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WO2002036762A1 - Procede de fabrication de peptides - Google Patents

Procede de fabrication de peptides Download PDF

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Publication number
WO2002036762A1
WO2002036762A1 PCT/JP2001/009476 JP0109476W WO0236762A1 WO 2002036762 A1 WO2002036762 A1 WO 2002036762A1 JP 0109476 W JP0109476 W JP 0109476W WO 0236762 A1 WO0236762 A1 WO 0236762A1
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WO
WIPO (PCT)
Prior art keywords
amino acid
seq
acid sequence
peptide
fusion protein
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
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PCT/JP2001/009476
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English (en)
Japanese (ja)
Inventor
Takao Yamada
Masato Suenaga
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Takeda Pharmaceutical Co Ltd
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Takeda Chemical Industries Ltd
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Filing date
Publication date
Application filed by Takeda Chemical Industries Ltd filed Critical Takeda Chemical Industries Ltd
Priority to AU2001296023A priority Critical patent/AU2001296023A1/en
Publication of WO2002036762A1 publication Critical patent/WO2002036762A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • 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
    • C07K14/575—Hormones
    • C07K14/635—Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones

Definitions

  • the present invention comprises an amino acid sequence identical or substantially identical to the amino acid sequence of the 1st to 34th from the N-terminus of PTH (parathyroid hormone) at the N-terminus of the target peptide
  • PTH thyroid hormone
  • PTH thyroid hormone
  • DNA gene
  • DNA recombination technology Many attempts have been made to integrate DNA (gene) into a vector using DNA recombination technology and express it by transforming it into a microorganism.
  • the peptide is susceptible to degradation by the action of proteases in the cells of the expressing bacteria, and the desired peptide is often not obtained. Therefore, it is often expressed in the form of a fusion protein.
  • a fusion protein in which somatosustin is linked to the C-terminus of j8-galactosidase at methionine via Escherichia coli is expressed in E. coli and then cleaved with bromocyan (Science, 98, 1056). , (1977)).
  • this chemical cleavage method cannot be applied to peptides having methionine residues in the molecule.
  • the expression level of the target protein is reduced depending on the nucleotide sequence encoding the N-terminal region thereof (Japanese Patent Application No. 2000-229920). ). Therefore, when the target peptide is placed in the N-terminal region of the fusion protein, the expression level of the fusion protein may be greatly affected by the nucleotide sequence encoding the N-terminal region. Furthermore, a methionine derived from the translation initiation codon may be added depending on the N-terminal amino acid sequence of the fusion protein (J. Bacteriol., 169, 751, 1987). Required. Disclosure of the invention
  • the present inventors have conducted intensive studies on a method for efficiently producing a target peptide.
  • the target peptide is expressed in Escherichia coli using DNA recombination technology
  • the N-terminal of parathyroid hormone having a relatively high expression level is expressed. It was expressed as a fusion protein in which the target peptide was linked to the C-terminus of PTH (1-34) consisting of 34 amino acid residues on the side through a cleavage site of a protease.
  • enterokinase is used as a proteolytic enzyme, the enzyme recognizes a specific amino acid sequence of Asp-Asp-Asp-Asp-Lys, which is located on the C-terminal side.
  • the present invention Since the peptide bond of the fusion protein was cleaved, it was considered to be highly versatile, and it was found that the peptide bond of the fusion protein was cleaved with a protease to efficiently produce the target peptide. Furthermore, as a result of earnest research based on these, the present invention was completed. That is, the present invention
  • the target peptide is located at the C-terminus of a peptide containing the same or substantially the same amino acid sequence as the amino acid sequence 1 to 34 from the N-terminus of PTH (parathyroid hormone) via a cleavage site of a protease.
  • a method for producing the peptide of interest or a salt thereof which comprises subjecting the fusion protein obtained by linking the above to a peptide bond cleavage reaction with a protease.
  • PTH thyroid hormone
  • the target peptide via the cleavage site of the protease at the C-terminus of a peptide containing the same or substantially the same amino acid sequence as the amino acid sequence at positions 1 to 34 from the N-terminus.
  • a transformant transformed with a vector containing a DNA coding for a fusion protein ligated with is cultivated to express the fusion protein, and the expressed fusion protein is subjected to a peptide bond cleavage reaction by a protease.
  • fusion protein according to (16) or a salt thereof, wherein apelin is a peptide containing the amino acid sequence represented by SEQ ID NO: 1.
  • fusion protein according to (15) or a salt thereof, wherein the fusion protein comprises the amino acid sequence represented by SEQ ID NO: 25;
  • the present invention relates to a method for producing the peptide of interest or a salt thereof, which comprises subjecting a peptide-linked fusion protein to a cleavage reaction of a peptide bond by the protease.
  • Figure 1 shows the results of analyzing the PTH-aperin-136 purification step from the cells by SDS-PAGE.
  • lane 1 is molecular weight marker 1
  • lane 2 is bacterial cells
  • lane 1 is molecular weight marker 1
  • lane 2 is bacterial cells
  • the DS-120T eluate is shown.
  • Figure 2 shows the results of treating PTH-apelin-36 with enterokinase and analyzing the apelin-36 purification step by SDS-PAGE.
  • lane 1 is the molecular weight marker
  • lane 2 is the PTH-apelin-36 fusion protein
  • lane 3 is the aperin-36 standard
  • lane 4 is the PTH-aperin-treated with enterokinase.
  • lane 5 is SP-5 PW eluate
  • lane 6 is ODS-12
  • the peptide of interest in the method of the present invention may be any peptide that does not have a cleavage site for the protease used in the method of the present invention in the molecule. May be.
  • the target peptide has about 10 to 100, preferably about 20 to 100 amino acid residues.
  • a peptide having a group is used. Specific examples thereof include apelin (Bioche Biophys. Res. Commun., 251, 471-476, (1998)), GPR8 ligand, ⁇ AQ ligand, insulin, endothelin, atrial natriuresis.
  • Peptides Peptides, somatosustin, vasopressin, calcitonin, bone morphogenetic factors, insiulinoto, oral pins, angiotensin, bradykinin, enkephalin, endorphin, various opioid peptides and the above peptide fragments.
  • Examples of the above apelin include a polypeptide having an ability to bind to a receptor protein having an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 3 described in WO 99/33976. Pseudo ".
  • polypeptide having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 described in WO 99/33976 a polypeptide having an amino acid sequence represented by SEQ ID NO: 1 and about 50 to 99. It contains an amino acid sequence having a homology of 9% (preferably 70 to 99.9%, more preferably 80 to 99.9%, and still more preferably 90 to 99.9%), and is represented by SEQ ID NO: 1.
  • a polypeptide having substantially the same activity as the polypeptide containing the amino acid sequence to be used is used.
  • polypeptide containing a partial sequence examples include: (1) a polypeptide containing a partial sequence of a precursor containing an amino acid sequence represented by SEQ ID NO: 15, SEQ ID NO: 38, SEQ ID NO: 40 or SEQ ID NO: 42 In addition to the peptide, (2) a polypeptide having substantially the same activity as the partial peptide of the precursor represented by SEQ ID NO: 15, (3) The same or the same as the amino acid sequence represented by SEQ ID NO: 38 Before containing a substantially identical amino acid sequence, a polypeptide having substantially the same activity as that of a partial peptide of a precursor, ⁇ identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 40 A polypeptide having substantially the same activity as the partial peptide of the precursor having the amino acid sequence of SEQ ID NO:
  • substantially the same activity examples include receptor binding activity, signal transduction activity and the like. “Substantially the same” means that the receptor binding activity and the like are the same in nature. Therefore, the quantitative factors such as the strength of the receptor binding activity and the molecular weight of the polypeptide may be different.
  • apelin examples include the amino acid sequence represented by SEQ ID NO: 1 or a partial sequence thereof described in WO99 / 33976, and the amino acid sequence represented by SEQ ID NO: 15. 3 a partial sequence of the amino acid sequence represented by SEQ ID NO: 38, 4 a partial sequence of the amino acid sequence represented by SEQ ID NO: 40, 5 a partial sequence of the amino acid sequence represented by SEQ ID NO: 42 Examples thereof include polypeptides derived from mouse brain, rat brain, mouse brain, mouse small intestine, mouse hypothalamus, mouse stomach, human hypothalamus or human lung containing partial sequences.
  • amino acid sequence represented by SEQ ID NO: 1 or a partial sequence thereof (2) the partial sequence of the amino acid sequence represented by SEQ ID NO: 15; (3) the amino acid sequence represented by SEQ ID NO: 38
  • a polypeptide containing an amino acid sequence in which one or more amino acids are substituted, deleted, added or inserted into a peptide or a partial peptide thereof is a polypeptide containing substantially the same amino acid sequence Examples include peptides.
  • amino acid sequence represented by SEQ ID NO: 1 or a partial sequence thereof For example: 1) the amino acid sequence represented by SEQ ID NO: 1 or a partial sequence thereof; 2) the partial distribution sequence of the amino acid sequence represented by SEQ ID NO: 15; 3) the partial sequence of the amino acid sequence represented by SEQ ID NO: 38 , 4 SEQ ID NO: 40 1 to 7 or less, preferably 1 to 5 or less, and more preferably 1 or more in the partial sequence of the amino acid sequence represented by or in the partial sequence of the amino acid sequence represented by SEQ ID NO: 42 Amino acid sequence in which three or less amino acids are deleted, (1) the amino acid sequence represented by SEQ ID NO: 1 or a partial sequence thereof, (2) partial sequence of the amino acid sequence represented by SEQ ID NO: 15, (3) SEQ ID NO: 1 or more of the partial sequence of the amino acid sequence represented by 38, 4 the partial sequence of the amino acid sequence represented by SEQ ID NO: 40, or 5 the partial sequence of the amino acid sequence represented by SEQ ID NO: 42 Or less, preferably 1 or more and
  • apelin or partial peptides include those in which the N-terminal side of G1n is cleaved in vivo and the Gln is pyroglutamine-oxidized.
