WO2002057443A1 - Methode de production de ligand zaq - Google Patents

Methode de production de ligand zaq Download PDF

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Publication number
WO2002057443A1
WO2002057443A1 PCT/JP2002/000378 JP0200378W WO02057443A1 WO 2002057443 A1 WO2002057443 A1 WO 2002057443A1 JP 0200378 W JP0200378 W JP 0200378W WO 02057443 A1 WO02057443 A1 WO 02057443A1
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Prior art keywords
leu
cys
arg
amino acid
gly
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PCT/JP2002/000378
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English (en)
Japanese (ja)
Inventor
Takao Yamada
Masato Suenaga
Osamu Nishimura
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Takeda Chemical Industries, Ltd.
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Publication of WO2002057443A1 publication Critical patent/WO2002057443A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • the present invention has an ability to bind to a protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 or a salt thereof, or to activate the protein or a salt thereof. Or a salt thereof (hereinafter sometimes simply referred to as “ZAQ ligand”.
  • the “peptide capable of binding to the salt or activating the protein or the salt thereof” is sometimes simply referred to as “ZAQ ligand.”) Is expressed in prokaryotic cells using genetic engineering technology.
  • the present invention relates to a method for producing a pharmacologically active ZAQ ligand by performing a refolding operation on the extracted ZAQ ligand. Background art
  • Proteins or peptides containing a large number of cysteine residues in the molecule and having disulfide bonds can be converted into Chinese hamster cells (eg, CHO), monkey cells (eg, COS-7), etc. by known genetic engineering techniques. Expression of a nuclear cell as a host yields a pharmacologically active protein or peptide.
  • the method of expressing the protein or peptide in eukaryotic cells generally has a lower expression level of the protein or peptide of interest than the method of expressing the protein or peptide in prokaryotic cells, and furthermore, the method for eukaryotic cells.
  • the medium is expensive and therefore not suitable for industrial scale production of the protein or peptide.
  • the ZAQ ligand has ligand activity for the orphan receptor ZAQ receptor (a protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 or a salt thereof). Digestion with peptides It is a useful peptide that has preventive and therapeutic effects on organ diseases (eg, enteritis, diarrhea, constipation, malabsorption syndrome, etc.).
  • the peptide can be produced by using known eukaryotic cells by a known genetic engineering technique.
  • pharmacologically active ZAQ ligands can be obtained by expressing ZAQ ligands in eukaryotic cells such as Chinese hamster cells (eg, CHO) and monkey cells (eg, COS-7).
  • eukaryotic cells such as Chinese hamster cells (eg, CHO) and monkey cells (eg, COS-7).
  • Chinese hamster cells eg, CHO
  • monkey cells eg, COS-7
  • the medium is expensive, and the expression level of the protein is low, so that production on an industrial scale is not suitable.
  • the present invention relates to a method for producing an active ZAQ ligand, comprising refolding a ZAQ ligand expressed in a prokaryotic host by genetic engineering in a redox buffer.
  • An amino acid identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 A peptide or a salt thereof capable of binding to, or activating a protein or a salt thereof containing, a nucleic acid sequence or a salt thereof is genetically engineered and expressed in a prokaryotic cell host; Binds to a protein having an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 or a salt thereof, or the protein or a salt thereof, wherein For producing an active peptide or a salt thereof having the ability to activate
  • (6) binds to, or binds to, a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a salt thereof;
  • the peptide capable of activating the salt thereof or the salt thereof is a peptide containing the amino acid sequence represented by SEQ ID NO: 30 or a salt thereof,
  • a peptide containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 21 or SEQ ID NO: 30 or a salt thereof is expressed in a prokaryotic host by genetic engineering. Refolding in a redox buffer, a method for producing the active form of the peptide or a salt thereof,
  • a peptide containing an amino acid sequence represented by SEQ ID NO: 21, SEQ ID NO: 19 or SEQ ID NO: 30 or a salt thereof is genetically engineered in a prokaryotic host, and is expressed in a redox buffer.
  • a peptide having the ability to activate a salt thereof or a salt thereof is genetically expressed in a prokaryotic host, and the cells are solubilized and then refolded in a redox buffer.
  • a peptide containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 21 or SEQ ID NO: 30 or a salt thereof is genetically expressed in a prokaryotic host, Is solubilized, and then refolded in a redox buffer, according to the above (7),
  • the salt is expressed in a prokaryotic host by genetic engineering, the cells are solubilized with a denaturing agent, and then a redox buffer (pH 7 to 9) further containing an amino acid having no mercapto group is used as a denaturing agent. Is diluted to an inactive concentration, the method according to the above (1),
  • a peptide containing the same or substantially the same amino acid sequence as the amino acid sequence represented by the sequence number: 21 or SEQ ID NO: 30 or a salt thereof is genetically expressed in a prokaryotic host.
  • the cells are solubilized with a denaturing agent, and the denaturing agent is diluted to an inactive concentration with a redox buffer of ⁇ 7 to 9 further containing an amino acid having no mercapto group.
  • a redox buffer of ⁇ 7 to 9 further containing an amino acid having no mercapto group.
  • a prokaryotic host which has genetically expressed a peptide or a salt thereof containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 21 or SEQ ID NO: 30,
  • the present invention relates to a method for producing an active peptide or a salt thereof, which is reacted with a buffer solution containing an amino acid having no mercapto group, reduced daltathione and oxidized daltathione, which is soluble with a denaturing agent.
  • the method of the present invention is a method for producing an active form of a ZAQ ligand, comprising expressing a ZAQ ligand by genetic engineering in a prokaryotic host and refolding it in a redox buffer.
  • the ZAQ ligand is expressed in a prokaryotic host by genetic engineering, and the ZAQ ligand inactivated by solubilizing the cell is refolded in a redox buffer to form an active form of the ZAQ ligand.
  • Produce ZAQ ligand comprising expressing a ZAQ ligand by genetic engineering in a prokaryotic host and refolding it in a redox buffer.
  • the ZAQ ligand used in the present invention includes a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a salt thereof (hereinafter, abbreviated as “ZAQ receptor”). Or a salt thereof, or a peptide or a salt thereof capable of activating the ZAQ receptor.
  • the ZAQ ligand comprises (1) an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 21, and having an ability to bind to the ZAQ receptor or to activate the ZAQ receptor.
  • the ability of a ZAQ ligand to bind to or activate a ZAQ receptor can be determined by a method for measuring a known ligand 'receptor binding activity or a method analogous thereto, or a known cell stimulating activity (intracellular Ca ion concentration). Change (eg, the method described in Example 1-6 below), cAMP synthesis inhibitory activity, etc.) or a method analogous thereto.
  • the ZAQ ligand produced by the method of the present invention is a peptide capable of binding to the ZAQ receptor or a peptide capable of activating the ZAQ receptor (hereinafter abbreviated as “active peptide”). Can be) or its salts. Specifically, (1) a peptide containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by the active form SEQ ID NO: 21 or a salt thereof, (2) an active form SEQ ID NO: 30 Or a salt thereof containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by
  • the ZAQ receptor (G protein-coupled receptor protein) is a receptor protein having an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 (WO 01/16309).
  • ZAQ ligands and ZAQ receptors include, for example, human and non-human mammals (eg, guinea pigs, rats, mice, rabbits, pigs, sheep, sheep, monkeys, monkeys, etc.), and all cells (eg, spleen cells, nerve cells, glial cells) 3 cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, endothelial cells, fibroblasts, fiber cells, muscle cells, fat cells, immune cells (eg, macrophages, T cells, B Cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes, bone cells, osteoblasts, osteoclasts, mammary cells, Hepatocytes or stromal cells, or precursors of these cells, stem cells or cancer cells, etc., or blood cells (eg, MEL, Ml, CTLL
  • Bone marrow adrenal gland, skin, muscle, lung, digestive tract (eg, large intestine, small intestine), blood vessels, heart, thymus, spleen, submandibular gland, peripheral blood, peripheral blood cells, prostate, testicle, testis, ovary, placenta, uterus It may be a peptide / protein derived from bones, joints, skeletal muscle, etc. (particularly, brain or various parts of the brain), or may be a synthetic peptide'synthetic protein.
  • amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 21 includes, for example, about 60 ° / 0 or more (preferably about 70% or more, Further preferred are amino acid sequences having a homology of about 80% or more, more preferably about 85% or more, particularly preferably about 90% or more, and most preferably about 95% or more.
  • Examples of the peptide having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 21 include, for example, a peptide having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 21; Peptides having substantially the same properties as the peptide containing the amino acid sequence represented by 21 are preferred.
  • the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 19 is, for example, about 60% or more (preferably An amino acid sequence having a homology of about 70% or more, more preferably about 80% or more, more preferably about 85% or more, particularly preferably about 90% or more, and most preferably about 95% or more. .
  • Examples of the peptide having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 19 include, for example, a peptide having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 19; Peptides having substantially the same properties as peptides containing the amino acid sequence represented by 19 are preferred.
  • the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 30 is, for example, about 60 ° / 0 or more (preferably, the amino acid sequence represented by SEQ ID NO: 30).
  • Examples of the peptide having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 30 include, for example, a peptide having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 30; Peptides having substantially the same properties as peptides containing the amino acid sequence represented by No. 30 are preferred.
  • ZAQ ligand examples include a peptide containing the amino acid sequence represented by SEQ ID NO: 19, a peptide containing the amino acid sequence represented by SEQ ID NO: 21 and SEQ ID NO: 30 And peptides containing an amino acid sequence.
