Process for producing peptide

专利摘要:
An object of the present invention is to provide a method capable of efficiently and mass-producing a target peptide by gene recombination. The method of the present invention combining the cleavage of the target peptide with the right-hand scissors (S-cyanolation reaction) and the left-hand scissors (Bromtian treatment, enterokinase, factor Xa treatment, etc.) and the serial iteration method are peptides by genetic recombination technology. And particularly useful for mass synthesis of low molecular weight peptides.
公开号:KR20040026654A
申请号:KR10-2003-7014828
申请日:2002-05-16
公开日:2004-03-31
发明作者:니시무라오사무；스에나가마사토；이토다카시；기타다치에코
申请人:가부시키가이샤 시마즈세이사쿠쇼；
IPC主号:

专利说明:

Process for producing peptide
[2] As a method of synthesizing a peptide, three kinds of methods are known, an organic chemical method, an enzyme method, and a method by genetic recombination technology.
[3] Among these, the synthesis by the direct expression method of a peptide is very difficult by the method using a genetic recombination technique. The reason for this is that peptides produced even by directly expressing peptides are rapidly degraded by protease in cells.
[4] Therefore, in synthesizing peptides using genetic recombination techniques, a fusion protein method that expresses a fusion protein with a protective protein is generally used. As the protective protein, protein A, β galactosidase, and the like, which can use affinity chromatography in order to perform subsequent purification quickly and efficiently, are advantageously used. Although the target peptide must be specifically cleaved from the fusion protein, the cleavage method may include chemical methods such as bromine and cleavage at the C-terminus of Met, and enterokinase cleavage sites (Asp-Asp-Asp-Asp-Lys Or an enzyme such as factor Xa (factor Xa) cleavage site (I-Glu-Gly-Arg) is cleaved immediately after the sequence).
[5] In addition, a novel peptide synthesis method has been reported in which FGF mutein (CS23) as a protective protein of the fusion protein is combined with the S-cyanolation reaction as a specific chemical cleavage method (EP0887417). This method takes advantage of the strong affinity of CS23 to heparin for efficient and easy purification of the fusion protein and furthermore, via the cysteine-mediated S-cyanoation reaction (C-N cleavage of the Cys). Specific cleavage by) is a useful method for cleaving the peptide of interest, and also a method for preparing a C-terminal amide which is essential for the expression of physiological activity in most peptides. This method is also applicable to all peptides that do not contain cysteine residues in the molecule.
[6] On the other hand, it is also true that the fusion protein method generally has a large molecular weight difference between the protective protein and the target peptide, so that it cannot necessarily be used efficiently of the microorganism (E. coli) protein synthesis ability. Therefore, tandem repeats have been attempted in which target peptide genes are linked in tandem in one molecule to be expressed as stable precursor proteins in cells, and then the target peptide is cleaved. The reason for this is conventionally a method of cutting and exposing the N-terminal side of the target peptide (say, left-handed scissors) is known as bromine treatment, factor Xa method, etc., but a method of specifically cutting the C-terminal to expose (say , Right handed scissors).
[7] An object of the present invention is to provide a method capable of efficiently and mass-producing a target peptide by gene recombination.
[1] The present invention relates to a method for producing a target peptide or a salt thereof by stably expressing to a microorganism as a precursor protein to which the target peptide is repeatedly linked, and then cleaving the peptide bond of the precursor protein.
[8] Disclosure of the Invention
[9] The present inventors have intensively studied how to specifically cleave and expose this C-terminal. As a result, the above-mentioned S-cyanoation reaction is unexpectedly similar to the application to the fusion protein method. The present invention was completed by discovering that it can be effectively used for the synthesis of.
[10] That is, the present inventors have succeeded in obtaining both left-handed scissors and right-handed scissors necessary for the serial repetition method, and in particular, efficient and large-scale preparation of small-molecular weight peptides that have been difficult to synthesize by genetic recombination is possible. (FIG. 1).
[11] In this way, by combining these two cleavage methods, it was possible to efficiently and largely prepare a peptide having a low molecular weight by the serial repetition method (FIG. 2).
[12] That is, the present invention
[13] (1) A method for producing a target peptide or salt thereof (hereinafter, preparation method A), wherein the precursor protein or the salt thereof is enzymatically or chemically cleaved by adding an enzyme or a chemical cleavage site to the N- and C-terminus of the target peptide and repeatedly connecting the precursor protein.
[14] (2) The production method according to (1), wherein the precursor protein obtained by repetitively connecting an enzyme or a chemical cleavage site to the N-terminus of the target peptide and a chemical cleavage site to the C-terminus is enzymatically or chemically cleaved. ,
[15] (3) a methionine residue or protease cleavage sequence at the N terminus of the target peptide, and a cysteine residue or cysteinyl peptide at the C terminus, provided that the peptide portion of the cysteinyl peptide is different from the target peptide and When adding a methionine residue, the peptide portion does not have a methionine residue) and the N-terminal side of each target peptide in the repeatedly linked precursor protein is cleaved with bromine or protease, and the cysteine or cystine at the C-terminal side A process for producing the desired peptide or its salt (hereinafter referred to as "preparation B"), characterized in that it is subjected to a cleavage reaction at the N-terminal side of the nil peptide
[16] (4) The method according to any one of (1) to (3), wherein the precursor protein is a recombinant precursor protein,
[17] (5) The production method according to (3), wherein the cleavage reaction is a reaction which is treated by an S-cyanolation reaction, followed by ammonolysis or hydrolysis reaction,
[18] (6) The process of (5), wherein the S-cyanoation reaction is carried out by 2-nitro-5-thiocyano benzoic acid (NTCB), 1-cyano-4-dimethylaminopyridium salt (DMAP-CN) or CN ^- ion. Manufacturing process in the presence of
[19] (7) The method according to (3), wherein the protease is enterokinase, factor Xa or thrombin,
[20] (8) In (3), when (a) bromine is used, the methionine residue is linked to the N terminus of each target peptide, and the target peptide does not have a methionine residue.
[21] (B) When the protease is enterokinase, enterokinase cleavage sites such as Asp-Asp-Asp-Asp-Lys are connected to the N terminus of each target peptide, and the target peptide is Asp-Asp-Asp-Asp-Lys etc. Does not have an amino acid sequence represented by
[22] (C) When the protease is factor Xa, an amino acid in which a factor Xa cleavage site such as Ile-Glu-Gly-Arg is connected to the N terminus of each target peptide, and the target peptide is represented by Ile-Glu-Gly-Arg, etc. Has no sequence,
[23] (D) when the protease is thrombin, a production method in which a thrombin cleavage site such as Gly-Pro-Arg is connected to the N-terminal of each target peptide, and the target peptide does not have an amino acid sequence represented by Gly-Pro-Arg,
[24] (9) The method according to any one of (1) to (3), wherein the target peptide is a KiSS-1 peptide,
[25] (10) The method according to any one of (1) to (3), wherein the target peptide is a GPR8 ligand,
[26] (11) An N-terminus of each GPR8 ligand in the precursor protein in which the enterokinase cleavage sequence was connected to the N-terminus of the GPR8 ligand and the cysteine residue was added to the C-terminus and linked three times, followed by enterokinase to cleave the C-terminal cysteine. Method for producing a GPR8 ligand or a salt thereof, characterized in that the treatment by the cleavage reaction at the N-terminal side of
[27] (12) The method according to (10) or (11), wherein the GPR8 ligand is a polypeptide containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 44,
[28] (13) The method of (10) or (11), wherein the GPR8 ligand is SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49 or SEQ ID NO: 50 Production method which is a polypeptide having an amino acid sequence represented by
[29] (14) The method according to (10) or (11), wherein the GPR8 ligand is a polypeptide having an amino acid sequence represented by SEQ ID NO: 44,
[30] (15) A methionine residue or protease cleavage sequence at the N terminus of the target peptide, and a cysteine residue or cysteinyl peptide at the C terminus, provided that the peptide moiety of the cysteinyl peptide differs from the target peptide, and at the N terminus. In the case of adding a methionine residue, the peptide portion does not have a methionine residue;
[31] (16) a recombinant vector containing the DNA of (15),
[32] (17) The transformant of (16) represented by FERM BP-8023: a recombinant vector carried in Escherichia coli MM294 (DE3) / pTCGPR3,
[33] (18) a transformant transformed with the recombinant vector of (16),
[34] (19) The transformant according to (18), which is represented by FERM BP-8023: A transformant which is Escherichia coli MM294 (DE3) / pTCGPR3,
[35] (20) A methionine residue or protease cleavage sequence at the N terminus of the target peptide, and a cysteine residue or cysteinyl peptide at the C terminus, provided that the peptide portion of the cysteinyl peptide differs from the target peptide, and at the N terminus. When adding a methionine residue, the peptide moiety does not have a methionine residue) and the precursor protein or salt thereof linked repeatedly;
[36] (21) The method according to (4), wherein the precursor protein is a recombinant precursor protein prepared by culturing the transformant of (18).
[37] Brief description of the drawings
[38] 1 shows a schematic of the peptide preparation of the invention.
[39] In the figure, the downward arrow is referred to as left-hand scissors (N-terminal cutting and exposure method) -A, and the upward arrow is referred to as right-hand scissors (C terminal cutting and exposure method) -B. Examples of the left-hand scissors include enzymes capable of exposing a new N-terminal by cleaving a peptide from the C-terminal side such as bromine, treatment, factor Xa method, enterokinase method and the like. As a method, the C terminus of Met or Ile-Glu-Gly-Arg, Asp-Asp-Asp-Asp-Lys is cleaved to expose the N terminus of the desired peptide.
[40] Right-handed scissors undergo S-cyanolation to cleave the N-terminus of Cys and expose the C-terminus of the desired peptide.
[41] 2 shows a schematic of the peptide preparation of the invention.
[42] 3 shows a schematic of the KiSS-1 peptide preparation using the peptide preparation of the invention.