  • the precursor may be any protein as long as it contains the above-mentioned apelin as its partial sequence, and is specifically represented by SEQ ID NO: 15, 38, 40 or 42. And a protein containing an amino acid sequence.
  • the molecular weight of array is about 1000 to 1000 daltons, preferably about 1000 to about 500 daltons, more preferably about 100 to about 300 daltons. 0 daltons.
  • polypeptide having a partial sequence of the amino acid sequence represented by SEQ ID NO: 15, 38, 40 or 42 is a polypeptide having a partial sequence of the amino acid sequence represented by SEQ ID NO: 15, 38, 40 or 42.
  • a peptide having the amino acid sequence of SEQ ID NO: 15, 38, 40 or 42 or a peptide having the amino acid sequence of SEQ ID NO: 15, 38, 40 or 42 Peptides having a sequence are preferably used.
  • the peptide represented by the 65th to 77th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 15, 38, 40 or 42 or the amino acid (G1n) at the N-terminal thereof is pyroglutamine-oxidized (PGlu Arg Pro Arg Leu Ser His Lys Gly Pro Met Pro Phe) is preferred.
  • a peptide having a partial amino acid sequence of the amino acid sequence represented by pGlu Arg Pro Arg Leu Ser His Lys Gly Pro Met Pro Phe is also preferably used as the (poly) peptide of the present invention.
  • apelin-36 described in Biodiem. Biophys. Res. Commun., 251, 471-476, (1998) (polypeptide represented by the amino acid sequence represented by SEQ ID NO: 1 in the present specification).
  • Peptide polypeptide represented by the amino acid sequence at positions 24 to 36 of SEQ ID NO: 1 in the present specification
  • any peptide having ligand activity may be used.
  • SEQ ID NO: 1 SEQ ID NO: 15, SEQ ID NO: 38, SEQ ID NO: 40 and SEQ ID NO: 42 described in WO 99/33976 are, respectively, SEQ ID NO: 61 and SEQ ID NO: 61 in the present specification. This corresponds to SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64 and SEQ ID NO: 65.
  • GPR8 ligand examples include a ligand activity for a seven-transmembrane receptor protein GPR8 (0, Dowd, BF et al., Genomics, 28, 84-91, 1995), for example, binding to GPR8.
  • Activity cell stimulating activity on GPR8 expressing cells (e.g., arachidonic acid release, acetylcholine release, intracellular Ca 2+ release, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein , Phosphorylation of c-fos, activation of c-fos, decrease in pH, activity to promote GTPrS binding activity, etc.).
  • an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 27 includes, for example, about 60% or more (preferably about 70%) of the amino acid sequence represented by SEQ ID NO: 27 More preferably, the amino acid sequence has 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: 27 include, for example, a peptide having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 27; : Peptides having substantially the same properties as those having the amino acid sequence represented by 27 are preferred.
  • (1) one or more (preferably about 1 to 20, more preferably about 1 to 10) amino acids in the amino acid sequence represented by SEQ ID NO: 27 are deleted 1 or 2 or more (preferably about 1 to 40, more preferably about 1 to 30, especially preferably about 1 to 20) in the amino acid sequence represented by SEQ ID NO: 27 ) Amino acid sequence to which amino acids are added, 3 one or more amino acids in the amino acid sequence represented by SEQ ID NO: 27 (preferably Preferably, about 1 to 20 amino acids, more preferably about 1 to 10 amino acids are substituted with another amino acid, or a peptide containing an amino acid sequence combining them is also used.
  • amino acids in the amino acid sequence represented by SEQ ID NO: 27 are deleted 1 or 2 or more (preferably about 1 to 40, more preferably about 1 to 30, especially preferably about 1 to 20) in the amino acid sequence represented by SEQ ID NO: 27 )
  • Amino acid sequence to which amino acids are added 3 one or more amino acids in the amino acid sequence represented by SEQ ID NO: 27 (preferably Prefer
  • a peptide having an amino acid sequence represented by SEQ ID NO: 27 is used as the GPR8 ligand.
  • the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 41 includes, for example, about 60% or more (preferably 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: 41 include, for example, a peptide having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 41; Peptides having substantially the same properties as those having the amino acid sequence represented by SEQ ID NO: 41 are preferred.
  • Substantially the same activity includes, for example, a binding activity to a ZAQ receptor, a signal transduction action via a ZAQ receptor, and the like. Substantially the same indicates that their activities are the same in nature. Accordingly, it is preferable that the activity of binding to the ZAQ receptor and the activity of signal transduction via the ZAQ receptor are equivalent (eg, about 0.5 to 2 times). Quantitative factors such as the molecular weight of the peptide and the peptide may be different.
  • ZAQ ligand examples include: (1) one or more (preferably about 1 to 30, more preferably about 1 to 20) amino acids in the amino acid sequence represented by SEQ ID NO: 41; 2 or more amino acid sequences represented by SEQ ID NO: 41 (preferably about 1 to 40, more preferably about 1 to 30, particularly preferably Is an amino acid sequence to which about 1 to 20 amino acids have been added, 3 1 or 2 or more (preferably about 1 to 30 amino acids, more preferably about 1 to 30 amino acids in the amino acid sequence represented by SEQ ID NO: 41) Is about 1 to 20 amino acids) Peptides containing an amino acid sequence substituted with an amino acid or an amino acid sequence obtained by combining them are also used.
  • a peptide having an amino acid sequence represented by SEQ ID NO: 41 is used.
  • the target peptide has the N-terminus at the left end (amino end) and the C-terminus at the right end (capillary terminal) according to the convention of peptide labeling.
  • Apelin_36 containing the amino acid sequence represented by SEQ ID NO: 1; GPR8 ligand containing the amino acid sequence represented by SEQ ID NO: 27; ZAQ ligand containing the amino acid sequence represented by SEQ ID NO: 41
  • the C-terminus is usually a sulfoxyl group (—COOH) or a carboxylate (—COO—).
  • PTH amino acid sequence identical or substantially identical to the amino acid sequence of the 1st to 34th from the N-terminal of PTH having the amino acid sequence represented by SEQ ID NO: 5 is specifically described as PTH (1-34).
  • Any peptide may be used as long as it contains the peptide, and more specifically, a peptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 3 and the like can be mentioned. .
  • PTH (1 ⁇ 34) in addition to the peptide containing the amino acid sequence represented by SEQ ID NO: 3, substantially the same properties as the peptide containing the amino acid sequence represented by SEQ ID NO: 3
  • the peptide having SEQ ID NO: As long as the peptide containing the amino acid sequence represented by 3 is used, any peptide may be used as long as it has the property of efficiently producing the target peptide.
  • amino acid sequence in which one or more and five or less, preferably one or more and three or less amino acids are added (or inserted) to the sequence, or one or more and five or less in the amino acid sequence represented by SEQ ID NO: 3 Preferably, a peptide containing an amino acid sequence in which one or more and three or less amino acids are substituted with another amino acid Throw.
  • a salt with a physiologically acceptable base eg, an alkali metal
  • an acid organic acid or inorganic acid
  • Addition salts are preferred.
  • Such salts include, for example, 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) , Tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, and benzenesulfonic acid).
  • 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
  • Tartaric acid citric acid, malic acid, oxalic acid
  • benzoic acid methan
  • the fusion protein (including the fusion peptide) used in the method of the present invention can be produced by culturing a transformant in a vector containing a DNA encoding the fusion protein described below and expressing the fusion protein.
  • the fusion protein may be any fusion protein, as long as the peptide of interest is bound to the C-terminus of PTH (1-34) via a cleavage site of a proteolytic enzyme.
  • the target peptide is aperin-36, specifically, for example, Ser Val Ser Glu ulie Gin Leu Met His Asn Leu Gly Lys His Leu Asn Ser Me t Glu Arg Val Glu u Trp Leu Arg Lys Lys Leu Gin Asp Val His Asn Phe Asp Asp Asp Asp Lys Le u Val Gin Pro Arg Gly Ser Arg Asn Gly Pro Gly Pro Trp Gin Gly Gly Arg Ar g Lys Phe Arg Arg Gin Arg Pro Arg Leu Ser His Lys Gly Pro Met Pro Phe (SEQ ID NO: : A protein containing the amino acid sequence represented by 25).
  • the protein containing the amino acid sequence represented by SEQ ID NO: 25 is the amino acid sequence of PTH (1-34) (Ser Val Ser Gluie Gin Leu Met His Asn Leu Gly Lys His Leu Asn Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gin Asp Val His Asn Phe; an amino acid sequence encoding an enterokinase cleavage site (Asp Asp Asp Lys; SEQ ID NO: 7) is bound to the C-terminal of SEQ ID NO: 3); Further, at the C-terminus, an amino acid sequence (Leu Val Gin Pro Arg Gly Ser Arg Asn Gly Pro Gly Pro Trp Gin Gly Gly Arg Ar g Lys Phe Arg Arg Gin Arg Pro Arg Leu Ser His Lys Gly Pro Met Pro Phe; This shows that SEQ ID NO: 1) is bound.
  • the protein containing the amino acid sequence represented by SEQ ID NO: 9 used in Example 1 was the amino acid sequence of PTH (1-34) (Ser Val Ser Glulie Gin Leu Met His Asn Leu Gly Lys His Leu Asn Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gin Asp Val His Asn Phe; C-terminal of SEQ ID NO: 3), enterokinase cleavage site via linker (Gly Ser Gly Ser Gly; SEQ ID NO: 57) Asp Asp Asp Lys; SEQ ID NO: 7) binds to the amino acid sequence encoding the target peptide (apelin-36) (Leu Val Gin Pro Arg Gly Ser Arg Asn Gly Pro Gly Pro Trp Gin Gly Gly Arg Arg Lys Phe Arg Arg Gin Arg Pro Arg Leu Ser His Lys Gly Pro Met Pro Phe;
  • the target peptide is a GPR8 ligand, specifically, for example, Ser Val Ser Glulie Gin Leu Met His Asn Leu Gly Lys His Leu Asn Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gin Asp Val His Asn Phe Asp Asp Asp Asp Lys Trp Tyr Lys His Val Ala Ser Pro Arg Tyr His Thr Val Gly Arg Ala Ala Gly Leu Leu Met Gly Leu (SEQ ID NO: 39) .