  • Substantially the same activity includes, for example, a binding activity to a ZAQ receptor, a signal transduction activity via a ZAQ receptor, and the like. Substantially the same indicates that their activities are the same in nature. Therefore, it is preferable that the activities such as the binding activity to the ZAQ receptor and the signal transduction via the ZAQ receptor are equivalent (for example, about 0.5 to 2 times). Quantitative factors such as molecular weight may be different.
  • the activity can be measured by a known method or a method analogous thereto (specifically, a method described in Example 11 to be described later or a method analogous thereto).
  • ZAQ ligand examples include: (1) one or two or more (preferably about 1 to 30 and more preferably 1 to 20) in the amino acid sequence represented by SEQ ID NO: 19 or SEQ ID NO: 21; Amino acid sequence in which amino acid has been deleted, (2) 1 or 2 or more (preferably, about 1 to 40 amino acids) in the amino acid sequence represented by SEQ ID NO: 19 or SEQ ID NO: 21; More preferably about 1 to 30 amino acids, particularly preferably about 1 to 20 amino acids), and 3 an amino acid sequence represented by SEQ ID NO: 19 or SEQ ID NO: 21.
  • amino acid sequence in which about 30 (more preferably about 1 to 20) amino acids have been substituted with another amino acid, or a peptide containing an amino acid sequence obtained by combining them, or the like can also be used.
  • amino acid sequence in which one or more (preferably about 1 to 30 and more preferably about 1 to 20) amino acids in the amino acid sequence represented by SEQ ID NO: 30 have been deleted (2) one or more amino acid sequences represented by SEQ ID NO: 30 (preferably about 1 to 40, more preferably about 1 to 30, particularly preferably 1 to 20) Amino acid sequence to which an amino acid sequence of about 30 or more (preferably about 1 to 30, more preferably 1 to 20) in the amino acid sequence represented by SEQ ID NO: 30 (About one amino acid) are substituted with another amino acid, or a peptide containing an amino acid sequence obtained by combining them is also used.
  • amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 encoding the ZAQ receptor include, for example, about 90% or more, preferably about 95%, of the amino acid sequence represented by SEQ ID NO: 1. Above, more preferably, an amino acid sequence having about 98% or more homology.
  • Examples of the protein having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 include, for example, an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1; A protein having substantially the same properties as the protein having the amino acid sequence represented by No. 1 is preferred.
  • Examples of the protein (ZAQ receptor) having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 of the present invention include, for example, the same or substantially the same as the amino acid sequence represented by SEQ ID NO: 1 Preferred are proteins which contain the same amino acid sequence and have substantially the same activity as the amino acid sequence represented by SEQ ID NO: 1.
  • substantially the same activity examples include a ligand binding activity and a signal transduction activity. Substantially the same indicates that their activities are the same in nature. Therefore, it is preferable that the activities such as the ligand binding activity and the signal transduction activity are equivalent (eg, about 0.5 to 2 times), but the quantitative factors such as the degree of these activities and the molecular weight of the protein are different. You may.
  • the activities such as ligand binding activity and signal information transduction can be measured according to known methods.
  • the ZAQ receptor includes: (1) one or more or more amino acids in the amino acid sequence represented by SEQ ID NO: 1 (preferably about 1 to 30, more preferably about 1 to 10, more preferably several ( Amino acid sequence in which 1 or 2 amino acids have been deleted, 2) 1 or 2 or more amino acids in the amino acid sequence represented by SEQ ID NO: 1 (preferably, about 1 to 30, more preferably 1 to 1) An amino acid sequence obtained by adding about 0 amino acids, more preferably several (1 or 2) amino acids; and 3 one or more (preferably, one or more) amino acids in the amino acid sequence represented by SEQ ID NO: 1. About 30 amino acids, more preferably about 1 to 10 amino acids, still more preferably several (one or two) amino acids substituted with another amino acid, or amino acids obtained by combining them.
  • peptides and proteins have N-terminus (amino terminus) at the left end and C-terminus (carboxyl terminus) at the right end according to the convention of peptide notation.
  • the peptides and proteins of the present invention including ZAQ ligands and ZAQ receptors have carboxyl groups (one COOH), carboxylate (—COO-1), amides (one CONH 2 ) or esters (one COOR ).
  • R in the ester e.g., methyl, Echiru, n- propyl, alkyl groups such as isopropyl, n- butyl, Shikurobe pentyl, C 3 _ 8 cycloalkyl group such as cyclohexyl, for example, phenyl , C such as a one-naphthyl 6 - 12 Ariru group, e.g., benzyl, full Eniru d_ 2 alkyl or ⁇ - naphthylmethyl etc. ⁇ - naphthyl one C ⁇ 2 Flip such as an alkyl group 7 _ 14 Ararukiru group such as phenethyl A piva mouth yloxymethyl group commonly used as an oral ester is used.
  • the peptides and proteins in the present specification have a carboxyl group (or carboxylate) other than the C-terminus, those in which the carboxyl group is amidated or esterified are also included in the peptides and proteins in the present specification. It is.
  • the ester in this case, for example, the above-mentioned C-terminal ester and the like are used.
  • the peptides and proteins in this specification the peptides • proteins described above, Amino group of the N-terminal amino acid protecting groups (e.g., formyl group, etc.
  • Ashiru groups such as C 2 _ 6 Arukanoiru group such Asechiru group
  • coercive Mamorumoto e.g., formyl group, C 2 such Asechiru group - C, such as 6 Arukanoiru groups - those are protected by like 6 Ashiru group
  • complex peptides such as so-called glycopeptides and glycoproteins to which sugar chains are bonded, and complex proteins.
  • ZAQ ligand examples include, for example, a peptide derived from human (more preferably from human brain) containing the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 21 or SEQ ID NO: 30 Is raised.
  • ZAQ receptor examples include, for example, human-derived (more preferably, human brain-derived) protein containing the amino acid sequence represented by SEQ ID NO: 1.
  • a physiologically acceptable acid addition salt is particularly preferable.
  • such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid) Acids, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and salts and hydrates are used.
  • inorganic acids eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid
  • organic acids eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid
  • the prokaryotic cells used in the present invention include Escherichia coli and other Escherichia bacteria, Bacillus subtilis and other Bacillus bacteria, and Serratia marcescens and other Serratia species. Among them, Escherichia coli and the like are preferable. Transformation and culture of these prokaryotic cells can be carried out by a conventional method (such as the method described in JP-A-3-204879) or a method analogous thereto, in addition to the following methods. it can.
  • the DNA encoding the ZAQ ligand may be any DNA as long as it contains the DNA encoding the ZAQ ligand described above. Also, It may be any of nome DNA, genomic DNA library, the above-mentioned cDNA derived from cells and tissues, the above-mentioned cDNA library derived from cells and tissues, and synthetic DNA.
  • the vector used for the library may be any of batteriophage, plasmid, cosmid, phagemid and the like.
  • amplification can be performed directly by reverse transcriptase polymerase chain reaction (hereinafter abbreviated as RT-PCR method) using a preparation of a total RNA or mRNA fraction from the above-mentioned cells.
  • the DNA encoding the ZAQ ligand includes, for example, (1) DNA containing the base sequence represented by SEQ ID NO: 20 or DNA containing the base sequence represented by SEQ ID NO: 20 It contains DNA that hybridizes under high stringent conditions and has substantially the same activity as a peptide containing the amino acid sequence represented by SEQ ID NO: 19 (eg, binding activity to ZAQ receptor, ZAQ receptor (2) DNA containing the nucleotide sequence represented by SEQ ID NO: 22, or DNA containing the nucleotide sequence represented by SEQ ID NO: 22 Which has DNA that hybridizes under high stringent conditions with DNA that binds to the DNA and has substantially the same activity as the peptide containing the amino acid sequence represented by SEQ ID NO: 21 (eg, ZAQ (3) DNA containing the nucleotide sequence represented by SEQ ID NO: 31, or DNA represented by SEQ ID NO: 31 Containing DNA that hybridizes under high stringent conditions with DNA containing the base sequence, and having substantially the same activity as a peptide
  • Examples of the DNA containing the nucleotide sequence represented by SEQ ID NO: 20 include DNA containing the nucleotide sequence represented by SEQ ID NO: 16.
  • Examples of the DNA containing the base sequence represented by SEQ ID NO: 22 include a DNA containing the base sequence represented by SEQ ID NO: 18.
  • Examples of the DNA containing the nucleotide sequence represented by SEQ ID NO: 31 include DNA containing the nucleotide sequence represented by SEQ ID NO: 41.
  • Examples of the DNA that hybridizes with the DNA having the nucleotide sequence represented by SEQ ID NO: 20, SEQ ID NO: 22 or SEQ ID NO: 31 under stringent conditions include, for example, SEQ ID NO: 20, DNA containing a nucleotide sequence having about 60% or more, preferably about 70% or more, more preferably about 80% or more homology with the nucleotide sequence represented by SEQ ID NO: 22 or SEQ ID NO: 31 Are used.
  • DNA that hybridizes under high stringent conditions with DNA containing the nucleotide sequence represented by SEQ ID NO: 22 specifically, DNA containing the nucleotide sequence represented by SEQ ID NO: 38, Or a DNA containing the DNA having the base sequence represented by SEQ ID NO: 38 and a DNA that hybridizes under high stringency conditions, and is substantially the same as the peptide containing the amino acid sequence represented by SEQ ID NO: 21 (Eg, binding activity to ZAQ receptor, signal transduction via ZAQ receptor, etc.), and DNA encoding a peptide.