[43] Xxx: Met (bromine) or Ile-Glu-Gly-Arg (factor Xa), Asp-Asp-Asp-Asp-Lys (entokinase) and the like.
[44] However, (Xxx) is generally added to the factor Xa or enterokinase in addition to the factor Xa or enterokinase when the target peptide contains Met. (1) Proline when the target peptide does not contain Pro. Proline Specific endopeptidase, ② Lysyl endopeptidase when the target peptide does not contain Lys, ③ Arginyl endopeptidase when the target peptide does not contain Arg. V8 protease when the peptide does not contain Glu and Asp, trypsin when the target peptide does not contain a basic amino acid, and chimotolipsin when the target peptide does not contain an aromatic amino acid.
[45] Figure 4 shows the results of measuring the GTPγS binding activity for GPR8 expressing CHO cells of hGPR8L (SEQ ID NO: 44) prepared in Examples 1-3. The horizontal axis shows the concentration of hGPR8L, and the vertical axis shows the GTPγS binding activity. -"-" Shows the result of using the synthesize | combined hGPR8L (SEQ ID NO: 44), and-(circle)-hGPR8L (SEQ ID NO: 44) manufactured by the peptide manufacturing method (serial repetition method) of this invention.
[46] Best Mode for Carrying Out the Invention
[47] In the method of the present invention, any peptide may be used as the peptide of interest (which may be referred to simply as "target peptide") as long as the peptide does not have a cleavage site in the molecule by the method used for cleavage. Anything that can be produced using genetic recombination techniques may be used.
[48] Although the number of amino acid residues of a target peptide is not specifically limited, Usually, about 10-100, Preferably it is about 10-50, More preferably, it is about 20-40.
[49] Although the molecular weight of the target peptide is not specifically limited, either, Usually, about 1000-10000 daltons, Preferably it is about 1000-5000 daltons, More preferably, it is about 2000-4000 daltons.
[50] Specific examples of the peptide of interest include KiSS-1 peptide (WO00 / 24890), RFRP-1 (WO00 / 29441), Apelin (WO99 / 33976), PrRP (WO97 / 24436), GALP (WO99 / 48920). ), GPR8 ligand (WO01 / 98494), Angiotensin, Bradykinin, Calcitonin, Calcotonin, Conotoxin, Corticotropin Release Factor, Dynorphin, Endorphin, Enkephalin ), Galanin, gastrin, glucagon, growth hormone releasing factor, FMRF-amide, neurokinin, neuromedin, neuropeptide, nociceptin, nocistatin, orexin-B, secretin, substance P, Eurocortin, VIP, PACAP, ACTH, various opioid peptides and the above peptide fragments may be mentioned. Among them, KiSS-1 peptide, RFRP-1, GPR8 ligand, apelin, PrRP, GALP and the like are preferable.
[51] The peptide of interest is the N terminus (amino terminus) at the left end and the C terminus (carboxyl terminus) at the right end, according to the conventions of peptide notation.
[52] As the KiSS-1 peptide, for example, the human KiSS-1 peptide described in WO00 / 24890 is used. Specifically, the amino acid sequence consisting of 54 amino acid residues represented by SEQ ID NO: 1 is the 47th to 54th place from the N terminus. Peptides containing an amino acid sequence of 8 to 54 amino acid residues; and the like.
[53] In the amino acid sequence represented by SEQ ID NO: 1, the "peptide containing the 47th to 54th amino acid sequence from the N-terminus and consisting of 8 to 54 amino acid residues" in the amino acid sequence represented by SEQ ID NO: 1 Any peptide may be used as long as the peptide contains the 47th to 54th amino acid sequence from the N-terminal and is composed of 8 to 54 amino acid residues. The peptide has activity (for example, binding activity between the peptide and the receptor and the peptide). Cell stimulatory activity of the receptor-expressing cells, etc.). Specifically, (1) the peptide represented by the amino acid sequence represented by SEQ ID NO: 1, (2) the amino acid sequence represented by SEQ ID NO: 1 of the present invention has a 47-54th amino acid sequence from the N terminus at the C terminus, Peptides of 8 to 15 amino acid residues and the like are used.
[54] More specifically, the KiSS-1 peptide is represented by the peptide represented by the amino acid sequence represented by SEQ ID NO: 1, the amino acid sequence from the N-terminus to the 40 th to 54 th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 1. Peptide, ③ peptide represented by the 45th to 54th amino acid sequence (amino acid sequence represented by SEQ ID NO: 2) from the N terminus of the amino acid sequence represented by SEQ ID NO: 1, ④ amino acid represented by SEQ ID NO: 1 Peptide represented by the 46th to 54th amino acid sequence from the N terminus of the sequence, ⑤ Peptide represented by the 47th to 54th amino acid sequence from the N terminus of the amino acid sequence represented by SEQ ID NO: 1, ⑥ SEQ ID NO: The peptide etc. which are represented by the 35th-54th amino acid sequence from the N terminal of the amino acid sequence shown by 1: are mentioned.
[55] The KiSS-1 peptide has ligand activity against receptor protein OT7T175 described in WO00 / 24890.
[56] Examples of RFRP-1 include RF amide-related peptides described in Hinuma et al., Nature Cell Biology, Vol 2, p703-708 (2000), polypeptides described in WO00 / 29441, and the like. And specifically for the receptor OT7T022, which contains the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 9 and is described in Hinuma et al., Nature Cell Biology, Vol 2, p703-708 (2000) or WO00 / 29441. Any peptide may be used as long as it has a ligand activity.
[57] More specifically, for example, a polypeptide having an amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11 is used.
[58] As apelin, for example, Biochem. Biophys. Res. Apelin-36 (polypeptide having an amino acid sequence represented by SEQ ID NO: 18) described in Commun., 251 , 471-476 (1998), Apelin-13 (amino acid sequence of Nos. 24 to 36 of SEQ ID NO: 18) And a peptide wherein the N-terminal amino acid (Gln) of apelin-13 is pyroglutaminated, and the receptor APJ (O'Dowd. BF, et al., Gene, 436 , 355-). 359, 1993) may be any peptide having ligand activity. Specifically, the "polypeptide which has a binding capacity with respect to the receptor protein containing the amino acid sequence identical or substantially identical to the amino acid sequence shown by SEQ ID NO: 3" described in WO99 / 33976, etc. are mentioned.
[59] As the PrRP (19P2 ligand), for example, the peptide described in WO97 / 24436 is used, specifically, bovine 19P2L (b19P2L or bovine PrRP) (SEQ ID NO: 20), rat 19P2L9 (r19P2L or rat PrRP) (SEQ ID NO: 21) And peptides identical to or substantially identical to human 19P2L (h19P2L or human PrRP) (SEQ ID NO: 22) or their amides, esters or salts thereof.
[60] "Substantially the same" means that the receptor binding activity and the like are homogeneous in nature. Therefore, the quantitative elements, such as the strength and the like of the receptor binding activity, the molecular weight of the peptide, and the like may be different.
[61] For example, in addition to the peptide containing the amino acid sequence represented by SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22, and the like, and the amino acid sequence represented by SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22, respectively Contains an amino acid sequence of about 50-99.9% (preferably 70-99.9%, more preferably 80-99.9%, more preferably 90-99.9%), SEQ ID NO: 20, SEQ ID NO: And peptides having substantially the same activity as the peptide containing the amino acid sequence represented by: 21 or SEQ ID NO: 22, provided that the peptide does not contain cysteine in the amino acid sequence.
[62] As a more specific example of a peptide substantially identical to the peptide containing the amino acid sequence represented by SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22, for example, SEQ ID NO: 23 (SEQ ID NO: 23 of 10) And Xa is Ala or Thr, the eleventh Xaa is Gly or Ser, and the twenty-first Xaa represents OH, Gly or Gly-Arg.
[63] More specifically, 1 or more and 15 or less, preferably 1 or more and 10 or less, more preferably 1 or more 5 or more of the amino acid sequences represented by SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22 1 or more and 80 or less, preferably 1 or more and 50 or less, more preferably the amino acid sequence represented by the following amino acid sequence having the amino acid sequence represented by SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22 1 or more and 15 or less, preferably 1 or more and 10 or less of the amino acid sequence represented by SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22 with one or more amino acids added More preferably, the peptide etc. which contain the amino acid sequence in which 1 or more and 5 or less amino acids were substituted by the other amino acid are mentioned.
[64] More specifically, for example, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: : 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: Peptides having an amino acid sequence represented by: 41, SEQ ID NO: 42 or SEQ ID NO: 43, and the like.
[65] In addition, the PrRP of the present invention includes the N-terminal side of Gln being cleaved in vivo and the Gln is pyroglutaminated.
[66] As the GALP, the peptide described in WO99 / 48920 is used.
[67] As the GPR8 ligand, ligand activity against the seven transmembrane receptor protein GPR8 (O'Dowd, BF et al., Genomics, Vol. 28, pp. 84-91, 1995), eg binding activity with GPR8, GPR8 expression Cell stimulatory activity against cells (eg, arachidonic acid free, acetylcholine free, intracellular Ca ²⁺ free, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential variation, intracellular proteins) Phosphorylation, activation of c-fos, lowering of pH, activity for promoting GTPγS binding activity, and the like) may be any polypeptide. For example, peptides described in WO01-98494 may be used.
[68] Specific examples of the ligand polypeptide for GPR8 include polypeptides containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 44.
[69] Examples of the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 44 include, for example, an amino acid sequence represented by SEQ ID NO: 45, an amino acid sequence represented by SEQ ID NO: 46, an amino acid sequence represented by SEQ ID NO: 47, The amino acid sequence shown by sequence number 48, the amino acid sequence shown by sequence number 49, and the amino acid sequence shown by sequence number 50 are mentioned.