  • the protein containing the amino acid sequence represented by SEQ ID NO: 39 is the amino acid sequence of PTH (1 -34) (Ser Val Ser Glu He Gin Leu Met His Asn Leu Gly Lys His Leu Asn Ser Met Glu Arg Val Glu Trp Leu
  • An amino acid sequence (Asp Asp Asp Asp Lys; SEQ ID NO: 7) encoding an enterokinase cleavage site is bound to the C-terminal of Arg Lys Lys Leu Gin Asp Val His Asn Phe;
  • the amino acid sequence encoding the target peptide (GPR8 ligand) Trp Tyr Lys His Val Ala Ser Pro Arg Tyr His Thr Val Gly Arg Ala Ala Gly Leu Leu Met Gly Leu; SEQ ID NO: 27). Show.
  • the protein containing the amino acid sequence represented by SEQ ID NO: A used in Example 2 was the amino acid sequence of PTH (1-34) (Ser Val Ser Glu He Gin Leu Met His Asn Leu Gly Lys His Leu Asn Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gin Asp Val His Asn Phe; (Gly Ser Gly Ser Gly; SEQ ID NO: 57) binds to an amino acid sequence encoding an enterokinase cleavage site (Asp Asp Asp Asp Lys; SEQ ID NO: 7), and further has a target peptide (GPR8 ligand) at its C-terminus.
  • PTH 1-34
  • Gly Ser Gly Ser Gly; SEQ ID NO: 57
  • the protein containing the amino acid sequence represented by SEQ ID NO: 53 is the amino acid sequence of PTH (1 to 34) (Ser Val Ser Glu He Gin Leu Met His Asn Leu Gly Lys His Leu Asn Ser Met Glu Arg Val Glu Trp Leu
  • An amino acid sequence (Asp Asp Asp Asp Lys; SEQ ID NO: 7) encoding an enterokinase cleavage site is bound to the C-terminal of Arg Lys Lys Leu Gin Asp Val His Asn Phe;
  • the amino acid sequence encoding the target peptide (ZAQ ligand) (A1 a Val lie Thr Gly Ala Cys Glu Arg Asp Val Gin Cys Gly Ala Gly Thr Cys Cys Ala lie Ser Leu Trp Leu Arg Gly Leu Arg Met Cys Thr Pro Leu Gly Arg Gl u Gly Glu Glu Cys His Pro Gly Ser His Lys Val Pro Phe Phe Arg Lys
  • the protein containing the amino acid sequence represented by SEQ ID NO: 43 used in Example 3 was the amino acid sequence of PTH (1-34) (Ser Val Ser Glulie Gin Leu Met His Asn Leu Gly Lys His Leu Asn Ser Met Glu Arg Val Glu Trp Leu Ar g Lys Lys Leu Gin Asp Val His Asn Phe; SEQ ID NO: 3), at the C-terminus, an amino acid sequence (Asp Asp) encoding an enterokinase cleavage site via a linker (Gly Ser Gly Ser Gly; SEQ ID NO: 57).
  • Asp Asp Lys SEQ ID NO: 7 binds, and further has an amino acid sequence encoding a target peptide (ZAQ ligand) (Ala Val Thr Thr Gly Ala Cys Glu Arg Asp Val Gin Cys Gly Ala Gly) Thr Cys Cys Ala He Ser Leu Trp Leu Arg Gly Leu Arg Met Cys Thr Pro Leu Gly Arg Glu Gly Glu Glu Cys His Pro Gly Ser His Lys Val Pro Phe Phe Arg Lys Arg Lys His His Thr Cys Pro Cys Leu Pro Asn Leu Leu Cys Ser Arg Phe Pro Asp Gly Arg Tyr Arg Cys Ser Met Asp Leu Lys Asn lie Asn Phe; SEQ ID NO: 41)
  • fusion protein when the fusion protein is cleaved with a protease, a molecular weight of Ala, Gly, Ser, etc.
  • a linker sequences selected from small amino acid residues eg, Gly Ser Gly Ser r Gly; SEQ ID NO: 57, etc.
  • the resulting fusion protein was used.
  • the DNA encoding the fusion protein (including the fusion peptide) used in the method of the present invention may be obtained by chemically synthesizing the entire nucleotide sequence.
  • a production method for example, a known phosphoramidite method, a phosphoric acid triester method, a diester method, an octahydrogen phosphonate method, or the like may be used. It can be created by ligation using 4 DNA ligase.
  • DNA encoding the fusion protein thus obtained is, if desired peptide is Aperin one 3 6, for example, TCTGTGTCCGAGATTCAGTTAATGCA TAACCTTGGCAAACATTTGAACTCGATGGAGCGTGTAGAATGGCTGCGTAAGAAGTTGCAGGATGTGCACAA TTTTGATGACGACGACAAGCTGGTGCAGCCCAGAGGGTCAAGGAATGGGCCAGGGCCCTGGCAGGGAGGTCG GAGGAAATTCCGCCGCCAGCGGCCCCGCCTCTCCCATAAGGGACCCATGCCTTTC (2 6 SEQ ID NO) And so on.
  • the DNA represented by the nucleotide sequence represented by SEQ ID NO: 26 represents PTH (1—34).
  • a base sequence encoding a enterokinase cleavage site (GATGACGi CGACAAG; This shows that the nucleotide sequence (CTGGTGCAGCCCAGAGGGTCAAGGAATGGGCCAGGGCCCTGGCAGG GAGGTCGGAGGAAATTCCGCCGCC AGCGGCCCCGCCTCTCCCATAAGGGACCCATGCCTTTC; SEQ ID NO: 2) encoding the peptide (apelin-36) is bound.
  • the target peptide is a GPR8 ligand, for example, TCTGTGTCGGAGCCTGCGTGCGTGCGTGCCTGGGCCTGGGCCTGCGTGCCTGGGCCTGCGTGCCTGGGCCTGCGTGCCTGGGCCTGCGTGCCTGGGCCTGCGTGCCTGGGCCTGCGTGCCTGGGCCTGCGCGCCTGGGCCTGCGCGCCTGGGCCTGCGCGCCTGGGCCTGCGCGCCTGGGCCTGCGCGCCTGGGCCTGCGCGCCTGGGCCTGCGCGCCTGGGCCTGCGCGCCTGGGCCTGCGCGCCTGGGCCTGCGCGCCTGGGCCTGCGCGCCTGGGCCTGCGCGCCTGGGCCTGCGCGCCTGGGCCTGCGCGCCTGGGCCTGCGCGCCTGGGCCTGCGCGCCTGGGCCTGCGCGCCTGGGCCTGCGCGCCTGGGCCTGCGCGCCTGGGCCTGCGCGCCTGGGCCTGCGCGCCTGGGCCTGCGCGCCTGGGCCTGCGCGCCTGGGCCTGCGCGCCTGGGCCTGCGCG
  • the DNA represented by the nucleotide sequence represented by SEQ ID NO: 40 is a nucleotide sequence coding for PTH (1-34) (TCTGTGTCCGAGATTCAGTTAATGCATAACCTTGGCAAACATTTGAACTCGA TGGAGCGTGTAGAATGGCTGCGTAAGAAGTTGCAGGATGTGCACAATTTT; The nucleotide sequence (GATGACGACGACAAG; SEQ ID NO: 8) is bound to the 3 ′ end, and the nucleotide sequence (TGGTATMACATGTGGCGAGCCCGCGTTATCATACCGTGGGCCG TGCGGCGGGCCTGCTGATGGGCCTG; SEQ ID NO: 34) is linked to the 3 ′ end of the nucleotide sequence.
  • peptide is Z AQ ligand, for example, TCTGTGTCCGAGATTCAGTTAATGCA TAACCTTGGCAAACATTTGAACTCGATGGAGCGTGTAGAATGGCTGCGTAAGAAGTTGCAGGATGTGCACAA TTTTGATGACGACGACAAGGCGGTGATTACCGGTGCGTGCGAACGTGATGTGCAGTGCGGTGCGGGTACCTG CTGCGCGATTAGCCTGTGGCTGCGTGGTCTGCGTATGTGCACCCCGCTGGGTCGTGAAGGTGAAGAATGCCA TCCGGGTAGCCATAAAGTGCCGTTCTTCCGTAAACGTAAACATCATACCTGCCCGTGCCTGCCGAACCTGCT GTGCAGCCGTTTCCCGGATGGTCGTTATCGTTGCAGCATGGATCTGAAAAACATTAACTTT (SEQ ID NO: 5 4) a D NA and the like containing the base sequence represented by.
  • the DNA represented by the nucleotide sequence represented by SEQ ID NO: 54 is a nucleotide sequence encoding PTH (1-34) (TCTGTGTCCGAGATTCAGTTAATGCATMCCTTGGCAMCATTTGMCTCGA TGGAGCGTGTAGAATGGCTGCGTAAGAAGTTGCAGGATGTGCACAATTTT; SEQ ID NO: 4).
  • nucleotide sequence encoding Enterokinaze cleavage site (GATGACGACGACAAG; SEQ ID NO: 8) is attached further to the 3 'end nucleotide sequence encoding the desired peptide (ZAQ ligand) (GCGGTGATTACCGGTGCGTGCGAACGTGATGTGCAGTGCGGTGCGG GTACCTGCTGCGCGATTAGCCTGTGGCTGCGTGGTCTGCGTATGTGCACCCCGCTGGGTCGTGAAGGTGAAG AATGCCATCCGGGTAGCCATAAAGTGCCGTTCTTCCGTAAACGTAAACATCATACCTGCCCGTGCCTGCCGA ACCTGCTGTGCAGCCGTTTCCCGGATGGTCGTTATCGTTGCAGCATGGATCTGAAAAACATTAACTTT; sequence No .: 42) indicates that they are linked.