  • DNA that hybridizes under high stringent conditions with a DNA containing the base sequence represented by SEQ ID NO: 31 specifically, a DNA containing the base sequence represented by SEQ ID NO: 39 Or a DNA comprising the DNA having the base sequence S sequence represented by SEQ ID NO: 39 and DNA hybridizing under high stringency conditions, and a peptide comprising the amino acid sequence represented by SEQ ID NO: 30
  • DNA encoding a peptide having substantially the same activity eg, binding activity to ZAQ receptor, signal transduction via ZAQ receptor, etc.
  • DNA encoding the peptide containing the amino acid sequence represented by SEQ ID NO: 19 a DNA containing the base sequence represented by SEQ ID NO: 20 is mentioned.
  • the DNA encoding the peptide containing the amino acid sequence represented by SEQ ID NO: 21 is a DNA containing the nucleotide sequence represented by the sequence number: 22 or SEQ ID NO: 38;
  • DNA encoding the peptide containing the amino acid sequence represented by SEQ ID NO: 30 includes SEQ ID NO: 31 or SEQ ID NO: 39.
  • the DNA encoding the ZAQ ligand may be (1) chemically synthesized by a known method or a method analogous thereto, or (2) DNA of the DNA encoding the ZAQ ligand.
  • the DNA may be amplified and obtained by polymerase chain reaction (hereinafter abbreviated as PCR) with reference to the sequence and the like.
  • Examples of plasmids for introducing DNA encoding the ZAQ ligand include, for example, PBR 322 derived from Escherichia coli (Gene, Vol. 2, p. 95 (1977)) and pBR 325 [Gene, Vol. 4, p. 121 (1978) )], PUC12 [Gene, 19, 259 (1982)], pUC13 [Gene, 19, 259 (1982)], pUB1 10 derived from Bacillus subtilis [Biochemical biophysical ⁇ Resate Communications (Biochemical ana Biophysical Research Communications), Vol. 112, p. 678 (1983)], etc., as long as they can be replicated and propagated in the host. , Can be used.
  • phage vectors incorporating DNA include: Lgt11 (Young, R. and Davis, R.), Proceedings of the National 'Academy of Ob', Science (Proc. Natl. Acad. Sci., USA), Vol. 80, p. 1194 (1983)], etc. Any substance that can proliferate can be used.
  • Escherichia bacteria examples include Escherichia coli K12DH1 [Proceding 'ob the national' academy ' ob science ( proc . Natl. Acad. Sci. USA) 60 vol. JM103 [Nucleic Acids Research], Vol. 9, 309 (1981)], JA221 [Journal of Molecular Biology], JA221 [Journal of Molecular Biology] ), 120, 517 (1978)], HB 101 (Journal of Molecular Biology 1, 41, 459 (1969)), C600 (Genetics, 39, 440 (1954)], MM 294 [Nature, 217, 111 (1968)], and the like.
  • Bacillus subtilis MI114 Gene, Vol. 24, p. 255 (1983)
  • 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, Co.;, 'Spring', "Cold Spring”. Harbor Laboratory), p. 249 (1982), calcium chloride method or calcium chloride Z rubidium chloride method.
  • a phage 'vector When a phage 'vector is used, it can be introduced into, for example, grown Escherichia coli using an in vitro packaging method.
  • the cloned ZAQ ligand co one de to DNA is plasmid if necessary, for example, p BR 322, pUC 12, pUC 13, pUC 18, UC 1 9 N pUC 1 18, pUC 1 1 9 Can be used after subcloning.
  • the nucleotide sequence of the DNA encoding the ZAQ ligand obtained in this manner was used, for example, by the Maxam-Gilbert method (Maxam, AM and Gilbert, W., Proceedings of the National Institute of Technology). Academy of Sciences of the USA (Proc. Natl. Acad. Sci., USA), 74 560 (1977)] or the dideoxy method (Messing, J. et al., Nucleic Acids Research, Vol. 9, 309 (1981)). The comparison of shows that DNA encoding the ZAQ ligand is present.
  • the DNA encoding the ZAQ ligand cloned as described above can be used as it is or after digestion with a restriction enzyme exonuclease, if desired.
  • a region to be expressed is cut out from the DNA encoding the cloned ZAQ ligand, and ligated downstream of a 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 may have TAA, TGA or TAG as a translation termination codon at its 3' end. These translation initiation codon and translation termination codon can also be added using an appropriate synthetic DNA adapter.
  • a promoter is connected upstream thereof.
  • Examples of the vector include the above-described plasmid derived from Escherichia coli (eg, pBR322, pBR325, pUC12, pUC13), and Bacillus subtilis-derived plasmid (eg, pUB110, TP5, pC194).
  • the promoter used in the present invention may be any promoter as long as it is appropriate for the host used for DNA expression.
  • the T7 promoter, trp promoter, lac promoter, recA promoter, promoter, 1 pp promoter, etc. are used when the host used for the transformation is Escherichia, and SP is used when the host is Bacillus. Ol promoter, SPO2 promoter, pen P promoter and the like are preferable. In particular, it is preferable that the host is a bacterium belonging to the genus Escherichia and the promoter is a T7 promoter, a trp promoter, or a LPL promoter.
  • a prokaryotic cell transformant is produced. Transformation of the above-mentioned Escherichia bacterium can be performed, for example, by the following procedure: Proc. Natl. Acad. Sci. USA, 69, 2110 (1972). And Gene, vol. 17, p. 107 (1982).
  • a prokaryotic cell transformant transformed with the expression vector containing the DNA encoding the ZAQ ligand is obtained.
  • the genus Escherichia is used as a host and the T7 promoter is used as a promoter
  • a ⁇ 7 lysozyme expression plasmid is used in addition to an expression vector containing DNA encoding ZAQ ligand to improve the expression efficiency of the T7 promoter. May be.
  • a liquid medium is suitable as the medium used for the culturing, and a carbon source necessary for the growth of the transformant is contained therein.
  • examples of the organic substance and the inorganic substance include calcium salt, sodium dihydrogen phosphate, magnesium chloride and the like.
  • yeast extract, vitamins and growth promoting factors may be added.
  • the pH of the medium is preferably about 5-8.
  • an M9 medium containing glucose and casamino acid (Miller, Journal 'Ob'Experiment'in'Molecuffe 1 —Journal of Experiments in Molecular Geometry netics), pp. 431-433, Cold Spring Harbor Laboratory, New York 1972], LB medium and the like.
  • a drug such as isopropynole j3-D-thiogalatatopyranoside (IPTG) or 3 / 3-indolyl ataryl acid can be added in order to make the promoter work efficiently.
  • cultivation is usually at about 15 to 43 ° C for about 3 to 24 hours. If necessary, ventilation and agitation can be added.
  • the cultivation is usually carried out at about 30 to 40 ° C for about 6 to 24 hours, and if necessary, aeration and stirring may be added.
  • the ZAQ ligand forms an insoluble component (inclusion body) in the host prokaryotic cells
  • collect cells by centrifugation or other methods, disrupt the cells, and allow the inclusion body to be denatured using a denaturant.
  • the ZAQ ligand can be extracted.
  • Cell framing can be carried out in a conventional manner, for example by sonication. It is preferable to use a suitable buffer (for example, a phosphate buffer or the like) adjusted to a neutral pH value (pH 6.5 to 7.5) as a suspension medium. At this time, EDTA may be added to promote cell disruption.
  • a suitable buffer for example, a phosphate buffer or the like
  • EDTA may be added to promote cell disruption.
  • the insoluble components are separated by centrifugation or filtration by any method.
  • washing with, for example, water or a phosphate buffer is preferable. In some cases, washing with about 4 M urea may be used.
  • a denaturing agent for solubilizing the obtained precipitate (pellet) using a denaturing agent a known denaturing agent, in particular, guanidine or urea can be used.
  • the denaturant is usually used as an aqueous solution, and the concentration of the denaturant in the aqueous solution is 4 to 8 mol Z liter, preferably about 6 to 7 mol Z liter for guanidine, and 5 to 9 mol / liter for urea, preferably about 8 mol / l.
  • Guanidine is usually used as an acid addition salt of guanidine such as guanidine hydrochloride.
  • the cells are lysed with a denaturing agent after culturing, harvested by centrifugation, etc.
  • the ZAQ ligand can be extracted by crushing and solubilizing with a mutagenic agent.
  • denaturing agents used for solubilizing the collected cells include guanidine and urea.
  • the denaturant is usually used as an aqueous solution, and the concentration of the denaturant in the aqueous solution is usually 4 to 8 mol / l, preferably about 6 to 7 mol / l for guanidine.
  • Guanidine is usually used as an acid addition salt of guanidine such as guanidine hydrochloride.
  • Cell disruption can be performed in a conventional manner, for example, by sonication or by French press. Wear.
  • a denaturant used for solubilization of the disrupted cells a known denaturant may be used, and guanidine or urea is preferably used.
  • the denaturant is usually used as an aqueous solution, and the concentration of the denaturant in the aqueous solution is 4 to 8 mol Z liter for guanidine, preferably about 6 to 7 mol Z liter, and 5 to 9 mol / liter for urea, preferably. Is about 8 mol / l.
  • Guanidine is usually used as an acid addition salt of guanidine such as guanidine hydrochloride.
  • the inclusion body is solubilized as described above, the cells are directly solubilized with a denaturing agent, or the cells are crushed and then solubilized with a denaturing agent. If necessary, subject the supernatant to a purification step, and then refold (activate and regenerate) the ZAQ ligand.