[70] That is, as a specific example of the polypeptide containing the amino acid sequence identical or substantially identical to the amino acid sequence shown by SEQ ID NO: 44,
[71] ① a GPR8 ligand having an amino acid sequence represented by SEQ ID NO: 44,
[72] ② GPR8 ligand having an amino acid sequence represented by SEQ ID NO: 45,
[73] ③ a GPR8 ligand having an amino acid sequence represented by SEQ ID NO: 46,
[74] ④ GPR8 ligand having the amino acid sequence represented by SEQ ID NO: 47,
[75] ⑤ GPR8 ligand having amino acid sequence represented by SEQ ID NO: 48,
[76] ⑥ GPR8 ligand having the amino acid sequence represented by SEQ ID NO: 49,
[77] ⑦ A GPR8 ligand having an amino acid sequence represented by SEQ ID NO: 50 is used.
[78] As the salt of the desired peptide, salts with physiologically acceptable bases (e.g., alkali metals) and acids (organic acids, inorganic acids) are used. Especially, physiologically acceptable acid addition salts are preferable. Such salts include, for example, salts with inorganic acids (e.g. hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (e.g. acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, Salts with oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.
[79] In the production method A of the present invention, as an enzymatic cleavage site that can be added to the N terminal of the target peptide, for example,
[80] (1) Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 58) (nucleotide sequence: encoded by DNA having GATGACGACGACAAG (SEQ ID NO: 59)), which is a cleavage site for enterokinase, a protease
[81] (2) Ile-Glu-Gly-Arg (SEQ ID NO: 60) (base sequence: encoded by DNA having ATTGAAGGCCGC (SEQ ID NO: 61)), which is a cleavage site of factor Xa, a protease
[82] (3) Gly-Pro-Arg (SEQ ID NO: 62) (base sequence: encoded by DNA having GGCCCGCGC (SEQ ID NO: 63)), which is a cleavage site for thrombin, which is a protease.
[83] As a chemical cleavage site which can be added to the N terminal of a target peptide, the methionine residue which is a cleavage site of bromine, etc. are mentioned, for example.
[84] As a chemical cleavage site which can be added to the C terminal of the target peptide, a cysteine residue, a cysteinyl peptide, etc. are mentioned, for example.
[85] The peptide portion of the cysteinyl peptide consists of one or two or more (eg, about 1 to 5) amino acid residues. Although the kind of amino acid residue is not specifically limited, Gly, Ala, Ser, Leu etc. are preferable. In addition, the peptide portion of the cysteinyl peptide differs from the desired peptide. Moreover, when adding a methionine residue at the N terminus of the desired peptide (using brominecine), the peptide portion of the cysteinyl peptide does not have a methionine residue.
[86] Precursor proteins (including precursor peptides) that are repeatedly linked by addition of an enzyme or chemical cleavage site to the N- and C-terminus of the target peptide include the enzyme or chemical cleavage site described above at the N- and C-terminus of the target peptide. Two or more added peptides (for example, about 2-100, preferably about 2-20, more preferably about 2-10) are the protein which connected repeatedly.
[87] In Preparation B of the present invention, the methionine residue or protease cleavage sequence at the N-terminal side of the target peptide, and the cysteine residue or cysteinyl peptide at the C-terminal side, provided that the peptide portion of the cysteinyl peptide differs from the target peptide. In addition, when the methionine residue is added to the N-terminal side (when bromine is used), the precursor protein (including the precursor peptide) which is repeatedly linked by addition of the peptide moiety does not have a methionine residue, means the above-mentioned target peptide. At least two proteins having a methionine residue or a protease cleavage site at the N-terminus, a cysteine residue or a cysteinyl peptide at the C-terminus, and an amino acid residue at the terminus. 100 pieces, preferably about 2 to 20 pieces, more preferably about 2 to 10 pieces) may be connected repeatedly.
[88] Protease cleavage sequences and cysteinyl peptides are the same as above.
[89] These precursor proteins used in Preparation A or B of the invention are preferably recombinant precursor proteins prepared using genetic engineering techniques.
[90] The recombinant precursor protein can be prepared, for example, by expressing the precursor protein by culturing a transformant containing a vector containing a DNA encoding a precursor protein described later.
[91] The DNA encoding the precursor protein may be any of those encoding the precursor protein, and may chemically synthesize the entire nucleotide sequence. As the production method in that case, for example, a known phosphoramidide method, a phosphate triester method, a diester method, a hydrogen phosphonate method or the like is used, and if it is short, the long one is divided and synthesized after T4DNA ligase. Can be connected and produced.
[92] As the DNA encoding each target peptide used for producing the DNA encoding the precursor protein, it is also possible to use a known DNA and can be cloned using a known method. The base sequence of DNA may be a natural base sequence as long as it codes each target peptide, or the base sequence which replaced the codon in the native base sequence with the other codon which codes for the same amino acid.
[93] DNA encoding the precursor protein repeatedly linked by adding an enzyme or a chemical cleavage site to the N- and C-terminal ends of the peptide of interest is enzyme or 5'-terminus and 3'-terminus of the DNA sequence encoding each target peptide. The base sequence encoding the chemical cleavage site is combined and constructed as a repeat sequence.
[94] The DNA encoding the precursor protein obtained by repetitively linking a methionine residue or protease cleavage sequence to the N-terminal side of the target peptide and a cysteine residue or cysteinyl peptide to the C-terminal side is obtained from the DNA sequence encoding the target peptide. The 5'-terminus binds the nucleotide sequence encoding the cleavage site of bromine or protease to be used for the cleavage reaction at the N-terminal side of the peptide bond, and further the 3'-terminus of the DNA nucleotide sequence encoding each target peptide. A base sequence encoding a site to be cleaved by ammonolysis or hydrolysis after S-cyanolation reaction using 1-cyano-4-dimethylaminopyridium salt (DMAP-CN), e.g. TGT or TGC, and the nucleotide sequence encoding the cysteinyl peptide) are combined and constructed as a repeat sequence.
[95] The C terminus of the precursor protein may be either a cysteine residue or a cysteinyl peptide.
[96] The DNA encoding the KiSS-1 peptide may be any DNA encoding the KiSS-1 peptide described above. For example, the DNA encoding the KiSS-1 peptide may be a DNA encoding a human KiSS-1 peptide having an amino acid sequence represented by SEQ ID NO: 1. DNA having a nucleotide sequence represented by SEQ ID NO: 3 is a nucleotide sequence represented by SEQ ID NO: 4 as a nucleotide sequence encoding a KiSS-1 (45-54) peptide having an amino acid sequence represented by SEQ ID NO: 2. DNA having the same or the like is used. Alternatively, the codon may be appropriately replaced with another codon encoding the same amino acid.
[97] As a specific example of DNA containing DNA which codes the KiSS-1 peptide used by the method of this invention, it is for example.
[98] GGTAGCGCGA TGTATAACTG GAACAGCTTT GGTCTGCGTT TTTGTGGCTC GGCGATGTAC
[99] AATTGGAATT CCTTCGGCCT GCGCTTCTGC GGCTCGGCGA TGTATAACTG GAACTCCTTT
[100] GGCCTGCGCT TTTGCGGTTC TGCT
[101] DNA containing the nucleotide sequence (SEQ ID NO: 5), and the like. This nucleotide sequence binds to the 5'-terminus of the nucleotide sequence represented by SEQ ID NO: 4 encoding the KiSS-1 peptide (45-54), and binds the nucleotide sequence (ATG) encoding the cleavage site of bromine. At the 3'-terminus, a base sequence (TGT) encoding a cleavage site of 1-cyano-4-dimethylaminopyridium salt (DMAP-CN) is bound. By repetitively linking the base sequences, DNA encoding the precursor protein of the KiSS-1 peptide can be prepared.
[102] The DNA encoding the RFRP-1 may be any DNA that codes the above RFRP-1. For example, the DNA encoding the RFRP-1 having the amino acid sequence represented by SEQ ID NO: 6 is SEQ ID NO: 12. DNA having a nucleotide sequence represented by SEQ ID NO: 2 is a DNA encoding RFRP-1 having an amino acid sequence represented by SEQ ID NO: 7 DNA having a nucleotide sequence represented by SEQ ID NO: 13 is represented by SEQ ID NO: 8 As DNA which codes RFRP-1 which has the displayed amino acid sequence, DNA which has the nucleotide sequence shown by SEQ ID NO: 14, and (4) DNA which codes RFRP-1 which has the amino acid sequence shown by SEQ ID NO: 9 is SEQ ID NO: : DNA having the nucleotide sequence represented by 15 is ⑤ DNA encoding the RFRP-1 having the amino acid sequence represented by SEQ ID NO: 10 is DNA having the nucleotide sequence represented by SEQ ID NO: 16 ⑥ SEQ ID NO: Marked with 11 As the DNA encoding RFRP-1 having an amino acid sequence, DNA having a nucleotide sequence represented by SEQ ID NO: 17 or the like is used.
[103] The DNA encoding the apelin may be any DNA so long as the above-mentioned apelin. For example, the DNA encoding the apelin 18 having the amino acid sequence represented by SEQ ID NO: 18 is represented by SEQ ID NO: 19. DNA having a nucleotide sequence to be used is used.
[104] The DNA encoding PrRP may be any DNA as long as the DNA encoding PrRP is mentioned. Specifically, the DNA described in WO97-24436 is used.
[105] As the DNA encoding the GALP, any DNA may be used as long as the DNA encodes the GALP. Specifically, the DNA described in WO 99-48920 is used.