  • the nucleotide sequence (SEQ ID NO: 10, SEQ ID NO: 30; SEQ ID NO: 44) used in the examples of the present invention is a nucleotide sequence encoding PTH (1-34) (TCTGTGTC CGAGATTCAGTTAATGCATAACCTTGGCAAACATTTGAACTCGATGGAGCGTGTAGAATGGCTGCGTAAGAA GTTGCAGGATGTGCACATT; : 4) via a base sequence (GGTTCTGGTTCTGGT; SEQ ID NO: 58) coding for a linker (Gly Ser Gly Ser Gly; SEQ ID NO: 57) at the 3 'end of the base sequence (coding for an enterokinase cleavage site) GATGAC GACGACAAG; SEQ ID NO: 8) binds, and further, a base sequence (SEQ ID NO: 2, SEQ ID NO: 34) encoding the target peptide (apelin-36, GPR8 ligand, ZAQ ligand) at its 3 '
  • Ala and Gly are used to prevent the fusion protein from being cleaved by the proteolytic enzyme due to steric hindrance of the fusion protein.
  • a DNA chain eg, GGTTCTG GTTCTGGT
  • a linker eg, about 1 to 5 linkers selected from amino acid residues having a low molecular weight such as, Ser (eg, Gy Ser Gly Ser Gly; SEQ ID NO: 57, etc.) SEQ ID NO: 58) was inserted at the 5 ′ end of the base sequence encoding the enterokinase cleavage site.
  • a DNA (plasmid) having an ATG at the 5 ′ end and a region encoding the fusion protein downstream thereof, and thus having a translation stop codon is a known DNA synthesized by chemical synthesis or genetic engineering. It can be produced by processing the cDNA of the protein or the DNA of the protein derived from the chromosome.
  • the target peptide (apelin-136, GRP8 ligand, ZAQ ligand, etc.) is linked to the C-terminal of PTH (1-34) via a protease cleavage site.
  • the DNA encoding the ligated fusion protein is converted to DNA encoding the mutin of the target peptide (apelin-36, GPR8 ligand, ZAQ ligand, etc.) using conventional gene technology, for example, site-directed mutagenesis technology.
  • Site-directed mutagenesis techniques that can be performed are well known and are described by R. F. Lazer (Lather, RF) and J. P. Lecoq (J.P.), Genetic Engineering. ), Academic Press (1983), pp. 31-50. Mutagenesis using oligonucleotides is described in M. Smith (M.) and S. Gillam (S.), Genetic Engineering: Principles and Methods, Plenum Pums, Inc. (1981), Volume 3, It is shown on pages 1-32.
  • plasmids used as vectors containing the DNA encoding the fusion protein include, for example, pBR322 [Gene, 2_, 95 (1977)], pBR313 [gene] derived from Escherichia coli.
  • the DNA preferably has a promoter upstream of the ATG, and the promoter may be any promoter as long as it is appropriate for the host used for the production of the transformant.
  • Trp promoter for example, in Escherichia coli, the Trp promoter, lac promoter, recA promoter, ⁇ PL promoter, lpp promoter, T7 promoter, and the like can be mentioned.
  • the T7 promoter When the T7 promoter system is used, the T7 promoter includes 17 types of promoters found on T7 DNA (J.L. Oakley et al., ⁇ ;. Nat.
  • a terminator operating in an Escherichia coli system preferably ⁇
  • a mineral factor [FW Studier et al., L Mol. Biol., 189: 113-130 (1986) ] Is used.
  • ⁇ 7 RNA polymerase DNA includes ⁇ 7 DNA CF. W. Studier et al., J. Mol. Biol., 189: 113-130 (1986)].
  • the vector is preferably constructed by incorporating the T7 promoter and T7 promoter into the above vector.
  • Such vectors include pET-1, ET-
  • the transformant of the present invention can be prepared by subjecting the expression plasmid obtained by the above method to a known method [e.g., Cohen S, N, et al., Processing.National “Academy” Op. “Science (Pro. Natl. Acad. Sci. USA.), 6_9, 2110 (1972)].
  • a known method e.g., Cohen S, N, et al., Processing.National “Academy” Op. "Science (Pro. Natl. Acad. Sci. USA.), 6_9, 2110 (1972).
  • Examples of the host of the microorganism to be transformed include Escherichia sp.
  • Escherichia coli examples include Escherichia coli (Escherichia coli). Specifically, Escherichia coli K12DH1 [Proceedings of National Academy of Sciences (Obs.)] Pro Natl. Acad. Sci.
  • T7 RNA polymerase DNA (T7DNA1) [FW Studier et al., J. Mol. Biol. 189: 113-130 (1986)] is used as a host for the transformant.
  • MM 294 strain and BL 21 strain in which ⁇ phage into which T 7 DNA1 has been incorporated are lysogenized are used.
  • the lac promoter whose expression is induced by isopropyl-11-thio3-D-galactopyranoside (sometimes abbreviated as IPTG) is used as the promoter of T7 DNA 1.
  • IPTG isopropyl-11-thio3-D-galactopyranoside
  • the fusion protein can be produced by culturing the above-described transformant in a medium and collecting the produced fusion protein.
  • the pH of the medium is preferably about 6-8.
  • M9 medium containing glucose and casamino acid M9 medium containing glucose and casamino acid
  • 2 XYT medium M9 medium containing.
  • a drug such as 3 jS-indolylacrylic acid or isopropyl-1-
  • cultivation is usually performed at about 15 to 43 ° C for about 3 to 24 hours, and if necessary, aeration and stirring can be applied.
  • the culturing is carried out at a temperature of about 15 to 36 ° C, preferably about 30 to 36 ° C.
  • the inactivation of the Aclts repressor is preferably carried out at about 37 ° C to 42 ° C.
  • a drug such as mitomycin C or naldixic acid, or irradiate with ultraviolet light, or culture.
  • the pH of the solution may be changed to the alkaline side.
  • the fusion protein can be produced by culturing the above transformant, producing and accumulating the fusion protein in a culture, and collecting this.
  • T7 promoter system When a T7 promoter system is used, (1) IPTG is added when expressing T7 DNA (RNA polymerase DNA) linked downstream of the lac promoter, or (2) ⁇ When expressing T7 DNA (RNA polymerase DNA) linked downstream of the PL promoter, the T7 phage generated by the T7 phage RNA polymerase 1 can be specifically increased by raising the culture temperature. Activate
  • the cells After culturing, the cells are collected by a known method, suspended in a buffer, for example, treated with a protein denaturant, treated with an enzyme such as lysozyme, sonicated, treated with glass beads, treated with French press, and frozen.
  • a buffer for example, treated with a protein denaturant, treated with an enzyme such as lysozyme, sonicated, treated with glass beads, treated with French press, and frozen.
  • the cells are disrupted by thawing or the like, and the supernatant is obtained by a known method such as centrifugation.
  • a generally known protein purification method may be used. For example, gel filtration, ion exchange chromatography It can be carried out by appropriately combining chromatography, adsorption chromatography, high performance liquid chromatography, affinity chromatography, hydrophobic chromatography, electrophoresis and the like.
  • the proteolytic enzyme used for the peptide bond cleavage reaction may be any of those known as proteolytic enzymes, and the "proteolytic enzyme" of the "proteolytic enzyme cleavage site" to be used may be used. Good to use.
  • any protease capable of cleaving the cleavage site of the protease may be a protease other than the “protease” of the “cleavage site of the protease” to be used.
  • novel proteases found in the future may be used.
  • enterokinase for example, enterokinase, factor-XA, thrombin and the like are preferable, and enterokinase is particularly preferably used.
  • the amount of proteolytic enzyme used per mg fusion protein is from 0.01 to 100 units, preferably from 0.1 to 10 units.
  • a sequence (Asp Asp Asp Asp Lys; SEQ ID NO: 7) indicating an entokinase cleavage site is ligated to the C-terminus of PTH (1-34).
  • the target peptide does not have the amino acid sequence represented by SEQ ID NO: 7.
  • Factor-I Xa a sequence showing a factor-Xa cleavage site at the C-terminal of PTH (1-34) (lie Glu Gly Arg (SEQ ID NO: 11) (base sequence: ATT GAAGGCCGC (SEQ ID NO: 12))
  • thrombin a sequence showing a thrombin cleavage site at the C-terminus of PTH (1-34) (G Pro Arg (SEQ ID NO: 13) (base sequence: GGCCCGCGC (Encoded by DNA having SEQ ID NO: 14))).
  • the target peptide When using factor Xa, the target peptide preferably does not have the amino acid sequence represented by SEQ ID NO: 11, and when thrombin is used, the target peptide is represented by the amino acid represented by SEQ ID NO: 13. Preferably, they have no sequence.
  • the reaction temperature of the peptide bond cleavage reaction by the protease is about 0 ° (: ⁇ 60 ° C). Any temperature between 0 ° C and 40 ° C is more preferable.
  • the reaction buffer used is not particularly restricted but includes, for example, Tris-HCl buffer, Tris-acetate buffer, phosphate buffer, borate buffer and the like.
  • the pH in the reaction may be anywhere from ⁇ 1 to 12, but is preferably between pH4 and 8.
  • a generally known peptide purification method may be used. For example, gel filtration, ion-exchange chromatography, high-performance liquid chromatography, affinity chromatography, hydrophobic chromatography, thin-layer chromatography, electrophoresis and the like can be appropriately combined.
  • the target peptide can be lyophilized to a powder if necessary. During lyophilization, a stabilizer such as solpitol, mannitol, dextrose, maltose, trehalose, or glycerol can be added.
  • the C-terminus of the target peptide obtained by the production method of the present invention is an amide (1-CONH). 2 ) It may be a carboxyl group (-COOH), a carboxylate (-COO-), an alkyl amide (one CONHR) or an ester (-COOR).
  • R of the ester le or alkyl amides such as methyl, Echiru, n- propyl, Flip E alkyl groups such as isopropyl or n- butyl, cyclopentyl, C 3, such as cyclohexyl cyclo - 8 cycloalkyl group, phenyl, alpha — C 6 such as naphthyl — 12 aryl group, phenyl such as benzyl, phenethyl, benzhydryl
  • C 7 ⁇ 4 aralkyl groups such as _ 2 alkyl or mono-naphthyl-C- 2 alkyl such as 1-naphthylmethyl, pivaloyloxymethyl group widely used as an oral ester and the like.