  • Reforno-reding is performed by adding a redox buffer to the purified ZAQ ligand or diluting the supernatant containing the ZAQ ligand with the redox buffer.
  • the redox buffer examples include a buffer containing oxidized daltathione (GSSG) and reduced daltathione (GSH), a buffer containing cysteine and cystine, and a buffer containing cysteamine and cystamine.
  • GSSG oxidized daltathione
  • GSH reduced daltathione
  • the buffer examples include a Tris buffer, a phosphate buffer, an acetate buffer, a citrate buffer and the like, and among them, a Tris buffer and the like are preferable.
  • the concentrations of the oxidizing agent (oxidized substance) and the reducing agent (reduced substance) in the redox buffer are, for example, 0.01 to 100 mmol Z liter and 0.01 to: L 00 mmol Z liter, respectively. . More specifically, when using 0330 chips & 311, the concentration of GS SG in the redox buffer is 0.01 to 100 millimoles, preferably 0.1 to 10 millimoles / liter, and preferably 0.1 to 10 millimoles / liter. 1-1.0 millimoles are used, the concentration of GSH is 0.01-: 100 millimoles Z liter, preferably 0.1-10 millimoles Z liter, preferably
  • an amino acid having no mercapto group is further contained in the redox buffer.
  • Supernatant containing AQ ligand When diluting with a redox buffer, it is preferable to dilute the denaturant to a non-active concentration at a neutral pH suitable for activation.
  • the concentration of guanidine in the diluent is 0 to 2.0 monoliters, preferably about 1 mol / liter or less.
  • urea is used as a denaturant, the diluent is used. It is desirable to dilute the concentration of urea in the solution to 0 to 4.0 mol / l, preferably to about 2 mol liter or less.
  • any amino acid having no mercapto group may be used as long as the object of the present invention is achieved, and arginine, aspartic acid, palin, lysine, and alanine And citrulline. Arginine and the like are preferred in terms of yield in refolding of ZAQ ligands.
  • the concentration of the amino acid added to the redox buffer is 0.1 to 1.0 mol / liter, preferably 0.2 to 0.8 mol / liter.
  • the temperature for the above reforming is 0 to 30 ° C, preferably 4 to 15 ° C.
  • the pH is 7-9, preferably ⁇ 7.5-8.5.
  • the time required for the refolding is usually 0.5 to 7 days, preferably 1 to 3 days, more preferably 1 to 2 days.
  • solubilization and before refolding known and commonly used purification steps such as extraction, salting-out, dialysis, distribution, crystallization, recrystallization, gel filtration, and chromatography can be introduced.
  • it can be purified, for example, by applying to Sephadex G-25 (Amersham Pharmacy Biotech) in a 0.1 mol / liter phosphate buffer solution. Separation of the denaturing agent can also be performed by dialyzing against a 0.1 mol Z liter phosphate buffer in some cases.
  • the purification step can also be performed following refolding.
  • such purification includes, for example, extraction, salting-out, dialysis, partitioning, crystallization, recrystallization, gel filtration, chromatography, and the like.
  • Preferred examples include dialysis and, for example, SP-Sepharose (Amersham Pharmacia Biotech (Amersham Pharmacia Biotech)).
  • CM-5PW Tosoh Corporation
  • DEAE-5PW Tosoh Corporation
  • C4P-50 Showa Denko Corporation
  • the ZAQ ligand (active ZAQ ligand) obtained in the present invention has the same action as the ZAQ ligand obtained from eukaryotic cells, and is used in the same manner as the method of using the ZAQ ligand obtained from eukaryotic cells. Can be used.
  • ZAQ ligand has an activity of controlling intestinal contraction and the like, and when DNA encoding ZAQ ligand is deficient or expression level is abnormally decreased, for example, gastrointestinal diseases ( For example, various diseases such as enteritis, diarrhea, constipation, and malabsorption syndrome occur.
  • gastrointestinal diseases For example, various diseases such as enteritis, diarrhea, constipation, and malabsorption syndrome occur.
  • the ZAQ ligand obtained by the production method of the present invention is useful for treating various diseases such as digestive diseases (eg, enteritis, diarrhea, constipation, malabsorption syndrome, etc.).
  • digestive diseases eg, enteritis, diarrhea, constipation, malabsorption syndrome, etc.
  • ZAQ ligand is administered to the patient.
  • the role of the ZAQ ligand in the patient can be sufficiently or normally exerted.
  • the ZAQ ligand When used as a therapeutic or prophylactic agent as described above, it should be purified to at least 90%, preferably 95% or more, more preferably 98% or more, and even more preferably 99% or more. Is preferred.
  • ZAQ ligands can be used, for example, as orally as tablets, capsules, elixirs, microcapsules, etc., optionally in sugar coating, or aseptic with water or other pharmaceutically acceptable liquids. It can be used parenterally in the form of injections such as solutions or suspensions. For example, mixing ZAQ ligand with physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, etc. in the unit dosage form generally required for the practice of the formulation It can be manufactured by The amount of the active ingredient in these preparations is such that a suitable dosage in the specified range can be obtained.
  • additives examples include binders such as gelatin, corn starch, tragacanth, and gum arabic; Excipients such as sesame, leavening agents such as corn starch, gelatin, alginic acid, etc., lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, peppermint, cocoa oil or tea. Flavoring agents are used.
  • the unit dosage form is a capsule, the above type of material may further contain a liquid carrier such as oil and fat.
  • a sterile composition for injection can be formulated according to a conventional pharmaceutical preparation such as dissolving or suspending an active substance in a vehicle such as water for injection, or a naturally occurring vegetable oil such as sesame oil or coconut oil.
  • aqueous solutions for injection include physiological saline, isotonic solutions containing pudose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.).
  • auxiliaries for example, alcohols (eg, ethanol, etc.), polyalcohols (eg, propylene glycol, polyethylene glycol, etc.), nonionic surfactants (eg, Polysorbate 80 TM, HCO-50, etc.) Is also good.
  • oily liquid examples include sesame oil and soybean oil, and may be used in combination with benzyl benzoate, benzyl alcohol, or the like as a solubilizing agent.
  • buffers for example, phosphate buffer, sodium acetate buffer, etc.
  • soothing agents for example, Shiridani benzalkonium, proforce hydrochloride, etc.
  • stabilizers for example, human serum albumin, polyethylene glycol, etc.
  • a preservative eg, benzyl alcohol, phenol, etc.
  • an antioxidant and the like.
  • the prepared injection solution is usually filled in an appropriate ampoule.
  • the preparations obtained in this way are safe and have low toxicity, and can be used, for example, in mammals (eg, humans, rats, mice, guinea pigs, egrets, sheep, pigs, pigs, dogs, cats, dogs, monkeys, etc.). ) Can be administered.
  • mammals eg, humans, rats, mice, guinea pigs, egrets, sheep, pigs, pigs, dogs, cats, dogs, monkeys, etc.
  • the dosage of the ZAQ ligand varies depending on the target disease, the administration target, the administration route, and the like.
  • the ZAQ ligand when orally administered for the purpose of treating digestive diseases, it is generally required for an adult (60 kg).
  • the single dose of the ZAQ ligand peptide varies depending on the administration target, target disease, etc.
  • ZAQ ligand is administered to adults ( Weighing 60 kg)
  • the dose can be administered in terms of 60 kg.
  • bases, amino acids, and the like are represented by abbreviations based on the abbreviations by the IUPAC-IUB Commission on Biochemical Nomenclature or the abbreviations in the art, and examples thereof are described below.
  • optical isomer for an amino acid the L-isomer is indicated unless otherwise specified.
  • DNA complementary deoxyribonucleic acid
  • RNA ribonucleic acid
  • H is (H): histidine
  • a s x A s + A s n
  • G 1x G 1 u + G 1 n
  • sequence numbers in the rooster s column table in the present specification indicate the following sequences.
  • the nucleotide sequence of the DNA encoding the ZAQ receptor is shown (ZAQC).
  • [SEQ ID NO: 8] 7 shows the nucleotide sequence of primer ZF2 used in Reference Example 3.
  • [SEQ ID NO: 34] 3 shows the nucleotide sequence of DNA fragment # 3 used in Example 2-1.
  • the nucleotide sequence of a synthetic DNA encoding the human ZAQ ligand represented by SEQ ID NO: 21 is shown.
  • the nucleotide sequence of the DNA encoding the human BV8 precursor peptide is shown.
  • the transformant Escherichia coli DH5 ⁇ / ⁇ CR2.1—ZAQC obtained in Reference Example 1 described below was obtained from Tsukuba, Ibaraki, Japan, on August 23, 1999 by Riki et al. Higashi 1-chome No. 1 1 Chuo No.
  • the transformant Escherichia coli TOP10 / pHMITA obtained in Reference Example 3 described below has been used since July 13, 2000, 1-1, Tsukuba-Higashi, Ibaraki, Japan 1 6 (Zip code 305-8566) National Institute of Advanced Industrial Science and Technology Patent Organism Depositary Center (formerly Ministry of International Trade and Industry, National Institute of Advanced Industrial Science and Technology, National Institute of Advanced Industrial Science and Technology (NI BH)) Patent, National Institute of Advanced Industrial Science and Technology Deposited with the Depositary No. FERM BP-7219 at the Organisms Depositary Center and deposited with the Fermentation Research Institute (IFO) under the deposit number IFO 16440 since May 26, 2000.