[106] The DNA encoding the GPR8 ligand may be any DNA that codes the GPR8 ligand described above. Specifically, the DNA encoding the GPR8 ligand having the amino acid sequence represented by SEQ ID NO: 44 is represented by SEQ ID NO: 51. DNA having a nucleotide sequence, ② DNA encoding a GPR8 ligand having an amino acid sequence represented by SEQ ID NO: 45, DNA having a nucleotide sequence represented by SEQ ID NO: 52, ③ amino acid sequence represented by SEQ ID NO: 46 DNA encoding a GPR8 ligand having a DNA having a nucleotide sequence represented by SEQ ID NO: 53 is a nucleotide represented by SEQ ID NO: 54 as a DNA encoding a GPR8 ligand having an amino acid sequence represented by SEQ ID NO: 47. DNA having a sequence is a base represented by SEQ ID NO: 55 as DNA encoding a GPR8 ligand having an amino acid sequence represented by The DNA having the sequence is a DNA encoding a GPR8 ligand having an amino acid sequence represented by ⑥ SEQ ID NO: 49 as the DNA having the nucleotide sequence represented by SEQ ID NO: 56, and the amino acid sequence represented by ⑦ SEQ ID NO: 50. As the DNA encoding the GPR8 ligand, DNA having the nucleotide sequence represented by SEQ ID NO: 57 and the like are used.
[107] As a specific example of DNA containing DNA which codes the GPR8 ligand used in the method of this invention, it is for example.
[108] GATGACGATG ACAAATGGTA TAAACATGTG GCGAGCCCGC GTTATCATAC CGTGGGCCGC GCGGCCGGTC TGCTGATGGG CCTGTGTCAA TTGGGTGGTG ATGACGATGA CAAATGGTAT AAACATGTGG CGAGCCCGCG TTATCATACC GTGGGCCGCG CGGCCGGTCT GCTGATGGGC CTGTGTGAGC TCGGCTCTGA CGACGATGAT AAATGGTACA AACACGTTGC CTCCCCGCGC TACCACACGG TTGGTCGTGC CGCGGGCCTG CTGATGGGTC TGTGCGGT
[109] DNA containing the nucleotide sequence shown by (SEQ ID NO: 64), etc. are mentioned. This nucleotide sequence encodes an enterokinase cleavage sequence (Asp-Asp-Asp-Asp-Lys; SEQ ID NO: 58) at the 5'-end of the nucleotide sequence represented by SEQ ID NO: 44 encoding a GRP8 ligand (23 residues). A nucleotide sequence (SEQ ID NO: 59) is bonded, and a nucleotide sequence (TGT) encoding a cleavage site of 1-cyano-4-dimethylaminopyridium salt (DMAP-CN) at its 3'-end is added. Combined. By repeating linking the base sequences, a DNA encoding the precursor protein of the GPR8 ligand (23 residues) can be produced.
[110] In addition, the DNA encoding the precursor protein to which the target peptide is repeatedly linked can be converted into DNA encoding the mutein of the target peptide using conventional genetic techniques such as site-specific mutagenesis.
[111] Site specific mutagenesis techniques are known and are described in RF Radar (Lather, RF) and J. Lecoq (JP), Genetic Engineering, Academic Press (1983), pages 31-50 and the like. It is. Mutagenesis directed at oligonucleotides is described in Smith, M. and Gilam, S., Genetic Engineering: Principles and Methods, Plenum Press, 1981 (Vol. 3, 1-32), and the like. have.
[112] As a plasmid used as a vector containing DNA encoding a precursor protein, for example, pBR322 [Gene, 2, 95 (1977)] and pBR313 [Gene, 2, 75 (1977) derived from Escherichia coli (E. coli) ], pBR324, pBR325 [Gene, 4, 124 (1978)], pBR327, pBR328 [Gene, 9, 287 (1980)], pBR329 [Gene, 17,79 (1982)], pKY2289 [Gene, 3, 1 ( 1978), pKY 2700 [biochemistry, 52, 770 (1980)], pACYC177, pACYC184 [Journal of Bacteriology, 134, 1141 (1978)], pRK248, pRK646, pDF [Methods in Enzymology, 68, 268 (1979)] , pUC18, pUC19 (Yanisperon et al., Gene, 33, 103 (1985)) and the like.
[113] In addition, λgt · λC [Proc. Nat. Acad. Sci. U.S.A. 71, 4579 (1974), t · λB [Proc. Nat. Acad. Sci. U.S.A. 72, 3461 (1975), [lambda] Dam [Gene, 1, 255 (1977)] or Sharon vectors [Science, 196, 161 (1977); Journal of Virology, 29, 555 (1979)], mp-based mp18 and mp19 (Yanisperon et al., Gene, 33, 103 (1985)) vectors using fibrous phage.
[114] It is preferable that the said DNA has a promoter upstream of ATG, and the said promoter may be any promoter as long as it is a suitable promoter corresponding to the host used for manufacture of a transformant. For example, when the host is Escherichia coli (E. coli), trp promoter, lac promoter, rec A promoter, λPL promoter, lpp promoter, T7 promoter and the like are used.
[115] In the case of using the system of the T7 promoter, as the T7 promoter, 17 kinds of promoters found on T7 DNA [J. L. Oakley et al., Proc. Natl. Acad. Sci. U.S.A, 74: 4266-4270 (1977), M. D. Rosa, Cell 16: 815-825 (1979), N. Panayotatos et al., Nature, 280: 35 (1979), J. J. Dunn et al., J. Mol. Biol., 166: 477-535 (1983)], although the Ø10 promoter [A. H. Rosenberg et al., Gene, 56: 125-135 (1987).
[116] As a transcription terminator, a terminator operating in the system of Escherichia coli, preferably a TØ terminator [F. W. Studier et al., J. Mol. Biol. 189: 113-130 (1986), and the like.
[117] T7 RNA polymerase DNA includes T7 DNA [F. W. Studier et al., J. Mol. Biol., 189: 113-130 (1986), and the like.
[118] The vector is preferably constructed by inserting a T7 promoter and a T7 terminator into the vector. Examples of such vectors include pET-1, pET-2, pET-3, pET-4, and pET-5 [A. H. Rosenberg, Gene 56: 125-135 (1987)], pTB960-2 [EP-A-499990], and the like, preferably pTB960-2.
[119] The transformant may be selected from the known plasmids obtained in the above-described methods by known methods [eg, Coen S. N. et al., Pro. Natl. Acad. Sci. U.S.A., 69, 2110 (1972).
[120] Examples of the host of the microorganism to be transformed include Escherichia spp.
[121] As an example of the said Escherichia genus, Escherichia coli is mentioned, Specifically, Escherichia coli K12DH1 [Pro. Natl. Acad. Sci. USA, 60, 160 (1968)], JM-103 [Nucleic Acids Research, 9, 309 (1981)], JA221 [Journal of Molecular biology, 120, 517 (1978)], HB101 [Journal of Molecular biology, 41, 459 (1969)], C600 [Genetics, 39, 440 (1954)], N4830 [Cell, 25, 713 (1981)], K-12MM294 [Pro. Natl. Acad. Sci. U.S.A., 73, 4174 (1976)] BL-21 and the like.
[122] When a system of the T7 promoter is used, the host of the transformant is T7 RNA polymerase DNA (T7 DNA1) [F. W. Studier et al., J. Mol. Biol. 189: 113-130 (1986)] Escherichia coli (e.g., MM294, DH-1, C600, JM109, BL21) and T7 RNA polymerase DNA (T7 DNA1) with E. coli Notes and the like are preferably used. Preferably, MM294 strains and BL21 strains in which λ phage into which T7 DNA1 is inserted are solubilized are used. In this case, as a promoter of T7 DNA1, a lac promoter in which expression is induced by isopropyl-1-thio-β-D-galactopyranoside (sometimes abbreviated as IPTG) is used.
[123] The recombinant precursor protein can be prepared by harvesting the recombinant precursor protein produced by culturing the above-described transformant in a medium.
[124] The pH of the medium is preferably about 6-8. As a medium for culturing Escherichia spp., For example, M9 medium containing glucose and casamino acid [Miller, Journal of Experiments in Molecular Genetics, 431-433, Cold Spring Harbor Laboratory, New York 1972] is preferable. . To this, a drug such as, for example, 3β-indolyl acrylic acid or isopropyl βD-thiogalactopyranoside may be added to efficiently operate the promoter as needed.
[125] When the host is Escherichia spp., The culture is usually carried out at about 15 to 43 ° C. for about 3 to 24 hours, and aeration or agitation can be added as necessary.
[126] When using a system of T7 promoter, (1) when expressing T7 DNA (RNA polymerase DNA) which is connected downstream of the lac promoter, IPTG or the like is added, or (2) which is downstream of the λPL promoter. When expressing T7 DNA (RNA polymerase DNA), the T7 promoter is specifically activated by T7 phage RNA polymerase 1 generated by raising the culture temperature.
[127] After incubation, the cells are collected by a known method and suspended in a buffer, for example, followed by protein denaturation, ultrasonic treatment, enzyme treatment such as lysozyme, glass beads treatment, French press treatment, and freeze thawing treatment. The cells are crushed to obtain an inclusion body or a soluble body (supernatant) by a known method such as centrifugation, but is preferably obtained as an inclusion body.
[128] The inclusion body obtained as mentioned above may be solubilized using a modifier and the following reaction process may be advanced. In order to isolate the recombinant precursor protein from the supernatant, purification of a known protein can be followed. For example, gel filtration, ion exchange chromatography, adsorption chromatography, high-performance liquid chromatography, affinity chromatography, hydrophobic chromatography, electrophoresis and the like can be appropriately combined. The precursor protein may not be purified or may be subjected to the next reaction step in a partially purified state.
[129] Bromine or protease is used for the cleavage reaction of the N-terminal side of each target peptide in the recombinant precursor protein obtained as described above.
[130] The protease may be any known as a protease, but for example, enterokinase, factor Xa, thrombin and the like are preferable, and enterokinase and factor Xa are particularly preferably used.
[131] The amount of protease per mg of recombinant polypeptide is usually about 0.01 unit to about 100 units, preferably about 0.1 unit to about 10 units.
[132] When bromine is used in the cleavage reaction, a methionine residue which is a bromine cleavage site is linked to the N terminus of the target peptide in the recombinant precursor protein. In addition, in this case, it is preferable that the target peptide does not have a methionine residue.