  • the peptide or its amide or its ester or its salt can be mixed with sterilized water, human serum albumin (HSA), physiological saline or other known physiologically acceptable carriers to provide a safe drug. It can be administered parenterally or topically to mammals (eg, humans, monkeys, mice, etc.). For example, the daily dose per person may be about 0. O.lmg-5 Omg, preferably about 0. lmg—l Onig can be administered parenterally, such as by intravenous or intramuscular injection.
  • HSA human serum albumin
  • the preparation containing the target peptide of the present invention also contains other physiologically acceptable active ingredients such as salts, diluents, adjuvants, other carriers, buffers, binders, surfactants, and preservatives. It may be.
  • Parenteral preparations can be sterile aqueous solutions or suspension ampules in physiologically acceptable solvents or sterile powders, which can be diluted before use with physiologically acceptable diluents (usually a peptide solution). It is provided as an ampoule (obtained by lyophilization).
  • DNA Deoxylipo nucleic acid
  • RNA Liponucleic acid
  • a 1 a Alanine
  • Th r Threonine Cy s
  • ATP triphosphate SEQ ID NOs in the sequence listing in the present specification indicate the following sequences.
  • amino acid sequence of aperin-36 is shown.
  • 1 shows the base sequence of synthetic DNA encoding apelin_36.
  • FIG. 3 shows the amino acid sequence of PTH (1-84).
  • Fig. 3 shows the nucleotide sequence of a synthetic DNA encoding PTH (1-84). [SEQ ID NO: 7]
  • FIG. 1 shows an amino acid sequence representing an enterokinase cleavage sequence.
  • FIG. 1 shows the nucleotide sequence of a synthetic DNA encoding an enterokinase cleavage sequence.
  • Example 1 shows the amino acid sequence of the PTH (1-34) -apelin-136 fusion protein used in Example 1 described later.
  • Example 1 shows the nucleotide sequence of a synthetic DNA encoding a PTH (1-34) -apelin-136 fusion protein used in Example 1 described later.
  • FIG. 1 shows the nucleotide sequence of a synthetic DNA encoding a factor Xa cleavage sequence.
  • FIG. 1 shows an amino acid sequence representing a thrombin cleavage sequence.
  • FIG. 1 shows the nucleotide sequence of a synthetic DNA encoding a thrombin cleavage sequence.
  • Example 1 shows the nucleotide sequence of DNA oligomer # 5 used in Example 1 described later.
  • [SEQ ID NO: 20] 3 shows the nucleotide sequence of DNA oligomer # 6 used in Example 1 described later.
  • Example 1 shows the nucleotide sequence of DNA oligomer # 9 used in Example 1 described later.
  • Example 1 shows the nucleotide sequence of DNA oligomer # 10 used in Example 1 described later.
  • FIG. 2 shows the amino acid sequence of a ligand polypeptide (human type ⁇ 1-23) for GPR8.
  • FIG. 1 shows the nucleotide sequence of synthetic DNA encoding a ligand polypeptide (human type ⁇ 1-23) for GPR8.
  • FIG. 7 shows the amino acid sequence of a PTH (1-34) -GPR8 ligand polypeptide (human type ⁇ 1-23) fusion protein used in Example 2 described later.
  • Example 5 shows the nucleotide sequence of a DNA oligomer used for production of a structural gene in Example 2-1.
  • Example 2-1 The nucleotide sequence of the DNA oligomer used for production of the structural gene in Example 2-1 is shown.
  • Example 2-1 The base sequence of the DNA oligomer used for production of the structural gene in Example 2-1 is shown.
  • Example 1 shows the nucleotide sequence of a DNA oligomer used in the production of a structural gene in Example 2-1.
  • Example 2-1 The nucleotide sequence of the DNA oligomer used for production of the structural gene in Example 2-1 is shown.
  • Example 2-1 The base sequence of the DNA oligomer used for production of the structural gene in Example 2-1 is shown.
  • Example 1 shows the nucleotide sequence of a DNA oligomer used in the production of a structural gene in Example 2-1.
  • Example 2-1 The base sequence of the DNA oligomer used for production of the structural gene in Example 2-1 is shown.
  • the base sequence of a synthetic DNA encoding the amino acid sequence represented by SEQ ID NO: 39 is shown.
  • 1 shows the nucleotide sequence of a synthetic DNA encoding a ZAQ ligand.
  • Example 3 shows the amino acid sequence of the PTH (1-34) -human ZAQ ligand fusion protein used in Example 3 described later.
  • Example 3 shows the nucleotide sequence of a synthetic DNA encoding PTH (1-34) -human ZAQ ligand fusion protein used in Example 3 described later.
  • SEQ ID NO: 50 The nucleotide sequence of the DNA oligomer used for the production of the structural gene in Example 3-1 is shown.
  • 5 shows the nucleotide sequence of a synthetic primer used in Examples 2-5.
  • 5 shows the nucleotide sequence of a synthetic primer used in Examples 2-5.
  • 1 shows an amino acid sequence representing a linker sequence.
  • 1 shows the nucleotide sequence of a synthetic DNA encoding a linker sequence.
  • Fig. 3 shows the nucleotide sequence of primer-ZAQC Sal used in Example 3-5-1.
  • Example 3 shows the nucleotide sequence of primer-ZAQC Spe used in Example 3-5-1.
  • 2 shows the amino acid sequence of the precursor of rat-derived apelin.
  • Example 1 shows the amino acid sequence of a precursor of apelin derived from Escherichia coli.
  • Escherichia coli MM294 (DE3) ZpTCPTHAl0L obtained in Example 1 described below has accession number FERM BP—7312 as of September 28, 2000 at 1-1-1 Tsukuba-Higashi, Ibaraki Prefecture. 1 Chuo No. 6 (postal code 305-8566) Incorporated administrative agency, National Institute of Advanced Industrial Science and Technology (AIST) It has been deposited with the Fermentation Research Institute (IFO) under the accession number IFO 16475 at 2-17-85 Jusanhoncho, Yodogawa-ku, Osaka-shi, Osaka (postal code 532-8686).
  • IFO Fermentation Research Institute
  • Escherichia coli MM294 (DE3) / pTCPTHhGPR8L obtained in Example 2 described below is accession number FERM BP-7586 and is located at 1-1-1 Tsukuba East, Ibaraki Prefecture on May 10, 2001. 1 Central Japan No. 6 (Zip code 305-8566), National Institute of Advanced Industrial Science and Technology (AIST), Patent Organism Depositary, and Yokogawa Ward, Osaka City, Osaka, under the accession number IFO 16589 on March 15, 2001. It has been deposited with the Fermentation Research Institute (IFO) at Jusanhoncho 2-chome 17-85 (zip code 532-8686).
  • IFO Fermentation Research Institute
  • IFO Fermentation Research Institute
  • Escherichia coli MM.29.4 (DE 3) / pTCPTHh 1 Z AQ obtained in Example 3 described below was assigned the accession number FERM BP-7584 on May 10, 2001 on Ibaraki Prefecture. 1-1 Tsukuba East Higashi, 1 Chuo No. 6 (Zip code 305-8566), National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary, and as accession number I FO 16586 on March 15, 2001 It has been deposited with the Fermentation Research Institute (IFO), located at 2-17-85, Jusanhoncho, Yodogawa-ku, Osaka-shi, Osaka (zip code 532-8686).
  • IFO Fermentation Research Institute
  • PTH (1-34) -aperin-I was prepared according to a known method as follows. Was prepared.
  • Each of the eight DNA oligomers (# 2 to # 9, SEQ ID NOs: 16 to 23) excluding # 1 (SEQ ID NO: 15) and # 10 (SEQ ID NO: 24) to be 5 ' / phosphorylation reaction of 1 [DNA oligomers 10 g, 5 OmM T ris -HC pH7. 6, 1 OmM MgC 1 2, lmM spermidine, 1 Omm Jichiosu Reitoru (hereinafter DTT abbreviated), 0.
  • BSA serum albumin
  • lmM ATP 10 units of T4 polynucleotide kinase (Takara Shuzo)] at 37 ° C for 1 hour, the 5 'end of each oligomer was phosphorylated.
  • 2 volumes of ethanol was added, and after cooling to -70 ° C, DNA was precipitated by centrifugation.
  • the DNA fragment obtained in (a) above and # 1 (SEQ ID NO: 15) and # 10 (SEQ ID NO: 24) were added to 1 OmM Tris-HC1, 2 mM EDTA (pH 8. And The mixture was kept at 90 ° C. for 10 minutes, gradually cooled to room temperature, annealed, and then subjected to a ligation reaction using DNA Ligation Kit ver.2 (Takara Shuzo). After 30 xl of solution II was added to annealing solution 301 and mixed well, 60 ⁇ 1 of solution I was added and reacted at 37 ° C. for 1 hour to perform ligation.
  • An expression vector was prepared as follows. After digesting pTC II (described in JP-A-2000-178297) with NdeI and BamHI (Takara Shuzo) at 37 ° C for 2 hours, a 4.3 kb DNA fragment was subjected to QIAduick Gel Extraction by 1% agarose gel electrophoresis. Extracted using Kit (Qiagen) and dissolved in 25 1 TE buffer. A ligation reaction was performed between the NdeI and BamHI fragments of pTCII and the structural gene of PT PT (1-34) -apelin-136 prepared as described above using DNA ligation kit ver.2 (Takara Shuzo). .
  • Escherichia coli JM109 competent cells (Toyobo) were transformed with 101 of this reaction solution, seeded on LB agar medium containing lOgZml of tetracycline, cultured at 37 ° C for 1 ⁇ , and the resulting tetracycline resistance was obtained. A colony was picked. The transformant was cultured overnight in an LB medium, a plasmid was prepared using QIAprep8 Miniprep Kit (Qiagen), and the obtained plasmid was named pTCPTHA 10 L.
  • the nucleotide sequence of the PTH (1-34) -apelin-36 structural gene in 0 L of pTCPTHAl was confirmed using an Applied Biosystems model 377 DNA sequencer.