  • IFO Fermentation Research Institute
  • the transformant Escherichia coli TOP 10 / pHMI TG obtained in Reference Example 3 described below has been used since July 13, 2000, 1-1-1 Tsukuba-Higashi, Ibaraki, Japan 1 6 (Zip code 305-8566) National Institute of Advanced Industrial Science and Technology Patent Organism Depositary Center (formerly Ministry of International Trade and Industry, National Institute of Advanced Industrial Science and Technology, National Institute of Advanced Industrial Science and Technology (NI BH)) Patent, National Institute of Advanced Industrial Science and Technology Deposited with the Depositary No. FERM BP-7220 under the deposit number IFO 16441 on May 26, 2000, at the Institute of Fermentation (IFO).
  • Escherichia coli MM 2 94 (DE 3) Zp TC h 1 ZAQ obtained in Example 1 described below was obtained from Tsukuba East 1-chome, Tsukuba, Ibaraki, Japan from April 27, 2001.
  • No. 1 1 Chuo No. 6 (Zip code 305-856 6) National Institute of Advanced Industrial Science and Technology (AIST) Patent Organism Depositary Center (formerly Ministry of International Trade and Industry, National Institute of Advanced Industrial Science and Technology, National Institute of Biotechnology and Industrial Technology (NI BH)) It has been deposited with the Institute of Fermentation Research (IFO) under the deposit number F ERM BP-7571 as the deposit number IFO 16527 since January 16, 2001 at the National Institute of Advanced Industrial Science and Technology.
  • IFO Institute of Fermentation Research
  • Escherichia coli MM 294 (DE 3) / pTCh 2 ZAQ obtained in Example 2 described below was obtained from April 27, 2001 at 1-1-1 Tsukuba East, Ibaraki, Japan 1 Chuo No. 6 (Zip code 305-8566) National Institute of Advanced Industrial Science and Technology (AIST) Patent Organism Depositary Center (formerly Ministry of International Trade and Industry, National Institute of Advanced Industrial Science and Technology (NI BH)) National Institute of Advanced Industrial Science and Technology It has been deposited with the Institute for Fermentation of Japan (IFO) under the deposit number FERM BP-7572 as the deposit number IFO 16587 on March 15, 2001 as the deposit number IFO 16587.
  • IFO Institute for Fermentation of Japan
  • the human pituitary gland cDNA (CLONTECH) was designated as type II, and two primers, primer 1 (5, one GTC GAC ATG GAG ACC ACC ATG GGG TTC ATG G-3 '; SEQ ID NO: 4) and primer 2 (5 PGR reaction was performed using '-ACT AGT TTA TTT TAG TCT GAT GCA GTC CAC CTC TTC-3;; SEQ ID NO: 5).
  • the composition of the reaction solution used in the reaction was 1/10 of the above cDNA as type III, 1/50 amount of Advantage2 Polymerase Mix (CLONTECH), primer 1 and primer 2 were each 0.2 ⁇ l, dNTPs 200
  • the attached buffer was added to ⁇ and the enzyme to make a liquid volume of 25 ⁇ l.
  • the PCR reaction is performed at 94 ° C for 2 minutes, followed by three cycles of 94 ° C for 20 seconds, 72 ° C for 100 seconds, and three cycles of 94 ° C for 20 seconds and 68 ° C for 100 seconds.
  • the cycle of 94 ° C for 20 seconds, 64 ° C for 20 seconds, and 68 ° C for 100 seconds was repeated 38 times, and the extension reaction was finally performed at 68 ° C for 7 minutes.
  • the reaction product after the PCR reaction was subcloned into a plasmid vector pCR2.1 (Invitrogen) according to the procedure of a TA cloning kit (Invitrogen).
  • the obtained solution I to solution IV were freeze-dried using a freeze dryer (12EL; VirTis).
  • a TSKgel ODS-80Ts reversed-phase high-performance liquid chromatography column (Tosoichi Co., Ltd., 4.6 mm x 25 cm) was applied at 40 ° C at a flow rate of 1 ml / min to solution A (0.1% trifluoroacetic acid / Distilled water) Volume 81.7% / B solution (0.1% trifluoroacetic acid / 60% acetonitrile) 8.3% volume was flowed and equilibrated.
  • the freeze-dried product of Solution I to Solution IV obtained in (2-3) above was dissolved in 4 ml of 1 M acetic acid, respectively, and subjected to chromatography.
  • the volume of solution A was increased to 67% / the volume of solution B to 33% over 1 minute at a flow rate of 1 ml / min, and then the solution A over 40 minutes.
  • Solution B concentration was increased in a linear gradient from 33% volume of 33% ZB solution to 100% of solution A volume of 0% ZB solution.
  • the eluate was fractionated by assigning a fraction number to each 1 ml, and 2 ⁇ l of each fraction was mixed with 150 ⁇ of 0.2% Bovine Serum Albumin (BSA) / distilled water and freeze-dried. The dried product was used as a sample for assay for measuring the activity of increasing the intracellular Ca ion concentration described in (2-5) below.
  • BSA Bovine Serum Albumin
  • the ZAQ stable expression cell line was prepared as follows. That is, one clone of DH5a / pCR2.1-ZAQC obtained in Reference Example 1 was shake-cultured in an LB medium containing ampicillin to obtain a plasmid pCR2.1-ZAQC. This was treated with the restriction enzymes Sal I and Spe I to cut out the insert coding for ZAQC. Similarly, pAKK0_l.11H treated with restriction enzymes Sal I and Spe I and the insert were ligated using a Ligation Express Kit (CL ONTECH Laboratories, Inc. (CA, USA)) and electroporated into E. coli DH10B. Introduced by law. The structure of the plasmid contained in the obtained clone was confirmed by restriction enzyme treatment and sequence analysis, and a correctly constructed plasmid was used as a plasmid pAK-ZAQC for CH0 cell expression.
  • This plasmid pAK- ZAQC obtained by introducing transformed Te Rere for CH0 / dhfr _ cells (American Type Culture Collect ion) ⁇ This CellPhect Transfection kit (Amersham Pharmacia Biotech, Inc.). First, add 120 ⁇ of Buffer A (attached to CellPhect Transfection Kit) to plasmid DNA 4 dissolved in distilled water 1201, stir and let stand for 10 minutes, then Buffer B (attached to CellPhect Transfection Kit) 240 ⁇ l was added, and the mixture was vigorously stirred to form a DNA-calcium phosphate complex containing the DNA.
  • Buffer A attached to CellPhect Transfection Kit
  • Buffer B attached to CellPhect Transfection Kit
  • the cells were dispersed by trypsin treatment, collected from a Petri dish, and 2 ⁇ 10 4 cells were inoculated in a 6-well plate, dialyzed 10% fetal bovine serum (JRH BIOSCIENCES), 1 mM MEM non-essential amino acid solution (Dainippon Pharmaceutical Co., Ltd.), 100 units / ml Penicillin ⁇ 100 ⁇ g / m 1 Dulbecco's modified Eagle medium (DMEM) medium containing Streptomycin (Nissui Pharmaceutical Co., Ltd.) at 37 ° C, 5% carbon dioxide Culture was started in gas.
  • JRH BIOSCIENCES 10% fetal bovine serum
  • 1 mM MEM non-essential amino acid solution Dainippon Pharmaceutical Co., Ltd.
  • ETA24 cell As a control, ETA (endothelin A receptor) -expressing CH0 cell clone 24 (hereinafter abbreviated as ETA24 cell; see Journal of Pharmacology and Experimental Therapeutics, Vol. 279, pp. 675-685, 1996) was used.
  • ZAQC-B1 cells and ETA 24 cells were measured using the FLIPR (Molecular Devices) with respect to the samples for atsey obtained in the above (2-4).
  • FLIPR Molecular Devices
  • dFBS dialyzed fetal bovine serum
  • ZAQC-B1 cells and ETA24 cells are each suspended in a medium (10% dFBS-DMEM) at a concentration of 15 ⁇ 10 4 cells / ml and dispensed into a 96-well plate for FLIPR (Black plate clear bottom, Coster).
  • H / HBSS Nasy Hanks 2 (Nissui Pharmaceutical Co., Ltd.) 9., sodium hydrogen carbonate 0.35 g, HEPES 4.77 g, pH 7.4 with sodium hydroxide solution, then filter sterilization) 20 ml, 250 mM Probenecid 200 ⁇ l, and 200 f / l fetal serum (FBS) were mixed.
  • sample plate For the atssay sample obtained in (2-4) above, dilute each fraction with 150 ⁇ H of H / HBSS containing 2.5 mM Probenecid, 0.2% BSA, and use it in a 96-well plate for FLI PR. (V-Bottom plate, Coster) (hereinafter referred to as sample plate). After dye loading on the cell plate, wash the cell plate 4 times with a washing buffer containing 2.5 mM Proben ecid in 3 ⁇ 4 ⁇ 33 using a plate washer (Molecular Devices), and wash 100 ⁇ after washing. The buffer was left. The cell plate and the sample plate were set on the FLIPR and assayed (50 samples were transferred from the sample plate to the cell plate by the FLIPR). As a result, the above (2-3) IV solution was transferred to the (2) — 4) Fraction No. 53 obtained by reversed-phase high-performance liquid chromatography showed an activity to increase intracellular Ca ion concentration specific to ZAQC-B1 cells.
  • TSKgel Super-Phenyl Reversed-Phase High-Performance Liquid Chromatography column (0.46 cm X 10 era, Tosoichi Co., Ltd.) .
  • Chromatography was performed on the fraction No. 53 obtained in the above (3-4).
  • the volume of solution A was increased to 75% / the volume of solution B at 25% over 1 minute at a flow rate of 1 ml / min, and then over 75 minutes. hand
  • the solution concentration was increased in a linear gradient to a solution volume of 33%.