[133] When enterokinase is used as a protease, a sequence (Asp-Asp-Asp-Asp-Lys; SEQ ID NO: 54) and the like that show an enterokinase cleavage site are linked to the N terminus of the target peptide in the recombinant precursor protein. In this case, it is preferable that the target peptide does not have an amino acid sequence represented by Asp-Asp-Asp-Asp-Lys or the like.
[134] When factor Xa is used as a protease, a sequence (Ile-Glu-Gly-Arg; SEQ ID NO: 56), etc., representing the factor Xa cleavage site, is linked to the N terminus of the target peptide in the recombinant precursor protein. In this case, it is preferable that the target peptide does not have an amino acid sequence represented by Ile-Glu-Gly-Arg or the like.
[135] When thrombin is used as a protease, a sequence (Gly-Pro-Arg; SEQ ID NO: 58) or the like indicating a thrombin cleavage site is linked to the N terminus of the target peptide in the recombinant precursor protein. In this case, it is preferable that the target peptide does not have an amino acid sequence represented by Gly-Pro-Arg or the like.
[136] The reaction temperature of the cleavage reaction of the peptide bond by the proteolytic enzyme is usually about 0 ° C to about 60 ° C, preferably about 0 ° C to about 40 ° C.
[137] Although it does not specifically limit as a solvent to be used, For example, a tris- hydrochloric acid buffer, a tris-acetic acid buffer, a phosphate buffer, a boric acid buffer, etc. are mentioned.
[138] The pH in the reaction is usually about 1 to about 12, preferably about 4 to about 8.
[139] The cleavage reaction at the C-terminal side of the desired peptide in the recombinant precursor protein can be carried out using, for example, the method described in EP887417. That is, the cleavage reaction is carried out at the N-terminal side of the C-terminal cysteine of the target peptide in the recombinant precursor protein.
[140] As said cleavage reaction, hydrolysis reaction is mentioned following S-cyanoation reaction, for example. When obtaining the amide of the target peptide or its salt as a final product, the said cleavage reaction, for example, performs an ammonolysis following an S-cyanoation reaction. Said S-cyanoation reaction is performed by making an S-cyanoation reagent react with a raw material compound.
[141] Examples of the S-cyanolation reagent include 2-nitro-5-thiocyano benzoic acid (NTCB), 1-cyano-4-dimethylaminopyridium salt (DMAP-CN), CN ^- ion, and the like. .
[142] The amount of the S-cyanolation reagent may be about 2 to 50 times the molar number of the total thiol groups, and preferably about 5 to 10 times the amount.
[143] As long as reaction temperature is a range of about 0 degreeC-80 degreeC, all are sufficient, and the range of about 0 degreeC-50 degreeC is more preferable. As the solvent to be used, any buffer may be used as long as it does not react with the S-cyanolation reagent. Examples thereof include tris-hydrochloric acid buffer, tris-acetic acid buffer, phosphate buffer, and boric acid buffer. The organic solvent may be present as long as it does not react with the S-cyanolation reagent.
[144] It is preferable to perform the said reaction in the range of pH 1-12. In particular, in the case of using NTCB, pH 7-10, in the case of using DMAP-CN, the range of pH 2-7 is preferable in order to prevent S-S exchange reaction. In the reaction solution, a modifying agent such as guanidine hydrochloride may be present.
[145] Examples of the ammonolysis or hydrolysis reaction include those which undergo alkali treatment.
[146] As said alkali treatment, it is performed by adjusting pH of the aqueous solution containing a raw material compound to 7-14.
[147] The pH is adjusted using, for example, ammonia, sodium hydroxide, amino compounds, Trizma Base (tris [hydroxymethyl] -aminomethane), dibasic sodium phosphate, potassium hydroxide, barium hydroxide and the like. Although appropriate amount is added to the aqueous solution containing a compound, especially ammonia etc. are preferable.
[148] As the concentration of the solution during the reaction, for example, about 0.01 to 15 N for ammonia or amino compounds, preferably about 0.1 to 3 N, about 0.01 to 2 N for sodium hydroxide, preferably about 0.05 to 1 N, about 1 mM to 1 M for Trisma base, preferably about 20 mM to 200 mM, about 1 mM to 1 M for disodium phosphate, preferably about 10 mM to 100 mM, hydroxide In the case of potassium, about 0.01-4 N, Preferably about 0.1-2 N is mentioned. As long as reaction temperature is the range of about -20 degreeC-80 degreeC, all are preferable, and the range of about -10 degreeC-50 degreeC is more preferable.
[149] The reaction time is preferably about 1 to 60 minutes, preferably about 15 to 30 minutes for the S-cyanoation reaction, about 5 to 100 hours for the hydrolysis reaction, preferably 10 minutes to 15 hours, Ammonolisis is about 5 minutes to 24 hours, preferably about 10 to 180 minutes.
[150] As the amino compound, for example, the formula R ^1- (NR ² ) -H (wherein R ¹ and R ² are the same or different, and (i) a hydrogen atom, (ii) a C _1-20 alkyl group, C _{3- 6} cycloalkyl group, C _6-14 aryl group, or C _6-14 aryl-C _1-3 alkyl group (they may have no substituent or may have _1-3 amino groups, hydroxyl groups, etc. on the carbon atom) and (iii) an amino group which may be substituted, (iv) a hydroxyl group or a C _1-6 alkoxy group.), etc. may be mentioned.
[151] In order to isolate the target peptide cut | disconnected by the said cleavage reaction, you may follow the purification method of the peptide normally known. For example, gel filtration, ion exchange chromatography, high-performance liquid chromatography, affinity chromatography, hydrophobic chromatography, thin layer chromatography, electrophoresis, and the like may be appropriately combined.
[152] In addition, the target peptide may be powdered by lyophilization if necessary. In lyophilization, it is possible to add stabilizers such as sorbitol, mannitol, dextrose, maltose, trehalose and glycerol.
[153] C-terminal of the target peptides obtained by the process of the invention is an amide (-CONH _2), carboxyl group (-COOH), carboxylate (-COO ^-) may be, alkylamide (-CONHR) or an ester (-COOR). As R of ester or alkylamide, for example, C _1-6 alkyl groups such as methyl, ethyl, n-propyl, isopropyl or n-butyl, C _3-8 cycloalkyl groups such as cyclopentyl, cyclohexyl, phenyl, α C such as C _6-12 aryl groups such as naphthyl, phenyl-C _1-2 alkyl such as benzyl, phenethyl, benzhydryl, or α-naphthyl-C _1-2 alkyl such as α-naphthylmethyl _In addition to the _7-14 aralkyl group, the pivaloyloxymethyl group etc. which are used as an oral ester are mentioned.
[154] The desired peptide, amide, ester or salt thereof obtained by the preparation of the present invention can be mixed with sterile water, human serum albumin (HSA), saline, other known physiologically acceptable carriers, Animals (eg, humans, monkeys, cattle, horses, sheep, pigs, rats, mice, morphotes, etc.) may be administered parenterally or topically. For example, the daily dose may be administered parenterally by intravenous injection, intramuscular injection or the like from about 0.01 mg to about 50 mg, preferably about 0.1 mg to about 10 mg per person.
[155] Preparations containing the peptide of interest of the present invention may also contain other physiologically acceptable active ingredients such as salts, diluents, adjuvants, other carriers, buffers, binders, surfactants, preservatives. Parenteral dosage forms are provided as suspension ampoules with sterile aqueous solutions or physiologically acceptable solvents, or as sterile powders (typically obtained by lyophilizing peptide solutions) that can be diluted when used in physiologically acceptable dilutions. .
[156] In the present specification and drawings, amino acids, peptides, protecting groups, activators, and other abbreviations are used as examples, and they are based on the abbreviation according to the Commission on Biochemical Nomenclature (IUPAC-IUB) or the common abbreviation in the relevant field. I put it next. In addition, when there exists an optical isomer with respect to an amino acid etc., unless otherwise indicated, L form shall be shown.
[157] DNA: deoxyribonucleic acid
[158] A: Adenine
[159] T: Thymine
[160] G: guanine
[161] C: cytosine
[162] RNA: ribonucleic acid
[163] EDTA: Ethylenediamine Saacetic Acid
[164] Gly: glycine
[165] Ala: Alanine
[166] Val: Valine
[167] Leu: Leucine
[168] Ile: Isoleucine
[169] Ser: Serine
[170] Thr: Threonine
[171] Met: Methionine
[172] Glu: glutamic acid
[173] Asp: Aspartic Acid
[174] Lys: Lysine
[175] Arg: Arginine
[176] His: histidine
[177] Phe: Phenylalanine
[178] Tyr: tyrosine
[179] Trp: Tryptophan
[180] Pro: Proline
[181] Asn: Asparagine
[182] Gln: Glutamine
[183] ATP: Adenosine Triphosphate
[184] T7P: T7 promoter
[185] T7T: T7 Terminator
[186] The sequence numbers of the present specification indicate the following.
[187] [SEQ ID NO: 1]
[188] The amino acid sequence of the human KiSS-1 peptide is shown.
[189] [SEQ ID NO: 2]
[190] The amino acid sequence of the human KiSS-1 (45-54) peptide is shown.
[191] [SEQ ID NO: 3]
[192] The base sequence of the DNA encoding the human KiSS-1 peptide represented by SEQ ID NO: 1 is shown.
[193] [SEQ ID NO: 4]
[194] The base sequence of the DNA encoding the human KiSS-1 (45-54) peptide represented by SEQ ID NO: 2 is shown.
[195] [SEQ ID NO: 5]
[196] The base sequence of the DNA containing DNA encoding the precursor protein of the human KiSS-1 (45-54) peptide is shown.
[197] [SEQ ID NO: 6]
[198] The amino acid sequence of RFRP-1 with 9 amino acid residues is shown.