  • E. coli (Escherichia coli) MM294 (DE3) was transformed with the plasmid pTCPTHAI0L to obtain PTH (1-34) _hA10L expression strain Escherichia coli MM294 (DE3) / pTCPTHA10L.
  • the obtained culture solution was added to a 19 L main fermentation medium (1.68% sodium monohydrogen phosphate, 0.3% potassium dihydrogen phosphate, 0.1% ammonium chloride, 0.05% sodium chloride, 0.05% % Magnesium sulfate, 0.02% antifoam, 0.00025% ferrous sulfate, 0.0005% thiamine hydrochloride, 1.5% glucose, 1 (5% high-performance amino) was transferred to a 50-liter fermenter, and aeration and stirring were started at 30 ° C.
  • Example 1-1 To 200 g of the cells obtained in Example 1-1, 400 ml of a 7 M guanidine hydrochloride, 50 mM Tris-HC1 (pH 8.0) solution was added, and the mixture was stirred for about 4 hours, followed by centrifugation (10000 rpm, 60 minutes), and the resulting supernatant was diluted with 15 L of 5 OmM phosphate buffer (pH 6.0).
  • FIG. 1 shows the results of analysis of the samples from each step of the purification step by SDS-PAGE (gel: PEPTIDE PAGE MINI (TEFC0); reduction conditions: 100 ° C for 1 minute).
  • Example 13 Preparation of apelin-36 by cleavage of proteolytic enzyme PTH obtained in Example 1 one 2 (1-34) Aperin one 36 lyophilized powder 20 m g of 20 ml 50 mM NaC l, was dissolved in 2mM CaC l 2, 2 OmM T ris one HC 1 (pH 7. 4) solution Thereafter, 13 units of enterokinase (Novagen) were added and reacted at 25 ° C for 17 hours.
  • the fraction was further passed through ODS-120T (21.5 mml DX 300 maraudal L, Showa Denko) equilibrated with 0.1% trifluoroacetic acid, and adsorbed and washed, followed by a flow rate of 5 ml / min. Elution was performed with a step gradient of 20-50 B (B: 80% acetonitrile Z0.1% trifluoroacetic acid) for 50 minutes. 36 fractions of apelin (elution time: about 40 minutes) were pooled, followed by lyophilization. Approximately 5 mg of aperin-136 lyophilized powder was obtained.
  • Fig. 2 shows the results of analysis of the samples at each stage by SDS-PAGE (gel: PEPTIDE PAGE MINI (TEF CO); reducing conditions: 100 ° C for 1 minute).
  • Example 1-4 Determination of the characteristics of Apelin-1
  • the amino acid composition was determined using an amino acid analyzer (Hitachi L-850 OA Amino Acid Analyzer) (acid hydrolysis (6N hydrochloric acid-14% thioglycolic acid, hydrolysis at 110 ° C for 24, 48 hours)). The results agreed with the amino acid composition expected from the DNA sequence of apelin-36 (Table 1). Base sequence of apelin per mole
  • N-terminal amino acid sequence is converted to a gas phase protein sequencer (PE Applied Biosystems). This was determined using the Stems model 492) (analysis was performed using apelin—36 lOO pmol). The results were consistent with the N-terminal amino acid sequence predicted from the DNA sequence of apelin-36 (Table 2).
  • Example 2 Production of human GPR 8 ligand (hGPR 8 L)
  • the DNA fragment obtained in (a) above was combined with # 1 (SEQ ID NO: 35) and # 8 (SEQ ID NO: 42) to give 1 201.
  • the mixture was kept at 90 ° C. for 10 minutes, cooled slowly to room temperature, annealed, and then subjected to a ligation reaction using DNA Ligation Kit ver.2 (Takara Shuzo).
  • the I solution 301 was added to the annealing solution 301 and mixed well, the I solution 601 was added and reacted at 37 ° C. for 1 hour to perform a ligation.
  • the aqueous layer was collected, two volumes of ethanol was added, the mixture was cooled to 170 ° C, and the DNA was precipitated by centrifugation.
  • the DNA fragment thus obtained was subjected to phosphorylation with T4 polynucleotide kinase (Takara Shuzo) and then subjected to the following (d).
  • pTCII described in WO 00Z40610 was digested with NdeI and BamHI (Takara Shuzo) for 2 hours at 37 ° C., and then 4.4% by 1% agarose gel electrophoresis.
  • the kb DNA fragment was extracted using QIAQUick Gel Extraction Kit (Qiagen) and dissolved in 251 TE buffer.
  • a ligation reaction was carried out using the NdeI and BamHI fragments of pCTII and the structural gene of PTH (1-34) -hGPR8L prepared as described above using DNA ligation kit ver.2 (Takara Shuzo).
  • E. coli JM109 competent cells (Toyobo) were transformed with 1 O 1 of this reaction solution, seeded on an LB agar medium containing 10 ⁇ g / ml tetracycline, and cultured at 37 with 1 ⁇ The resulting tetracycline resistant colonies were selected.
  • This trait The transformant was cultured overnight in LB medium, and 8 L of plasmid pTCPTHhGPR was prepared using QIAprep8 Miniprep Kit (Qiagen).
  • the nucleotide sequence of the PTH (1-34) hGPR8 L structural gene was confirmed using an Applied Biosystems model 377 DNA sequencer.
  • Escherichia coli MM294 (DE3) was transformed with the plasmid pTCPTHhGPR8L to obtain PTH (1-34) -hGPR8L expression strain MM294 (DE3) / pTCPTHhGPR8L.
  • Example 2-2 obtained in PTH (1-34) one hGPR 8 L lyophilized powder 0. 8m g to 0. 8 ml of 50mM NaC l, 2mM CaC l 2 , 2 OmM T ris -HC 1 (pH7. 4 ) After dissolution in the solution, 0.8 units of enterokinase (Novagen) was added, and the mixture was reacted at 25 ° C for 20 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 predicted from the DNA base sequence of hGPR8L (Table 3).
  • the analysis was performed using 100 pmol.
  • Reverse transcription was performed using human brain-derived poly (A) ⁇ RNA (Clontech) as type ⁇ using a random primer.
  • A human brain-derived poly
  • RNA PCR ver 2.1 kit Tet al.
  • the synthetic primer was constructed so that the gene in the region translated into the receptor protein was amplified.At this time, the nucleotide sequence recognized by the restriction enzyme C1aI was added to the 5 'side of the gene, and the 3' Recognition sequences for the respective restriction enzymes were added to the 5 'and 3' sides so that the base sequence recognized by the restriction enzyme Spe I was added to the side.
  • the composition of the reaction mixture was cDNA type 5/21, synthetic DNA primers 0.4 ⁇ m each, 0.8 mM dNTPs, Pfu polymerase (Stratagene) 0.51, and the buffer supplied with the enzyme. was set to 50 n 1.
  • the cycle for amplification uses a thermal cycler (PE Biosyst ems), and after heating at 94 ° C for 60 seconds, 94 ° C for 60 seconds, 65 ° C for 60 seconds, 72 ° C for 150 seconds. The cycle was repeated 35 times. Confirmation of the amplification product was carried out by 0.8% agarose gel electrophoresis followed by ethidium umbamide staining.
  • the PCR reaction solution performed in (1) was separated by 0.8% low-melting point agarose gel electrophoresis, and the band was cut out with a razor, followed by fragmentation, phenol extraction, phenol-cloth form extraction, and ethanol.
  • the DNA was recovered by the operation of precipitation.
  • the recovered DNA was subcloned into the plasmid vector pCR-SCripT A immediate SK (+) according to the prescription of the PCR-SCripT TM A immediate SK (+) cloning kit (Stratagene). This was introduced into Escherichia coli DH5a competent cells (Toyobo), transformed, and clones containing the cDNA insert were selected on LB agar medium containing ampicillin, IPTG and X-Ga1.
  • Plasmid DNA was prepared from Plasmid Midi KIT (Qiagen) from E. coli clones transformed with the plasmid into which the gene to which the I recognition sequence was added was introduced. Digestion excised the insert DNA. After electrophoresis, the insert DNA was cut out of the agarose gel with a razor, then recovered by fragmentation, phenol extraction, phenol-chloroform extraction, and ethanol precipitation.
  • This insert DNA was digested with C1aI and SpeI and vector plasmid pAKKO-111H for animal cell expression (S. HinumA eT ⁇ 1, BioCim. Biophys. ACTA, 1219, 251-259, 1994)
  • vector plasmid pAKKO-111H for animal cell expression
  • S. HinumA eT ⁇ 1, BioCim. Biophys. ACTA, 1219, 251-259, 1994 the same vector and plasmid as described above
  • T4 ligase Takara Shuzo
  • Escherichia coli transformed with this plasmid pAKKO-GPR8 was named DH5 ⁇ / pAKKO-GPR8 (Escherichia coli DH5 ⁇ / ⁇ -GPR8).
  • pAKKO-GPR8 plasmid DNA was prepared using Plasmid Midi Kit (Qiagen). This was introduced into CHO dh fr-cells using CellPect Transfection Kit (Amersham Pharmacia Biotech) according to the attached protocol. 4. The DNA of step 4 was used as a coprecipitation suspension with calcium phosphate, and added to a 6 cm diameter petri dish seeded with 5 ⁇ 10 5 or 1 ⁇ 10 6 CHO dhfr_ cells 24 hours before.
  • the expression level of the full-length GPR8 protein mRNA of 47 clones of the CHO / GPR8 cell line established in (3) was measured using Cytostar T Plate (Amersham Pharmacia Biotech) as follows according to the attached protocol did. Each clone of the CHO / GPR8 cell line was seeded on a Cytostar T Plate at 2.5 ⁇ 10 4 cells / well, cultured for 24 hours, and then fixed with 10% formalin. After adding 0.25% Triton X-100 to each well to increase cell permeability, 35 S-labeled riboprbe of SEQ ID NO: 5 was added and hybridized.