  • the eluate was fractionated with a fraction number of 500 1 each. From the fractionated fractions, each 25 1 was mixed with 0.2% BSA1501 and freeze-dried with a freeze dryer (12EL; VirTis). To this dried product, 150 ⁇ l of H / HBSS containing 2.5 mM Probenecid, 0.2% BSA was added and dissolved, and 50 / l of this solution was used according to the test method of (2-5) above. By measuring the intracellular Ca ion concentration increasing activity, ZAQC-B1 cells Receptor activity was measured. As a result, it was found that the target component having a receptor activating effect on ZAQC-B1 cells, that is, the ZAQ-active component was mainly eluted in fraction No. 103-105. (2-7) 3 ⁇ 4 3 (2/83 > 14.6 / 100 Reversed-phase high-performance liquid chromatography)
  • AiRPC C2 / C18 ST 4.6 / 100 Reversed-phase high-performance liquid chromatography column (Amersham Pharmacia Biotech, 0.46 cm x 10 cm) was applied at 40 ° C at a flow rate of 1 ml / min to liquid A (heptafluorobutyric acid / distilled). (Water) 95% volume / B solution (0.1% heptafluorobutyric acid / 100% acetonitrile) 5% volume was flowed to equilibrate.
  • the eluate was fractionated by assigning a fraction number of 500 ⁇ , and each 10 ⁇ 1 of each fraction was mixed with 150 ⁇ % of 0.2% BSA and freeze-dried with a freeze dryer (12EL; VirTis).
  • H / HBS S 150 / zl containing 2.5 mM Probenecid and 0.2% BSA was added to the dried product to dissolve the ZAQC-B1 cells using this solution 501 according to the test method described in (3-5) above.
  • the receptor activating effect on was measured.
  • the target component having a receptor activating effect on ZAQC-B1 cells that is, the ZAQ activating component, was eluted in fraction Nos. 82-84.
  • This activity peak completely coincided with the ultraviolet absorption peak at 210 nm, and it was determined that the peptide had been purified to a single peptide.
  • a Blast search of the database using the N-terminal amino acid sequence of the peptide that activates ZAQ purified from the milk obtained in Reference Example 2 (SEQ ID NO: 6) as a query shows that it is SEQ ID NO: 6.
  • Human EST 0W0467 containing a sequence equivalent to the nucleotide sequence of DNA encoding a peptide having an amino acid sequence was found. Since this sequence did not have a full-length open reading frame, the sequence of the undetermined portion was determined by the RACE method, and a cDNA clone having the full-length open reading frame was obtained. did.
  • ZF1 5'-GGTGCCACGCGAGTCTCAATCATGCTCC-3 '(SEQ ID NO: 7)
  • ZF2 5'-GGGGCCTGTGAGCGGGATGTCCAGTGTG-3 '(SEQ ID NO: 8)
  • ZF3 5'-CTTCTTCAGGAAACGCAAGCACCACACC-3 '(SEQ ID NO: 9)
  • the reaction solution was 1 ⁇ l of 50 X Advantage 2 Polymerase Mix (CL0NTECH) and the attached 10 x Advantage 2 PCR buffer (400 mM Tricine-K0H, 150 mM KOAc, 35 mM Mg (0Ac) 2 , 37.5 ⁇ g / ml 5 ⁇ l of BSA, 0.05% Tween-20, 0.05% Nonidet-P40), 4 ⁇ l of dNTP mixture (2.5 mM each, Takara Shuzo), 10 primer ZF2 1 ⁇ 1, 10 ⁇ ⁇ primer ⁇ 2 (primer ⁇ 2 1 jl of type III DNA (attached to Marathon-Ready cDNA Kit from CL0NTECH), 5 ⁇ l of type I DNA (50-fold dilution of the PCR reaction solution), and 33 / l of distilled water were prepared.
  • CL0NTECH X Advantage 2 Polymerase Mix
  • 10 x Advantage 2 PCR buffer 400 mM Tricine-K0H, 150 mM K
  • the reaction conditions were as follows: after initial denaturation at 94 ° C for 60 seconds, a cycle reaction of 94 ° C for 30 seconds to 72 ° C for 4 minutes was performed 5 times, and a cycle reaction for 94 ° C for 30 seconds to 70 ° C for 4 minutes Were repeated 5 times, and a cycle reaction of 94 ° 030 seconds-68 ° 044 minutes was performed 25 times.
  • a second nested PCR was performed using the reaction solution of the PCR reaction as a type II.
  • the reaction solution was 1 / xl of 50 Advantage 2 Polymerase Mix (CL0NTECH), and the attached 10 ⁇ Advantage 2 PCR buffer (400 mM Tricine—KOH, 150 mM KOAc, 35 mM Mg (OAc) 2 , 37.5 // g / ral 5 ⁇ l of BSA, 0.05% Tween-20, 0.05% Nonidet-P40), 4 ⁇ l of dNTP mixture (2.5 mM each, Takara Shuzo), 10 ⁇ m of primer ZF3 1 ⁇ l, 10 ⁇ m of primer ⁇ 2 (primer ⁇ 2 is CL0NTECH 1 ⁇ l), 5 ⁇ l of type I DNA (50-fold dilution of the PCR reaction solution), and 33 of distilled water were prepared.
  • CL0NTECH Advantage 2 Polymerase Mix
  • the reaction conditions were as follows: after initial denaturation at 94 ° 060 seconds, 5 cycles of 94 ° C-30 seconds-72 ° C'4 minutes, 5 cycles of 94 ° ⁇ 30 seconds-70 ° C'4 minutes, A cycle reaction of 94 ° C. for 30 seconds—68 ° C. for 44 minutes was performed 25 times.
  • the obtained DNA fragment was cloned using a T0P0 TA Cloning Kit (Invitrogen) according to the method described in the attached manual.
  • the nucleotide sequence of the cloned DNA was decoded using ABI377 DNA sequencer to obtain a 3′-end sequence (SEQ ID NO: 10).
  • primers ZAQL-CF SEQ ID NO: 11
  • ZAQL-XR1 SEQ ID NO: 12
  • ZAQL-CF 5 '-CCACCATGAGAGGTGCCACG- 3' (SEQ ID NO: 1 1)
  • ZAQL-XR1 5, -CTCGAGCTCAGGAAAAGGATGGTG-3 '(SEQ ID NO: 12)
  • PCR reaction solution 1 ⁇ l of PfuTurbo DNA polymerase (Stratagene), 5 ⁇ l of attached 10x PCR buffer, 4 ⁇ l of 2.5 mM dNTP mixture, 10 zM primer ZAQL-CF and ZAQL-XR1 Were prepared by mixing 2.5 ⁇ each, 5 ⁇ 1 of type I DNA, and 30 of distilled water.
  • the reaction conditions were: 95 ° C for 1 minute initial denaturation, 95 ° C for 1 minute-60 ° C, 1 minute to 72 ° C, 1 minute cycle 40 times, and final extension at 72 ° 010 minutes
  • the obtained DNA fragment was cloned using the TOPO ⁇ A Cloning Kit (Invitrogen) according to the method described in the attached manual.
  • the plasmid having the DNA fragment having the nucleotide sequence represented by SEQ ID NO: 13 was named pHMITA
  • the plasmid having the DNA fragment having the nucleotide sequence represented by SEQ ID NO: 14 was named pHMITG.
  • Escherichia coli was transformed with plasmids pHMITA and pHMITG, and named Escherichia coli ⁇ / ⁇ and Escherichia coli TOPlO / pHMITG, respectively.
  • the DNA fragment represented by SEQ ID NO: 13 was converted to the human ZAQ ligand precursor peptide represented by SEQ ID NO: 15 (A type, 105 amino acid residues).
  • the DNA fragment containing the coding DNA (SEQ ID NO: 16), the DNA fragment represented by SEQ ID NO: 14 is a human ZAQ ligand precursor peptide represented by SEQ ID NO: 17 (G type, 105 amino acid residues) (SEQ ID NO: 18).
  • the base sequence represented by SEQ ID NO: 16 and SEQ ID NO: 17 has a typical signal sequence
  • the DNA having the base sequence represented by SEQ ID NO: 16 is represented by SEQ ID NO: 19
  • the DNA having the sequence is represented by SEQ ID NO: 21. It was found that the DNA contained 258 base pairs of DNA (SEQ ID NO: 22) encoding the human-type ZAQ ligand mature peptide (G type, 86 amino acid residues).
  • Reference Example 4 Production of human ZAQ ligand peptide in mammalian cells
  • Plasmid pHMITG was digested with EcoRI and XhoI, and the resulting DNA was electrophoresed on a 1.5% agarose gel. Cut out. DNA fragments were recovered from the gel pieces using a Gene Clean spin DNA extraction kit (BIO 101). The obtained DNA fragment was cloned in accordance with a standard method at a cleavage site for EcoRI and XhoI restriction enzymes with respect to a mammalian cell expression vector PCAN618 using CMV-IE enhancer and chicken beta-actin promoter as expression promoters.
  • the mammalian cell expression vector having the DNA encoding the human ZAQ ligand precursor peptide was named pCANZAQLg2.
  • C0S7 cells used were purchased from ATCC and subcultured using DMEM medium (supplemented with 10% FBS).
  • Human type ZAQ ligand precursor peptide expression plasmid (pCANZAQLg2) 2 ⁇ (2 ⁇ l of ⁇ Noffer t ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ) ⁇ N ⁇ offer EC (Effectene transfection reagent, Q IAGEN) 298 ⁇ was added, and Enhancer 16 was added.