[199] [SEQ ID NO: 7]
[200] The amino acid sequence of RFRP-1 with 12 amino acid residues is shown.
[201] [SEQ ID NO: 8]
[202] The amino acid sequence of RFRP-1 with 20 amino acid residues is shown.
[203] [SEQ ID NO: 9]
[204] The amino acid sequence of RFRP-1 with 37 amino acid residues is shown.
[205] [SEQ ID NO: 10]
[206] The amino acid sequence of RFRP-1 with 9 amino acid residues is shown.
[207] [SEQ ID NO: 11]
[208] The amino acid sequence of RFRP-1 with 17 amino acid residues is shown.
[209] [SEQ ID NO: 12]
[210] The base sequence of DNA which codes the polypeptide shown by the amino acid sequence of SEQ ID 6 is shown.
[211] [SEQ ID NO: 13]
[212] The base sequence of the DNA encoding the polypeptide represented by the amino acid sequence of SEQ ID 7 is shown.
[213] [SEQ ID NO: 14]
[214] The base sequence of the DNA encoding the polypeptide represented by the amino acid sequence of SEQ ID NO: 8 is shown.
[215] [SEQ ID NO: 15]
[216] The base sequence of the DNA encoding the polypeptide represented by the amino acid sequence of SEQ ID NO: 9 is shown.
[217] [SEQ ID NO: 16]
[218] The base sequence of the DNA encoding the polypeptide represented by SEQ ID NO: 10 is shown.
[219] [SEQ ID NO: 17]
[220] The base sequence of the DNA encoding the polypeptide represented by SEQ ID NO: 11 is shown.
[221] [SEQ ID NO: 18]
[222] The amino acid sequence of Apelin-36 is shown.
[223] [SEQ ID NO: 19]
[224] The base sequence of DNA encoding apelin-36 is shown.
[225] [SEQ ID NO: 20]
[226] The amino acid sequence of bovine PrRP is shown.
[227] [SEQ ID NO: 21]
[228] The amino acid sequence of rat PrRP is shown.
[229] [SEQ ID NO: 22]
[230] The amino acid sequence of human PrRP is shown.
[231] [SEQ ID NO: 23]
[232] Specific examples of peptides substantially the same as those having the amino acid sequence represented by SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22 are shown.
[233] [SEQ ID NO: 24]
[234] The amino acid sequence obtained from the analysis of the N terminal sequence of the P-3 fraction by refine | purifying the bovine hypothalamic derived ligand polypeptide of WO97 / 24436 is shown.
[235] [SEQ ID NO: 25]
[236] The amino acid sequence obtained from the analysis of the N terminal sequence of the P-2 fraction by refine | purifying the bovine hypothalamic derived ligand polypeptide of WO97 / 24436 is shown.
[237] [SEQ ID NO: 26]
[238] The amino acid sequence of the bovine hypothalamic derived ligand polypeptide described in WO97 / 24436 is shown.
[239] [SEQ ID NO: 27]
[240] The amino acid sequence of the bovine hypothalamic derived ligand polypeptide described in WO97 / 24436 is shown.
[241] [SEQ ID NO: 28]
[242] The amino acid sequence of the bovine hypothalamic derived ligand polypeptide described in WO97 / 24436 is shown.
[243] [SEQ ID NO: 29]
[244] The amino acid sequence of the bovine hypothalamic derived ligand polypeptide described in WO97 / 24436 is shown.
[245] [SEQ ID NO: 30]
[246] The amino acid sequence of the bovine hypothalamic derived ligand polypeptide described in WO97 / 24436 is shown.
[247] [SEQ ID NO: 31]
[248] The amino acid sequence of the bovine hypothalamic derived ligand polypeptide described in WO97 / 24436 is shown.
[249] [SEQ ID NO: 32]
[250] The amino acid sequence of the rat ligand polypeptide described in WO97 / 24436 is shown.
[251] [SEQ ID NO: 33]
[252] The amino acid sequence of the rat ligand polypeptide described in WO97 / 24436 is shown.
[253] [SEQ ID NO: 34]
[254] The amino acid sequence of the rat ligand polypeptide described in WO97 / 24436 is shown.
[255] [SEQ ID NO: 35]
[256] The amino acid sequence of the rat ligand polypeptide described in WO97 / 24436 is shown.
[257] [SEQ ID NO: 36]
[258] The amino acid sequence of the rat ligand polypeptide described in WO97 / 24436 is shown.
[259] [SEQ ID NO: 37]
[260] The amino acid sequence of the rat ligand polypeptide described in WO97 / 24436 is shown.
[261] [SEQ ID NO: 38]
[262] The amino acid sequence of the humanoid ligand polypeptide described in WO97 / 24436 is shown.
[263] [SEQ ID NO: 39]
[264] The amino acid sequence of the humanoid ligand polypeptide described in WO97 / 24436 is shown.
[265] [SEQ ID NO: 40]
[266] The amino acid sequence of the humanoid ligand polypeptide described in WO97 / 24436 is shown.
[267] [SEQ ID NO: 41]
[268] The amino acid sequence of the humanoid ligand polypeptide described in WO97 / 24436 is shown.
[269] [SEQ ID NO: 42]
[270] The amino acid sequence of the humanoid ligand polypeptide described in WO97 / 24436 is shown.
[271] [SEQ ID NO: 43]
[272] The amino acid sequence of the humanoid ligand polypeptide described in WO97 / 24436 is shown.
[273] [SEQ ID NO: 44]
[274] The amino acid sequence of the ligand polypeptide (human type 1-23) with respect to GPR8 is shown.
[275] [SEQ ID NO: 45]
[276] The amino acid sequence of the ligand polypeptide (swine 1-23) with respect to GPR8 is shown.
[277] [SEQ ID NO: 46]
[278] The amino acid sequence of the ligand polypeptide (rat mouse type 1-23) with respect to GPR8 is shown.
[279] [SEQ ID NO: 47]
[280] The amino acid sequence of the ligand polypeptide (human type 1-30) with respect to GPR8 is shown.
[281] [SEQ ID NO: 48]
[282] The amino acid sequence of the ligand polypeptide (swine type 1-30) with respect to GPR8 is shown.
[283] [SEQ ID NO: 49]
[284] The amino acid sequence of the ligand polypeptide (rat type 1-30) with respect to GPR8 is shown.
[285] [SEQ ID NO: 50]
[286] The amino acid sequence of the ligand polypeptide (mouse type 1-30) with respect to GPR8 is shown.
[287] [SEQ ID NO: 51]
[288] The base sequence of the DNA encoding the ligand polypeptide (human type 1-23) for GPR8 is shown.
[289] [SEQ ID NO: 52]
[290] The base sequence of the cDNA encoding the ligand polypeptide (swine type 1-23) for GPR8 is shown.
[291] [SEQ ID NO: 53]
[292] The base sequence of the DNA encoding the ligand polypeptide (rat mouse type 1-23) for GPR8 is shown.
[293] [SEQ ID NO: 54]
[294] The base sequence of the DNA encoding the ligand polypeptide (human type 1-30) for GPR8 is shown.
[295] [SEQ ID NO: 55]
[296] The nucleotide sequence of the DNA encoding the ligand polypeptide (swine type 1-30) for GPR8 is shown.
[297] [SEQ ID NO: 56]
[298] The base sequence of the DNA encoding the ligand polypeptide (rat type 1-30) for GPR8 is shown.
[299] [SEQ ID NO: 57]
[300] The base sequence of the DNA encoding the ligand polypeptide (mouse type 1-30) for GPR8 is shown.
[301] [SEQ ID NO: 58]
[302] The amino acid sequence which represents an enterokinase cleavage sequence is shown.
[303] [SEQ ID NO: 59]
[304] The base sequence of the DNA encoding the enterokinase cleavage sequence is shown.
[305] [SEQ ID NO: 60]
[306] An amino acid sequence representing the factor Xa cleavage sequence is shown.
[307] [SEQ ID NO: 61]
[308] The base sequence of DNA encoding factor Xa cleavage sequence is shown.
[309] [SEQ ID NO: 62]
[310] An amino acid sequence representing the thrombin cleavage sequence is shown.
[311] [SEQ ID NO: 63]
[312] The base sequence of the DNA encoding the thrombin cleavage sequence is shown.
[313] [SEQ ID NO: 64]
[314] The base sequence of the DNA containing DNA encoding the precursor protein of the human GPR8 ligand (SEQ ID NO: 44) is shown.
[315] [SEQ ID NO: 65]
[316] The base sequence of the DNA oligomer used for manufacture of a structural gene in Example 1 is shown.
[317] [SEQ ID NO: 66]
[318] The base sequence of the DNA oligomer used for manufacture of a structural gene in Example 1 is shown.
[319] [SEQ ID NO: 67]
[320] The base sequence of the DNA oligomer used for manufacture of a structural gene in Example 1 is shown.
[321] [SEQ ID NO: 68]
[322] The base sequence of the DNA oligomer used for manufacture of a structural gene in Example 1 is shown.
[323] [SEQ ID NO: 69]
[324] The base sequence of the DNA oligomer used for manufacture of a structural gene in Example 1 is shown.
[325] [SEQ ID NO: 70]
[326] The base sequence of the DNA oligomer used for manufacture of a structural gene in Example 1 is shown.
[327] [SEQ ID NO: 71]
[328] The base sequence of the DNA oligomer used for manufacture of a structural gene in Example 1 is shown.
[329] [SEQ ID NO: 72]
[330] The base sequence of the DNA oligomer used for manufacture of a structural gene in Example 1 is shown.
[331] [SEQ ID NO: 73]
[332] The base sequence of the DNA oligomer used for manufacture of a structural gene in Example 1 is shown.
[333] [SEQ ID NO: 74]
[334] The base sequence of the DNA oligomer used for manufacture of a structural gene in Example 1 is shown.
