), Lys1.13 (1), His0.99
(1), Trp1.88 (2) Here, the SS binding position is mouse mature endothelin β
And the combination of 1-15 and 3-11.

[Brief description of the drawings]

FIG. 1 is a simple restriction map of DNA including mouse endothelin (α and β) precursors and mature peptide DNA. FIG. 2 shows the nucleotide sequences of the mouse endothelin (α and β) precursor and the mature peptide DNA, and the amino acid sequences of a part of the mouse endothelin (α and β) precursor and the mature peptide deduced therefrom.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12N 5/10 C12P 21/02 C C12P 21/02 C12N 5/00 B // A61K 38/00 AER A61K 37/02 AER (72 Inventor Kaname Saida 1-3-1 Higashi, Tsukuba City, Ibaraki Pref.Institute of Microbiology, National Institute of Advanced Industrial Science and Technology (72) Inventor Masahiko Fujino 2-10-7 Hibarigaoka, Takarazuka-shi, Hyogo Examiner Mayumi Saito (56) Reference Document JP-A-2-72877 (JP, A) FEBS LETTERS, Vol. 2, (April 1988), p. 440-444 Nature, Vol. 332 (31 March 1988); 411-415 (58) Fields investigated (Int.Cl. ⁶ , DB name) C12N 15/00-15/90 C07K 1/00-19/00 C12N 1/00-5/28 C12P 21/00-21 / 08 BIOSIS (DIALOG) WPI (DIALOG) DDBJ / GenBank / EMBL (Genseq)

Claims (7)

(57) [Claims] 1. The amino acid sequence Cys Ser Cys Asn Ser Trp Le
u Asp Lys Glu Cys Val Tyr Phe Cys His Leu Asp Ile
Contains DNA encoding the peptide represented by Ile Trp
DNA. 2. The DNA encoding the peptide has a base sequence. The DNA according to claim 1, which is represented by: 3. The DNA encoding the peptide has the following nucleotide sequence: The DNA according to claim 1, which is represented by the following formula: 4. An amino acid sequence A peptide represented by 5. An amino acid sequence The precursor of the peptide according to claim 4, which is represented by the following formula: 6. A transformant carrying the DNA according to claim 1. 7. The transformant according to claim 6, which is cultured, and the amino acid sequence Cys Ser Cys Asn Ser Trp Leu Asp is added to the culture.
Lys Glu Cys Val Tyr Phe Cys His Leu Asp Ile Ile Tr
A method for producing a peptide represented by p, comprising producing and accumulating a peptide represented by p, and collecting the peptide.