  • the culture supernatant of the C0S7 cell expressing human ZAQ ligand precursor peptide was collected, and the following operation was performed to prepare an extract.
  • 1.1 ml of acetic acid was added dropwise to the cell culture supernatant (about 18.5 ml) to a final concentration of 1 M, followed by stirring for 1 hour. Further Aseto down twice that capacity addition, 4 ° C at stirring for 30 min, then a high-speed centrifugal separator (CR26H, 23 type rotor: Hitachi, Ltd.) using a 15, 000 r centrifugal P m, 30 minutes Then, a supernatant was obtained. The resulting supernatant was applied to an evaporator to remove acetone, and then lyophilized with a freeze dryer (12EL; VirTis).
  • a freeze dryer (12EL; VirTis
  • Solution A (0.1% trifluoroacetic acid / distilled water) was applied to a TSKgel Super ODS reversed-phase high-performance liquid chromatography column (Tosoichi, 0.46 cm x 10 cm) at 40 ° C at a flow rate of 1 ml / min. ) And allowed to equilibrate. After applying the SepPak C18-5 g force fraction obtained in (4-3-2) to the servant, the mixture was applied to Super ODS reversed-phase high-performance liquid chromatography, and the solution A (0.1%) was applied at a flow rate of 1 ml / min for 60 minutes.
  • the eluate was fractionated by adding fraction No. to each 1 ml, and the entire fraction fraction was freeze-dried with a freeze dryer (12EL; VirTis). H / HBSS plus 2.5mM Probenecid and 0.2% BSA ⁇ ⁇ 1 was added to the dried product and dissolved. Using this solution, ZAQC-B1 cells were tested by the following test method (4-1-3-4). The receptor activating effect was measured.
  • F LIPR was used to measure the intracellular Ca ion concentration increasing activity of the ZAQ-expressing cells (ZAQC-B1) obtained in Reference Example 2 (2-5). It was performed using.
  • h0T7T175-expressing cells h0T7T175-16; described in WO 00/24890.
  • ZAQC-B1 cells and h0T7T175-16 cells that had been subcultured in DMEM supplemented with 10% dialyzed fetal bovine serum (hereinafter referred to as dFBS) were used.
  • dFBS dialyzed fetal bovine serum
  • ZAQC-B1 cells and MT7T175-16 cells were each suspended in a medium (10% dFBS-DMEM) at a concentration of 15 ⁇ 10 4 cells / ml, and distributed in a 96-well plate for FLIPK (Black plate clear bottom. Coster).
  • a device seeded by 200 ⁇ 1 each Ueru using (3.0 ⁇ 10 4 ⁇ 113 / 200 / ⁇ 1 / Uweru), and 5% C0 2 was incubated in a coater 37 ° C De ⁇ culture, using the (hereafter cell plates ).
  • H / HBSSCHAN KS '9.8 g, sodium hydrogen carbonate 0.35 g, HEPES 4.77 g, pH with sodium hydroxide After adjusting to 7.4, filter sterilization treatment) 21 ml, 210 mM Probenecid 210 ⁇ l, and 210 ⁇ l of mouse fetal serum (FBS) were mixed.
  • each fraction was dissolved by adding 150 ⁇ l of H / HBSS to which 2.5 mM Probenecid and 0.2% BSA were added, and dissolving in a 96-well plate for FLIPR ( (V-Bottom plate, Coster) (hereinafter referred to as sample plate).
  • sample plate 96-well plate for FLIPR ( (V-Bottom plate, Coster)
  • the cell plate was washed four times with a washing buffer containing 2.5 mM Probenecid added to H / HBSS using a plate washer (Molecular Devices), and after washing, 100 washing buffers were left.
  • the DNA7 fragment obtained in (b) above was combined with # 1 and # 6 above to give 1 201.
  • the mixture was kept at 90 ° C. for 10 minutes, gradually cooled to room temperature, annealed, and subjected to a ligation reaction using TaKaRa DNA Ligation Kit ver.2 (Takara Shuzo).
  • the II solution 301 included in the kit was added and mixed well, and then 60 ⁇ l of the I solution included in the kit was added.
  • the mixture was reacted at 37 ° C. for 1 hour to perform ligation.
  • pTCII As an expression vector, pTCII (described in JP-A-2000-178297) was digested with NdeI and BamHI (Takara Shuzo) at 37 ° C for 2 hours, and then 4.3 kb by 1% agarose gel electrophoresis. was extracted using a QIAquick Gel Extraction Kit (Qiagen) and dissolved in 25 ju1 of TE buffer. The ligation reaction was carried out using the Ndel and BamHI fragments of pTCII and the structural gene (SEQ ID NO: 38) of the ZAQ ligand prepared as described above using TaKaRa DNA ligation kit ver.2 (Takara Shuzo).
  • Escherichia coli JM109 Combinant Cell was transformed with 101 of this reaction solution, seeded on LB agar medium containing 10 ⁇ g / m1 of tetracycline, and cultured at 37 ° C for 1 ⁇ . Tetracycline resistant colonies The transformant was over ⁇ cultured in LB medium, plasmids were prepared ptch 1 ZAQ using QIA P rep8 Miniprep Kit (Qiagen). The base sequence of this ZAQ ligand DNA was confirmed using an Applied Biosystems model 377 DNA sequencer.
  • the plasmid pTCh1ZAQ was transformed into Escherichia coli MM294 (DE3) to obtain a ZAQ ligand expression strain Escherichia coli MM294 (DE3) / pTChlZAQ.
  • Example 1-2 Production of ZAQ ligand
  • the above Escherichia coli MM294 (DE3) / pTChlZAQ was added to LB medium containing 5.Omg / L tetracycline ⁇ 1 L (1% peptone, 0.5% yeast extract, 0.5% sodium salt).
  • the culture was shake-cultured at 37 ° (: 8 hours in a 2 L flask.
  • the obtained culture was mixed with 19 L of the main fermentation medium (1.68% sodium monohydrogen phosphate, 0.3% phosphoric acid 2%).
  • Potassium hydrogen, 0.1% ammonium chloride, 0.05% sodium chloride sodium, 0.05% magnesium sulfate, 0.02% defoamer, 0.00025% ferrous sulfate, 0.0005% hydrochloric acid Thiamine, 1.5% budou sugar, 1.5% high-case amino) were introduced into a 5 L fermenter, and aeration and stirring were started at 30 ° C.
  • isopropyl-1- ⁇ -D —Tiogalactopyranoside was added to a final concentration of 12 mg / L, and the cells were further cultured for 4 hours. After completion of the culture, the culture was centrifuged to obtain about 200 g of wet cells and stored at 180 ° C.
  • Example 1-3 The regenerating solution after the completion of the activation in Example 1-3 was adjusted to pH 6.0, and the SP-Sepharose column (11.3 cm ⁇ 15 cm) equilibrated with 50 mM phosphate buffer (pH 6.0) was used. ), And eluted with 600 mM NaCl / 50 mM phosphate buffer (pH 6.0), and the fractions containing the ZAQ ligand were pooled.
  • Example 14 The ZAQ ligand obtained in 1-4 was suspended in Sam1 ebuffer [Laemmli, Nature, 227, 680 (1979)] supplemented with 100 mM DTT, heated at 95 ° C for 1 minute, and then multi-gel Electrophoresis was performed with 15 25 (Daiichi Kagaku). The gel after electrophoresis was stained with Coomassie 'Brilliant' Benolay (Coomassie brillian t blue), and the same position as the recombinant ZAQ ligand sample derived from COS 7 cells obtained in Reference Example (4-3-3) was obtained. A single protein band was observed. From this, the recombinant ZAQ ligand preparation derived from E. coli obtained in Examples 14 to 14 is extremely high in purity and has the same molecular weight as the recombinant ZAQ ligand prepared from COS 7 cells. I found it.
  • the amino acid composition was determined using an amino acid analyzer (Hitachi L-1 8500A Amino Acid Analyzer). As a result, the amino acid composition was consistent with the amino acid composition deduced from the DNA base sequence of the ZAQ ligand (peptide comprising the amino acid sequence represented by SEQ ID NO: 21) (Table 1).
  • Tr_09 acid hydrolysis (6 N HC 1-1% phenol, 110. C, average of 24 and 48 hours hydrolysis)
  • the N-terminal amino acid sequence was determined using a gas phase protein sequencer (PE Applied Biosystems model 492). As a result, it was consistent with the N-terminal amino acid sequence of the ZAQ ligand deduced from the nucleotide sequence of the DNA of the obtained ZAQ ligand (Table 2).
  • the C-terminal amino acid was determined using an amino acid analyzer (Hitachi L-8500A Amino Acid Analyzer). The obtained ZAQ ligand was in agreement with the C-terminal amino acid deduced from the DNA base sequence (Table 3).
  • Mass spectrometry was performed using an LCQ ion trap mass spectrometer (manufactured by ThermoQuest) equipped with a nanoESI ion source. As a result, a molecular weight% of 57.55 ⁇ 0.89 was obtained, and the theoretical molecular weight of the ZAQ ligand of SEQ ID NO: 21 and 10 residues of Cys of SEQ ID NO: 21 in which 5 pairs of disulfide bonds were formed (9657 , 3).