[335] The transformant Escherichia coli MM294 (DE3) / pTCGPR3 obtained in Example 1 below was 1-1-1 Chuo Higashi 1-1-1, Tigakushi Ibarakiken from April 17, 2002 (Zip No. 305). (Friority No. FERM BP-8023), the Independent Administrative Institution of the Institute of Industrial Technology, Japan Institute for Research on Patents and Biotechnology, March 14, 2002, 2-17-85, Juusanbonmachi, Yodogawa-ku, Osaka, Japan. -8686, deposited as IFO 16773 at the Fermentation Research Institute (IFO).
[336] Although an Example is shown to the following and this invention is demonstrated in more detail, this invention is not limited to these.
[337] Example 1
[338] (a) Preparation of a gene encoding the human GPR8 ligand (hGPR8L; SEQ ID NO: 44) three times in series
[339] Structural genes encoding hGPR8L three times in series were prepared using ten types of DNA fragments shown below (SEQ ID NOs: 65 to 74).
[340] #One
[341] 5'-TATGGATGACGATGACAAATGGTATAAACATGTGGCGAGCCCGCGTTATCATACCG
[342] (SEQ ID NO: 65)
[343] #2
[344] 5'-GCGCGGCCCACGGTATGATAACGCGGGCTCGCCACATGTTTATACCATTTGTCATCGTCATCCA
[345] (SEQ ID NO: 66)
[346] # 3
[347] 5'-TGGGCCGCGCGGCCGGTCTGCTGATGGGCCTGTGTCAATTGGGTTTGAACTTCTCTGTCTCCGC
[348] CGCCGGAG (SEQ ID NO: 67)
[349] #4
[350] 5'-GATCCTCCGGCGGCGGAGACAGAGAAGTTCAAACCCAATTGACACAGGCCCATCAGCAGACCGGCC
[351] (SEQ ID NO: 68)
[352] # 5
[353] 5'-AATTGGGTGGTGATGACGATGACAAATGGTATAAACATGTGGCGAGCCCGCGTTATCATACCG
[354] (SEQ ID NO: 69)
[355] # 6
[356] 5'-CGGCCCACGGTATGATAACGCGGGCTCGCCACATGTTTATACCATTTGTCATCGTCATCACCACCC
[357] (SEQ ID NO: 70)
[358] # 7
[359] 5'-TGGGCCGCGCGGCCGGTCTGCTGATGGGCCTGTGTGAGCTCGGCTCTGACGACGATGATAAATGGT
[360] AC (SEQ ID NO: 71)
[361] #8
[362] 5'-CAACGTGTTTGTACCATTTATCATCGTCGTCAGAGCCGAGCTCACACAGGCCCATCAGCAGACCGG
[363] CC (SEQ ID NO: 72)
[364] # 9
[365] 5'-AAACACGTTGCCTCCCCGCGCTACCACACGGTTGGTCGTGCCGCGGGCCTGCTGATGGGTCTGTGC
[366] GGTTGAG (SEQ ID NO: 73)
[367] # 10
[368] 5'-GATCCTCAACCGCACAGACCCATCAGCAGGCCCGCGGCACGACCAACCGTGTGGTAGCGCGGGGAG
[369] G (SEQ ID NO: 74)
[370] DNA oligomers of # 2 and # 3 were respectively diluted with 25 μl of phosphorylation reaction [10 μg of DNA oligomer, 50 mM Tris-HCl, pH 7.6, 10 mM MgCl ₂ , 1 mM spermidine, 10 mM dithiosray Toluene (dithiothreitol, then abbreviated to DTT), 0.1 mg / ml bovine serum albumin (hereinafter abbreviated to BSA), 1 mM ATP, 10 units T4 polynucleotide kinase (Takara Casting)] for 1 hour at 37 ° C. for each oligomer The 5 'end was phosphorylated. After phenol treatment, twice the amount of ethanol was added, cooled to -70 ° C, and the DNA was precipitated by centrifugation.
[371] The DNA fragments obtained above were combined with # 1 and # 4 to obtain 120 µl. The mixture was kept at 90 ° C. for 10 minutes, then slowly cooled to room temperature and annealed, followed by ligation reaction using TaKaRa DNA Ligation Kit ver. 2 (Takara Casting). 30 μl of solution II was added to 30 μl of the annealing solution, and the mixture was well mixed. After phenol treatment, the aqueous layer was recovered, twice the amount of ethanol was added, cooled to -70 ° C, and the DNA was precipitated by centrifugation. The DNA fragment thus obtained was phosphorylated by T4 polynucleotide kinase (Takara Co., Ltd.) to prepare a structural gene.
[372] As the expression vector, pTCII (WO00 / 20643) was digested with NdeI and BamHI (Takara) for 4 hours at 37 ° C, and then 4.4 kb of DNA fragments were subjected to 1% agarose gel electrophoresis. Extraction was performed and dissolved in 25 μl of TE buffer. The NdeI, BamHI fragment of pTCII and the structural gene prepared as described above were subjected to ligation reaction using TaKaRa DNA ligation kit ver. 10 μl of the reaction solution was transformed into E. coli JM109 competent cells (Toyobo), and sprinkled on LB agar medium containing 10 μg / ml of tetracycline and incubated at 37 ° C. overnight. Cyclin resistant colonies were selected. This transformant was incubated overnight in LB medium, and plasmid pTCGPR1 was prepared using QIAprep8 Miniprep Kit (Qiagen). This pTCGPR1 was digested with MunI and BamHI for 4 hours at 37 ° C., followed by extraction of 4.5 kb of DNA fragments using QIAquick Gel Extraction Kit (Qiagen) by 1% agarose gel electrophoresis and 25 μl of TE. Dissolved in buffer.
[373] DNA oligomers of # 6, # 7, # 8 and # 9 were each diluted with 25 μl of phosphorylation reaction [10 μg of DNA oligomer, 50 mM Tris-HCl, pH 7.6, 10 mM MgCl ₂ , 1 mM spermidine, 10 mM DTT, 0.1 mg / ml BSA, 1 mM ATP, 10 unit T4 polynucleotide kinase (Takara Co., Ltd.)] was reacted at 37 ° C. for 1 hour to phosphorylate the 5 ′ end of each oligomer. After phenol treatment, twice the amount of ethanol was added, cooled to -70 ° C, and the DNA was precipitated by centrifugation. The DNA fragments # 5 and # 10 were combined to make 120 µl. The mixture was kept at 90 ° C. for 10 minutes, then slowly cooled to room temperature and annealed, followed by ligation reaction using TaKaRa DNA Ligation Kit ver. 2 (Takara Casting). 30 μl of solution II was added to 30 μl of the annealing solution, followed by well mixing. After phenol treatment, the aqueous layer was recovered, twice the amount of ethanol was added, cooled to -70 ° C, and the DNA was precipitated by centrifugation. The DNA fragment thus obtained was phosphorylated by T4 polynucleotide kinase (Takara). The structural gene portion and the MunI and BamHI fragments of pCTGPR1 were ligated using TaKaRa DNA ligation kit ver. 10 μl of the reaction solution was transformed into E. coli JM109 competent cells (Toyobo), and sprinkled on LB electrostatic medium containing 10 μg / ml tetracycline and incubated at 37 ° C. overnight to produce tetracycline resistant colonies. Was selected. This transformant was incubated overnight in LB medium, and plasmid pTCGPR3 was prepared using QIAprep8 Miniprep Kit (Qiagen). The base sequence of the precursor protein structural gene portion of pTCGPR3 was confirmed using an Applied Biosystems Model 377 DNA sequencer. Plasmid pTCGPR3 was transformed into Escherichia coli MM294 (DE3) to obtain an expression strain of precursor protein MM294 (DE3) / pTCGPR3.
[374] (b) Preparation of Precursor Protein
[375] MM294 (DE3) / pTCGPR3 in 30 ml (1% peptone, 0.5% yeast extract, 0.5% sodium chloride) in LB medium containing 10 mg / L tetracycline for 8 hours at 37 ° C. in a 200 ml flask. Shake culture was performed. 15 ml of the obtained culture was added to 300 ml of 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 Shake culture was initiated at 37 ° C. by implanting into a 1000 ml flask filled with% casamino acid. When the turbidity of the culture solution became 150 cleats (Klett) units, the final concentration of isopropyl-β-D-thiogalactopyranoside was added to 10 mg / L and further incubated for 3 hours. After completion of the culture, the culture solution (300 ml) was centrifuged to obtain about 2 g of a wet cell.
[376] Example 2
[377] 10 g of 10 mM EDTA (pH 6.0) was added to 2 g of the cells obtained in Example 1, followed by sonication (BRANSON SONIFIER MODEL450), followed by centrifugation (15000 rpm, 15 minutes). The same operation was carried out again on the precipitate. 5 ml (pH 5.0) of 7 M guanidine solution was added to the precipitate, followed by stirring for 2 hours, followed by centrifugation (15000 rpm, 15 minutes). 17 mg of tris (2-carboxyethyl) -phosphine hydrochloride (TCEP-HCl) was added to the supernatant, and reduced for 10 minutes at 50 ° C., followed by C4P-50 (1 cm × 25 cm, equilibrated with 0.1% TFA). After adsorbing and washing the liquid through Wadenko), the step gradient of 20-60% B (B: 80% acetonitrile / 0.1% trifluoroacetic acid) was eluted at a flow rate of 2 ml / min. Protein fractions (elution time about 27 minutes) were collected and lyophilized to obtain lyophilized powder of precursor protein.