  • Free riboprobe was digested by adding 20 g / ml of RNase A to each well, the plate was washed well, and the radioactivity of the hybridized riboprobe was measured with a Topcounter. Cell lines with high radioactivity have high mRNA expression levels. Three clones with high mRNA expression levels (# 17, 41 and 46) were used in the following experiments, and in particular, clone number 17 was used.
  • the CHO / GPR 8 cells and mock CHO cells prepared in (4) were seeded on a 24-well plate at 5 ⁇ 10 4 cells and cultured for 48 hours. Remove cells to 0.
  • the plate was washed with Hanks buffer (pH 7.4) containing 2 mM 3 3-f-sobutyl-methylxanthine, 0.05% BSA and 20 mM HEPS (hereinafter referred to as 0.2 mM 3-isobutyl-methylxanthine and 0.05%).
  • a Hanks buffer (pH 7.4) containing% BSA and 20 mM HEPS is called a reaction buffer).
  • 5 ml of a reaction buffer was added, and the mixture was kept warm in an incubator for 30 minutes. After removing the reaction buffer and adding a new 0.25 ml reaction buffer to the cells, add 0.25 ml 1 reaction buffer containing the sample and 2 M forskolin to the cells, and add them at 37 ° C.
  • Example 3 Production of human ZAQ ligand (hZAQ ligand)
  • Each of the six DNA oligomers (# 2 to # 7) excluding # 1 (SEQ ID NO: 45) and # 8 (SEQ ID NO: 52) to be 5 ' 10 g, 5 OmM Tris—HC1, pH7.6, 1 OmM MgC l 2, lmM spermidine, 1 OmM Jichiosurei!
  • DTT ⁇ 1l
  • BSA 0.1 lmg / ml ⁇ serum albumin
  • lmM ATP 10 units T4 polynucleotide kinase (Takara Shuzo)
  • the DNA fragment obtained in (a) above was combined with # 1 (SEQ ID NO: 45) and # 8 (SEQ ID NO: 52) to give 1201.
  • the mixture was kept at 90 ° C for 10 minutes, cooled slowly to room temperature, annealed, and then subjected to a ligation reaction using DNA Ligation Kit ver.2 (Takara Shuzo).
  • the I solution 60 ⁇ 1 was added and reacted at 37 ° C. for 1 hour to perform ligation.
  • the aqueous layer was collected, two volumes of ethanol was added, the mixture was cooled to 170 ° C, and the DNA was precipitated by centrifugation.
  • the DNA fragment thus obtained was subjected to phosphorylation with T4 polynucleotide kinase (Takara Shuzo), and then subjected to the following (d).
  • pTCh1 ZAQ was digested with NdeI and KpnI (Takara Shuzo) at 37 ° C for 2 hours, and a 4.4 kb DNA fragment was subjected to QIAduick Gel Extraction Kit (Qiagen) by 1% agarose gel electrophoresis. Extract using
  • the reaction solution was used to transform E. coli JM109 competent cells (Toyobo) using 101 and inoculated on LB agar medium containing 10 g / m1 tetracycline.
  • the transformant was cultured in an LB medium, and the plasmid pTCPTHh1ZAQ was prepared using the QIAprep8 Miniprep Kit (Qiagen).
  • This PTH (1-34) — hZ The nucleotide sequence of the AQL-1 structural gene was confirmed using an Applied Biosystems Model 377 DNA sequencer. Escherichia coli MM294 (DE3) was transformed with the plasmid pTCPTHh1ZAQ to obtain a PTH (1-34) -hZAQL-1 expression strain MM294 (DE3) / pTCPTHh1ZAQ.
  • E. coli MM294 (DE 3) Zp TCP THh 1 Z AQ 5.Om1 (1% peptone, 0.5% yeast extract, 0.5% sodium chloride) in LB medium containing Omg / L tetracycline And shake-cultured at 37 ° C for 8 hours in a 20-Om 1-volume flask. 1.5 ml of the obtained culture solution was replaced with 30 ml of the main fermentation medium (1.68% sodium monohydrogen phosphate, 0.3% potassium dihydrogen phosphate, 0.1% ammonium chloride, 0.05 % Sodium chloride, 0.025% magnesium sulfate, 0.00025%, thiamine hydrochloride, 1.5% glucose, 1.5% casamino acid), and shaken at 37 ° C.
  • the main fermentation medium 1.68% sodium monohydrogen phosphate, 0.3% potassium dihydrogen phosphate, 0.1% ammonium chloride, 0.05 % Sodium chloride, 0.025% magnesium sulfate, 0.00025%, thiamine hydrochlor
  • Example 3-1 To 0.6 g of the cells obtained in Example 3-1 was added 2 ml of a 7 M guanidine hydrochloride, 50 mM Tris-HC1 (pH 8.0) solution, and the mixture was stirred for about 2 hours, followed by centrifugation (15 OOO r pm 15 minutes). To the supernatant, 50 M 1 of 0.4 M arginine, 50 mM Tris_HCl, 0.2 mM GS SG, ImM GSH (pH 8.0) was added, and activated at 4 ° C. overnight.
  • a 7 M guanidine hydrochloride 50 mM Tris-HC1 (pH 8.0) solution
  • the activated regenerating solution was adjusted to pH 6.0 and adsorbed to a SP-Sepharose column (1 cm x 3 cm) equilibrated with 50 mM phosphate buffer (pH 6.0). Then, it was eluted with 600 mM NaC 1Z5 OmM phosphate buffer (pH 6.0), and the fractions containing PTH (1-34) -ZAQL-1 were pooled. This fraction was purified from C 4 P—50 (21.5 thigh ID) equilibrated with 0.1% trifluoroacetic acid.
  • Example 3 2 obtained in PTH (1-34) -hZAQ ligand lyophilized powder 40/1 of 50 mM NaC l, was dissolved in 2mM CaC l 2, 2 OmM Tr is- HC 1 (pH7. 4) solution Thereafter, 0.8 units of enterokinase (Novagen) was added and reacted at 25 ° C for 20 hours.
  • the N-terminal amino acid sequence was determined using a gas phase protein sequencer (PE Applied Biosystems model 492). The results agreed with the N-terminal amino acid sequence predicted from the DNA sequence of the ZAQ ligand (Table 4).
  • Example 3-3 The ZAQ ligand obtained in Example 3-3 was suspended in Sam1e buffer (Laemmli, Nature, 227, 680 (1979)) supplemented with 10 OmM DTT, and heated at 95 ° C for 1 minute. Electrophoresis was performed on Multigel 15/25 (Daiichi Pure Chemicals). The gel after electrophoresis was stained with Coomassie brilliant blue (Coomassie brilliant blue), and as a result, a single protein band was observed at 10 kDa, indicating that this sample was extremely pure. In addition, comparison with a recombinant ZAQ ligand sample obtained using a direct expression system in E. coli showed that both were the same in molecular weight.
  • Example 3-5 Measurement of biological activity (measurement of intracellular Ca ion concentration increasing activity using FLIPR)
  • the human pituitary gland cDNA (CLONTECH) was designated as type II, and two primers, primer 1 (5′- GTCGACATGGAGACCACCATGGGGTTCATGG-3 ′; SEQ ID NO: 59) and primer 2 (5′-ACTAGTTTATTTTAGTCTGATGCAGTCCACCTCTTC-3 ′; The PCR reaction was performed using No. 60).
  • the composition of the reaction solution used in the reaction was 1/10 volume of the above-mentioned cDNA, and the 1/50 volume of Advantage2 Polymerase Mix (CLONTECH), Primer-1 and Primer-2 were each 0.2 xM.
  • DNTPs, 200 M, and the enzyme and the attached buffer were added to make a liquid volume of 251.
  • 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.
  • a cycle of 94 ° C for 20 seconds, 64 ° C for 20 seconds, and 68 ° C for 100 seconds was repeated 38 times, and an 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 prescription of a TA cloning kit (Invitrogen).
  • a double volume of acetone was added to the supernatant while stirring, followed by stirring at 4 ° C for 3 hours.
  • the obtained supernatant was filtered with a gauze to remove insolubles.
  • the obtained supernatant was subjected to rotary evaporation overnight to remove acetone, and finally concentrated to 135 Om1.
  • the obtained concentrated liquid was mixed with 338 ml of getyl ether every 675 ml, and mixed vigorously in a separating funnel to obtain an aqueous phase after separation of two phases. The same operation was repeated once more for the obtained aqueous phase to obtain a clear aqueous phase.
  • the obtained aqueous phase was concentrated to 80 Om1 by using a liquor evaporator to obtain a final extract.
  • Each of the obtained solutions I to IV was freeze-dried with a freeze dryer (12EL; Virtis).
  • Solution A (0.1% trifluoroacetic acid) at a flow rate of 1 m1 / min at 40 ° C at 40 ° C using TSKGel ODS-80Ts reversed-phase high-performance liquid chromatography column (Tosoichi Co., Ltd., 4.6 band x 25 cm). / Distilled water) Volume 81.7% / Liquid (0.1% trifluoroacetic acid / 60% acetonitrile) Equilibrated by flowing 8.3% volume.
  • the freeze-dried products of Solution I to Solution IV obtained in the above (3-5-2-3) were each dissolved in 4 ml of 1M acetic acid and subjected to one chromatographic operation.
  • solution A was increased to 67% ZB volume over 33 minutes at a flow rate of 1 mlZmin, and then the solution A volume was reduced to 67% over 40 minutes.
  • Solution B concentration was increased in a linear gradient from 33% solution volume to 0% solution A volume and 100% ZB solution volume.
  • the eluate was fractionated by assigning a fraction number to each lm1, and each fraction 21 was mixed with 150 1 of 0.2% Bovine Serum Albumin (BSA) / distilled water and freeze-dried.
  • BSA Bovine Serum Albumin
  • the dried product was used as an assay sample for measuring the intracellular Ca ion concentration increasing activity described in (3-5-2-5) below.