  • Example 11 Measurement of Activity of 6 Z AQ Ligand Measurement of Cell-Caion Concentration Elevation Activity Using FLIPR
  • Example 14 Using the purified recombinant E. coli-derived ZAQ ligand sample obtained in 1-4, the method of Reference Example (4-1-3-4) was used to measure the activity (intracellular Ca ion using FLIPR). Measurement of concentration increasing activity). As a result, it had an activity equivalent to that of the C0S7 cell-derived recombinant sample (purified ZAQ ligand) obtained in Reference Example (4-1-3-3).
  • a human Bv8 structural gene was prepared using the following six DNA fragments # 1 to # 6.
  • Each of the four DNA oligomers (# 2 to # 5) excluding # 1 and # 6 above, which should be on the 5 'side, was added to 25 ⁇ l of phosphorylation reaction solution (DNA oligomer 10 ⁇ g, 50 mM Tris-HCl , pH 7.6, 10 mM MgCl 2 , ImM spermidine, 10 mM dithiothreitol (hereinafter abbreviated as DTT), 0.1 mg / m 1 ⁇ serum album Min (hereinafter abbreviated as BSA), ImM ATP, 10 units T4 polynucleotide kinase (Takara Shuzo)] at 37 ° C for 1 hour to phosphorylate the 5 'end of each oligomer. After the phenol treatment, 2 volumes of ethanol was added, the mixture was cooled to 170 ° C, and DNA was precipitated by centrifugation.
  • the DNA fragment obtained in (b) above was combined with # 1 and # 6 to give 120 ⁇ l.
  • the mixture was kept at 90 ° C. for 10 minutes, cooled slowly to room temperature, annealed, and subjected to a ligation reaction using TaKaRa DNA Ligation Kit ver.2 (Takara Shuzo).
  • a ligation reaction using TaKaRa DNA Ligation Kit ver.2 (Takara Shuzo).
  • 3 ⁇ m of the II solution attached to the kit was added, and the mixture was thoroughly mixed.
  • the solution of 601 included in the kit was added, and reacted at 37 ° C for 1 hour to perform ligation.
  • pTCII As an expression vector, pTCII (described in JP-A-2000-178297) was digested with NdeI and BamHI (Takara Shuzo) at 37 ° C for 2 hours, and then subjected to 1% agarose gel electrophoresis. The 4.3 kb DNA fragment was extracted using the QIAquick Gel Extraction Kit (Qiagen) and dissolved in 25 ⁇ l of TE buffer. A ligation reaction was carried out between the Ndel and BamHI fragments of pTC II and the human Bv8 structural gene (SEQ ID NO: 39) prepared above using TaKaRa DNA ligation kit ver.2 (Takara Shuzo).
  • Escherichia coli JM109 competent cells (Toyobo) were transformed with 10 1 of this reaction solution, seeded on LB agar medium containing 10 ⁇ g Zm 1 of tetracycline, cultured at 37 ° C for 1 ⁇ , and the resulting tetracycline-resistant colonies were selected. It is.
  • This transformant was cultured once in an LB medium, and plasmid pTCh2ZAQ was prepared using QIAprep8 Miniprep Kit (Qiagen). The nucleotide sequence of this human Bv8 DNA was confirmed using an Applied Biosystems model 377 DNA sequencer.
  • the plasmid pTCh2ZAQ was transformed into Escherichia coli MM294 (DE3), and the human Bv8 expression strain Escherichia coli was transformed. Obtained 294 (DE3) I pTCh2ZAQ.
  • Example 2-2 Production of Human Bv8
  • Escherichia coli MM294 (DE3) / pTCh2ZAQ was added to LB medium containing 5.0 mg / L tetracycline in 1 L (1% peptone, 0.5% yeast extract, 0.5% sodium chloride). The cells were shake-cultured at 37 ° C for 8 hours in an L-volume flask. The obtained culture solution was added to a 19 L main fermentation medium (1.6% sodium monohydrogen phosphate, 0.3% potassium dihydrogen phosphate, 0.1% ammonium chloride, 0.05% sodium chloride).
  • Example 2-4 Purification of human Bv8
  • a type Bv8 fraction (elution time: about 40 minutes) was obtained.
  • the fraction was further passed through C4P-50 (21.5 mm IDX, 300 mL, Showa Denko) equilibrated with 0.1% trifluoroacetic acid, and adsorbed and washed.
  • B Elution was performed with a step gradient (60 minutes) of 80% acetonitrile / 0.1% trifluoroacetic acid.
  • lyophilization was performed. Approximately 25 m of lyophilized BV8 lyophilized powder was obtained.
  • Human BV8 obtained in Example 2-4 was suspended in Sampiebuffer [Laemmli, Nature, 227, 680 (1979)] supplemented with 10 OmM DTT, and heated at 95 ° C for 1 minute. Electrophoresis was performed on 15/25 (Daiichi Pure Chemicals). Genole after electrophoresis was stained with Coomassie brilliant blue, and as a result, a single protein band was observed at 9 kDa. From this, it was found that the recombinant human BV8 preparation derived from Escherichia coli obtained in Examples 2 to 4 was extremely high in purity.
  • Amino acid composition was determined using an amino acid analyzer (Hitachi L-8500A Amino Acid Analyzer). As a result, it was consistent with the amino acid composition deduced from the DNA base sequence of human Bv8 (a peptide consisting of the amino acid sequence represented by SEQ ID NO: 30) (Table 4). .
  • Trp09.1 Acid hydrolysis (6 N HC 1-4% thioglycolic acid, average value of hydrolysis at 110 ° C, 24 and 48 hours)
  • the N-terminal amino acid sequence was determined using a gas phase protein sequencer (PE Applied Biosystems model 492). As a result, it was consistent with the N-terminal amino acid sequence of human BV8 deduced from the nucleotide sequence of the obtained human BV8 DNA (Table 5).
  • Th r Th r
  • the C-terminal amino acid was determined using an amino acid analyzer (Hitachi L-8500A Amino Acid Analyzer).
  • the obtained human BV8 coincided with the C-terminal amino acid deduced from the nucleotide sequence of the DNA (Table 6).
  • the production method of the present invention is useful for industrially producing a pharmacologically active ZAQ ligand in a large amount and efficiently as compared with the conventional production method.

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Abstract

La présente invention concerne une méthode permettant d'obtenir une protéine possédant la même activité que celle d'un ligand ZAQ isolé de cellules eucaryotes. Ladite méthode comprend la restauration efficace d'un ligand ZAQ inactif produit dans des cellules procaryotes au moyen d'une technique de manipulation génétique. Précisément, l'invention concerne une méthode permettant de produire un ligand ZAQ actif, caractérisée en ce qu'elle consiste à exprimer un ligand ZAQ par génie génétique dans un hôte procaryote et à le replier ensuite dans un tampon redox.
PCT/JP2002/000378 2001-01-22 2002-01-21 Methode de production de ligand zaq WO2002057443A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7052674B2 (en) 2000-11-03 2006-05-30 The Regents Of The University Of California Prokineticin polypeptides, related compositions and methods

Citations (7)

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Publication number Priority date Publication date Assignee Title
US5453363A (en) * 1985-10-23 1995-09-26 Boehringer Mannheim Gmbh Process for the activation of t-PA or Ing after genetic expression in prokaryotes
JPH10191989A (ja) * 1996-11-12 1998-07-28 Takeda Chem Ind Ltd ベータセルリン類の製造方法
WO2000034334A1 (fr) * 1998-12-10 2000-06-15 Synaptic Pharmaceutical Corporation Adn codant pour un recepteur mammalien (fb41a) et ses applications
WO2000052022A1 (fr) * 1999-03-01 2000-09-08 Millennium Pharmaceuticals, Inc. Proteines secretees et acides nucleiques codant pour elles
WO2000053753A2 (fr) * 1999-03-08 2000-09-14 Genentech, Inc. Activation ou inhibition de l'angiogenese et de la cardiovascularisation
WO2000070049A2 (fr) * 1999-05-19 2000-11-23 Incyte Genomics, Inc. Molecules de signalisation extracellulaires
WO2001016309A1 (fr) * 1999-08-27 2001-03-08 Takeda Chemical Industries, Ltd. Proteine recepteur couplee a une proteine g et adn correspondant

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5453363A (en) * 1985-10-23 1995-09-26 Boehringer Mannheim Gmbh Process for the activation of t-PA or Ing after genetic expression in prokaryotes
JPH10191989A (ja) * 1996-11-12 1998-07-28 Takeda Chem Ind Ltd ベータセルリン類の製造方法
WO2000034334A1 (fr) * 1998-12-10 2000-06-15 Synaptic Pharmaceutical Corporation Adn codant pour un recepteur mammalien (fb41a) et ses applications
WO2000052022A1 (fr) * 1999-03-01 2000-09-08 Millennium Pharmaceuticals, Inc. Proteines secretees et acides nucleiques codant pour elles
WO2000053753A2 (fr) * 1999-03-08 2000-09-14 Genentech, Inc. Activation ou inhibition de l'angiogenese et de la cardiovascularisation
WO2000070049A2 (fr) * 1999-05-19 2000-11-23 Incyte Genomics, Inc. Molecules de signalisation extracellulaires
WO2001016309A1 (fr) * 1999-08-27 2001-03-08 Takeda Chemical Industries, Ltd. Proteine recepteur couplee a une proteine g et adn correspondant

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Title
CHRISTAIN WECHSELBERGER ET AL.: "The mammalian homologues of frog Bv8 are mainly expressed in spermatocytes", FEBS LETTERS, vol. 462, 1999, pages 177 - 181, XP002173442 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7052674B2 (en) 2000-11-03 2006-05-30 The Regents Of The University Of California Prokineticin polypeptides, related compositions and methods

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