  • a ZAQ stable expression cell line was prepared as follows. That is, one clone of DH5a / pCR2.1-ZAQC obtained in (3-5-1-1) above was shake-cultured in an LB medium containing ampicillin to obtain plasmid pCR2.1-ZAQC. This was treated with restriction enzymes Sa1I and SpeI to cut out an insert encoding ZAQC. Similarly, pAKKO-1.11H treated with restriction enzymes Sa1I and SpeI and the insert were ligated using a Ligation Express Kit (CLONTECH Laboratories, Inc. (CA, USA)) to form Escherichia coli DH10B. Was introduced by the electro-volatilization method. The structure of the plasmid contained in the obtained clone was confirmed by restriction enzyme treatment and sequence analysis. Used as Smid pAK—ZAQC.
  • the plasmid pAK-ZAQC was obtained by transducing CHO / dh fr-cells (American Type Culture Collection) using CeUPhect Transfection kit Okie sham Pharmacia Biotech. First, Buffer 4 (attached to CellPhect Transfection Kit) is added to 4 g of plasmid DNA dissolved in distilled water 1201, stirred, and allowed to stand for 10 minutes. Buffer B (attached to CellPhect Transfection KiU) 240/21 was added, and the mixture was vigorously stirred to form a DNA-calcium phosphate complex containing the DNA.
  • the cells After culturing for 6 hours in 5% carbon dioxide, the cells are washed twice with serum-free Ham's F-12 medium, and a buffer containing 15% glycerol on the cells in a Petri dish It was treated (140mM N aC l, 25mM HEPES , 1. 4mM Na 2 P_ ⁇ 4, pH7. 1) 1. added 2 m 1 2 min. This was washed again twice with serum-free Ham's F-12 medium, and then in Ham's F-12 medium containing 10% fetal calf serum at 37 ° C, 5% carbonate. Cultured overnight in gas.
  • the cells were dispersed by trypsin treatment and recovered from the Petri dish, and 2 ⁇ 10 4 cells were inoculated into 6-we 11 p 1 ate, dialyzed 10% fetal serum (JRH BIOSCIENCES), ImM MEM Non-essential amino acid solution (Dainippon Pharmaceutical Co., Ltd.) containing 100 units / ml Penicillin, 100 / ig / ml Streptomycin in Dulbecco's modified Eagle medium (DM EM) medium (Nissui Pharmaceutical Co., Ltd.) 37 ° C. Culture was started in 5% carbon dioxide gas.
  • Transformed CHO cells into which the plasmid has been introduced grow in the medium, but non-transfected cells gradually die, so the medium is changed on the first and second days of culture to replace the dead cells. Removed. Approximately 21 colonies of transformed CHO cells that grew 8 to 10 days after the start of culture were selected. RNA is recovered from each of the selected cells using a commercially available RNA isolation kit, and ZAQ-expressing ZAQ is highly expressed by a known RT-PCR method. Abbreviated as cell Was selected.
  • ETA endothelin A receptor
  • CHO cell clone 24 (hereinafter abbreviated as ETA 24 cell; see Journal of Pharmacology and Experential Therapeutics, Vol. 279, pp. 675-685, 1996) was.
  • ZAQC-B1 cells and ETA24 cells were both subcultured in DMEM supplemented with 10% dialyzed fetal bovine serum (hereinafter referred to as dFBS).
  • dFBS dialyzed fetal bovine serum
  • ZAQC—B1 cells and ETA24 cells are each suspended in a medium (10% dFB S-DMEM) to a concentration of 15 ⁇ 10 4 Ce 11 sZml and placed in a 96-well plate for FLIPR (Black plate Clear bot tom, Coster).
  • H / HBS S (Nissy Hanks 2 (Nissui Pharmaceutical Co., Ltd.) 9.8 g, sodium hydrogen carbonate 0.35 g, HEPES 4.77 g, pH 7.4 with sodium hydroxide solution, then fill 20 ml, 25 OmM Probenecid 200 ⁇ ⁇ fetal serum (FBS) 200/1 was mixed.
  • sample plate V-Bottom plate, Coster
  • sample plate After the dye loading of the cell plate was completed, the cell plate was washed four times using a plate washer (Molecular Devices) with a washing buffer prepared by adding 2.5 mM Proben ecid to HZHBS S. After washing, 100 1 of the washing buffer was left. .
  • the cell plate and the sample plate were set on the FLIPR and assembled (the FLIPR transfers a sample of 50 ⁇ 1 from the sample plate to the cell plate).
  • Example 3-3 For the recombinant ZAQ ligand preparation obtained in Example 3-3, the intracellular Ca ion concentration in the ZAQ-expressing cell (ZAQC-B1) obtained in (3-5-2-5) above was increased. Activity was measured using FLIPR. As a control, hOT7Tl75-expressing cells (hOT7T175-16; described in WO00 / 24890) were used.
  • ZAQC-B1 cells and hOT7T175-16 cells were used that had been subcultured in DMEM supplemented with 10% dialyzed fetal bovine serum (hereinafter referred to as dFBS).
  • Z AQ CB 1 cells were suspended in culture medium (10% dFBS- DMEM) such that W3T7T175- 16 cells each 15 xl 0 4 Ce ll sZml, ?
  • sow 200 n 1 into each 96-well plate Black plate Clear bottom. Coster
  • H / HBSS (9.8 g of HANKS ', 0.35 g of sodium bicarbonate, 4.77 g of HEPES, adjusted to pH 7.4 with sodium hydroxide solution, and then sterilized with a filter) 21 m and 25 OmM Probenecid 210 xl and fetal calf serum (FBS) 210 1 were mixed. Also, 2 vials (50 ⁇ g) of Fluo3-AM were dissolved in 42 ⁇ 1 of dimethylsulfoxide, 20% Pluronic Acid 421, and added to the above H / HBSS-Probenecid-FBS.
  • the production method of the present invention does not require removal of the N-terminal met residue of the target peptide, and uses a fusion protein with PTH (1-34), so that the target peptide can be highly expressed. This is advantageous for industrially producing a large amount of peptides for pharmaceutical use.

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Abstract

L'invention concerne un procédé de fabrication efficace d'un peptide cible etc. Selon ledit procédé, on emploie une protéine fusionnée avec PTH (1-34). Par conséquent, il est possible d'exprimer largement un peptide cible sans retirer le résidu Met N-terminal du peptide cible. Ainsi, le procédé selon l'invention se révèle être avantageux dans le cas d'une fabrication industrielle sur une échelle massique d'un peptide d'usage médical ou similaire.
PCT/JP2001/009476 2000-10-30 2001-10-29 Procede de fabrication de peptides Ceased WO2002036762A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003082907A1 (fr) * 2002-03-29 2003-10-09 Euroscreen Sa Ligands du recepteur couple aux proteines g gpr7 et utilisations de ces derniers
JP2009542203A (ja) * 2006-06-27 2009-12-03 コリア リサーチ インスティチュート オブ バイオサイエンス アンド バイオテクノロジー システインタグ付きブドウ球菌タンパク質ｇ変異体
WO2014152955A1 (fr) * 2013-03-14 2014-09-25 Regeneron Pharmaceuticals, Inc. Protéines de fusion d'apéline et leurs utilisations
US9644018B2 (en) 2013-11-20 2017-05-09 Regeneron Pharmaceuticals, Inc. Antibody modulators of APLNR

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WO2000000625A1 (fr) * 1998-06-26 2000-01-06 Ppl Therapeutics (Scotland) Limited Procedes d'obtention de polypeptides de recombinaison
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WO2000000625A1 (fr) * 1998-06-26 2000-01-06 Ppl Therapeutics (Scotland) Limited Procedes d'obtention de polypeptides de recombinaison
WO2000052022A1 (fr) * 1999-03-01 2000-09-08 Millennium Pharmaceuticals, Inc. Proteines secretees et acides nucleiques codant pour elles

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KAZUHIKO TATEMOTO ET AL.: "Isolation and characterization of a novel endogenous peptide ligand for the human APJ receptor", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 251, no. 2, 1998, pages 471 - 476, XP002947594 *
O'DOWD ET AL.: "The cloning and chromosomal mapping of two novel human opioid-somatostatin-like receptor genes, GPR7 and GPR8, expressed in discrete areas of the brain", GENOMICS, vol. 28, no. 1, 1995, pages 84 - 91, XP002942500 *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003082907A1 (fr) * 2002-03-29 2003-10-09 Euroscreen Sa Ligands du recepteur couple aux proteines g gpr7 et utilisations de ces derniers
JP2009542203A (ja) * 2006-06-27 2009-12-03 コリア リサーチ インスティチュート オブ バイオサイエンス アンド バイオテクノロジー システインタグ付きブドウ球菌タンパク質ｇ変異体
US8541005B2 (en) 2006-06-27 2013-09-24 Korea Research Institute Of Bioscience And Biotechnology Cysteine-tagged streptococcal protein G variant
WO2014152955A1 (fr) * 2013-03-14 2014-09-25 Regeneron Pharmaceuticals, Inc. Protéines de fusion d'apéline et leurs utilisations
US9353163B2 (en) 2013-03-14 2016-05-31 Regeneron Pharmaceuticals, Inc. Apelin fusion proteins and uses thereof
US9751921B2 (en) 2013-03-14 2017-09-05 Regeneron Pharmaceuticals, Inc. Apelin fusion proteins and uses thereof
US9644018B2 (en) 2013-11-20 2017-05-09 Regeneron Pharmaceuticals, Inc. Antibody modulators of APLNR
US10155811B2 (en) 2013-11-20 2018-12-18 Regeneron Pharmaceuticals, Inc. APLNR modulators and uses thereof
US10189901B2 (en) 2013-11-20 2019-01-29 Regeneron Pharmaceuticals, Inc. Methods for improving cardiac function by administering an antibody that activates APLNR
US10626173B2 (en) 2013-11-20 2020-04-21 Regeneron Pharmaceuticals, Inc. Method for treating pathological angiogenesis by administering an antibody that inhibits APLNR
US10947310B2 (en) 2013-11-20 2021-03-16 Regeneron Pharmaceuticals, Inc. Fusion protein comprising apelin and an anti-APLNR antibody
US11642390B2 (en) 2013-11-20 2023-05-09 Regeneran Pharmaceuticals, Inc. Method of treatment with a fusion protein comprising apelin and an anti-APLNR antibody

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