[378] Example 3
[379] After dissolving the precursor lyophilized powder obtained in Example 2 with 1 ml of 0.1 M acetic acid and 6 M urea solution, 3.5 mg of DMAP-CN was added and reacted at 25 ° C. for 15 minutes. After completion of the reaction, the solution was adsorbed and washed through C4P-50 (1 cm × 25 cm, Showa Denko) equilibrated with 0.1% TFA, followed by 20-60% B (B: 80% acetonitrile / 0.1% trifluoro). Step gradient of roacetic acid) was eluted at a flow rate of 2 ml / min to collect the S-cyanoated precursor protein fraction (elution time about 27 minutes) and then lyophilized to obtain a lyophilized powder of the S-cyanoated precursor protein. . The mobile-dried powder was dissolved in 0.8 ml of 6 M urea, and 0.2 ml of 1 N caustic soda solution was added and reacted at 0 ° C. for 15 minutes. After completion of the reaction was prepared with pH 7.4 with acetic acid. 9 ml of 50 mM NaCl, 2 mM CaCl ₂ , 20 mM Tris / HCl (pH 7.4) solution was added to the cleavage reaction solution, and 10 units of enterokinase (Novagen) were added thereto and reacted at 25 ° C. for 20 hours. After completion of the reaction, the solution was adsorbed and washed through C4P-50 (1 cm × 25 cm, Showa Denko) equilibrated with 0.1% trifluoroacetic acid, followed by 20-60% B (B: 80% acetonitrile / 0.1 % Trifluoroacetic acid) was eluted at a flow rate of 2 ml / min to collect hGPR8L fractions (elution time about 22 minutes) and then lyophilized to obtain about 70 μg of hGPR8L lyophilized powder.
[380] Example 4 (Characteristic Determination of hGPR8L)
[381] a) N-terminal amino acid sequence analysis
[382] The N terminal amino acid sequence was determined using a gaseous protein sequencer (PE Applied Biosystems Model 491). As a result, it was consistent with the N-terminal amino acid sequence expected from the DNA base sequence of hGPR8L (Table 1).
[383]
[384] 1) Phenylthiohydantoin
[385] b) mass spectrometry
[386] As a result of analyzing the mass of the obtained hGPR8L, it became 2583.7 Da (theoretical value: 2584.0).
[387] c) active
[388] Using the same method as in Example 6 of WO01 / 98494, GTPγS binding activity using the membrane fraction of GPR8 expressing CHO cells was confirmed to be equivalent to the chemical synthesis product.
[389] Example 5 Construction of KiSS-1 Peptide Precursor Protein Expression Plasmids in E. Coli
[390] Precursor protein expression plasmids of KiSS-1 (35-54) peptides corresponding to the 35th to 54th amino acid sequences represented by SEQ ID NO: 1 were constructed as follows.
[391] In the same manner as in Example 1, 10 kinds of DNA fragments were used to prepare a structural gene encoding the KiSS-1 (35-54) peptide in series three times. This structural gene is ligated using the NdeI and BamHI fragments of the pTCII vector and the structural gene using TaKaRa DNA ligation kit ver.2 (Takara Casting). E. coli JM109 competent cells (Toyobo) were transformed using 10 µl of the reaction solution, sprinkled on LB agar medium containing 10 µg / ml of tetracycline, and cultured overnight at 37 ° C. Select. This transformant is incubated overnight in LB medium, and plasmid pTCKiSS3554 is prepared using QIAprep8 Miniprep Kit (Qiagen). The base sequence of this polypeptide DNA was confirmed using the Applied Biosystems Model 377 DNA sequencer. Plasmid pTCKiSS 3554 is transformed into E. coli (E. coli) MM294 (DE3) to obtain the precursor protein expression Escherichia coli MM294 (DE3) / pTCKiSS3554.
[392] Example 6 Preparation of Precursor Protein
[393] Escherichia coli MM294 (DE3) / pTCKiSS3554 of Example 5 was dosed with 2 L using LB medium containing 1 mg (1% peptone, 0.5% yeast extract, 0.5% sodium chloride) containing 5.0 mg / L tetracycline. Shake culture was carried out at 37 ℃ for 8 hours in a flask of. The resulting culture was prepared with 19 L of 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 Aeration was initiated at 30 ° C. by implanting into a 50 L fermenter filled with% thiamine hydrochloride, 1.5% glucose, 1.5% Hy-Case Amino (tradename). When the turbidity of the culture solution reaches 500 cleats, add the final concentration of isopropyl-β-D-thiogalactopyranoside to 10 mg / L and incubate for another 4 hours. After completion of the culture, the culture solution is centrifuged to obtain wet cells and stored at -80 ° C.
[394] Example 7 Acquisition of KiSS-1 (35-54)
[395] 200 ml of 10 mM EDTA (pH 6) was added to 100 g of the cell obtained in Example 6, followed by sonication (BRANSON SONIFIER MODEL 450) and centrifugation (10000 rpm, 60 minutes). The same operation is carried out again on the precipitate. 50 ml of 0.1 M acetic acid and 8 M urea solution were added to the precipitate, which was stirred for 2 hours and centrifuged (10000 rpm, 60 minutes). 200 mg of 1-cyano-4-dimethylaminopyridinium salt (DMAP-CN) was added to the supernatant and allowed to react at room temperature for 15 minutes. After completion of the reaction, the reaction solution was passed through a Sephadex G-25 column (48 mmID × 600 mmL, Parmacia) equilibrated with 10% acetic acid, and 10% acetic acid used for equilibration was developed at a flow rate of 6 ml / min. Obtain a cyanoylated polypeptide. The eluate is concentrated and desalted with a Pellicon Mini Cassette (Millipore), urea is added to a final concentration of 6 M, and 25% ammonia water is added to a concentration of 3 M, followed by 15 minutes at 15 ° C. React. After completion of the reaction, the mixture was adjusted to pH 6.0 with acetic acid. The reaction solution was adsorbed and washed with ODS-120T (21.5 mm x 300 mm, Tosoh) equilibrated with 0.1% trifluoroacetic acid (TFA), followed by 20-60% B (B: 80% acetonitrile). Elution in a step gradient of 0.1% TFA) to collect peptide fractions amidated at the C terminus, and the fractions are lyophilized to obtain lyophilized powder of peptides. The peptide is dissolved in 10 ml of 70% formic acid solution, and 10 mg of brominecine is added and reacted at room temperature for 24 hours. After completion of the reaction, the solution was adsorbed and washed through ODS-120T (21.5 mm × 300 mm, Toso) equilibrated with 0.1% trifluoroacetic acid (TFA), and then 20-60% B (B: 80% acetonitrile / Eluate in a step gradient of 0.1% TFA) to collect KiSS-1 (35-54) fractions and lyophilize to obtain lyophilized powder of KiSS-1 (35-54).
[396] Example 8 Preparation of KiSS-1 (45-54)
[397] According to the method described in Example 1, the precursor protein expression plasmid pTCKiSS4554 of the KiSS-1 (45-54) peptide corresponding to the 45th to 54th amino acid sequence represented by SEQ ID NO: 1 was constructed. This plasmid has a structural gene encoding KiSS-1 (45-54) nine times in series. Plasmid pTCKiSS4554 is transformed into E. coli (E. coli) MM294 (DE3) to obtain the precursor protein expression Escherichia coli MM294 (DE3) / pTCKiSS4554. The expression strain was cultured, and KiSS-1 (45-54) was obtained in the same manner as in Example 6 from the cells after expression induction with isopropyl-β-D-thiogalactopyranoside.
[398] The method of the present invention combining the cleavage of the target peptide with the right-hand scissors (S-cyanolation reaction) and the left-hand scissors (Bromtian treatment, enterokinase, factor Xa treatment, etc.) and the serial iteration method are peptides by genetic recombination technology. And particularly useful for mass synthesis of low molecular weight peptides.

权利要求:
Claims (21)
[1" claim-type="Currently amended] A method for producing a target peptide or a salt thereof, characterized by enzymatically or chemically cleaving a precursor protein repeatedly linked by adding an enzyme or a chemical cleavage site to the N- and C-terminal ends of the target peptide.
[2" claim-type="Currently amended] The method according to claim 1, wherein the precursor protein obtained by repetitively connecting the enzyme or chemical cleavage site to the N-terminal of the target peptide and the chemical cleavage site to the C-terminal is enzymatically or chemically cleaved.
[3" claim-type="Currently amended] A methionine residue or protease cleavage sequence at the N terminus of the target peptide, a cysteine residue or a cysteinyl peptide at the C terminus, provided that the peptide portion of the cysteinyl peptide is different from the target peptide and a methionine residue at the N terminus. When added, the peptide moiety does not have a methionine residue), and the N-terminal side of each target peptide in the repeatedly linked precursor protein is cleaved with bromine or protease, and the C-terminal cysteine or cysteinyl peptide Process for the preparation of the desired peptide or salt thereof, which is subjected to cleavage reaction at the N-terminal side.
[4" claim-type="Currently amended] The method according to any one of claims 1 to 3, wherein the precursor protein is a recombinant precursor protein.
[5" claim-type="Currently amended] The process according to claim 3, wherein the cleavage reaction is a reaction which is subjected to an S-cyanolation reaction followed by an amonolisis or a hydrolysis reaction.
[6" claim-type="Currently amended] The process of claim 5 wherein the S-cyanoation reaction is carried out in the presence of 2-nitro-5-thiocyanobenzoic acid (NTCB), 1-cyano-4-dimethylaminopyridium salt (DMAP-CN) or CN ⁻ ions. The manufacturing method to perform.
[7" claim-type="Currently amended] The method of claim 3, wherein the protease is enterokinase, factor Xa or thrombin.
[8" claim-type="Currently amended] The method of claim 3,
(1) When brominecine is used, the methionine residue is linked to the N terminus of each target peptide, and the target peptide does not have a methionine residue,
(2) When the protease is enterokinase, Asp-Asp-Asp-Asp-Lys is linked to the N terminus of each target peptide, and the target peptide does not have an amino acid sequence represented by Asp-Asp-Asp-Asp-Lys. ,
(3) When the protease is factor Xa, Ile-Glu-Gly-Arg is linked to the N terminus of each target peptide, and the target peptide does not have an amino acid sequence represented by Ile-Glu-Gly-Arg,
(4) When the protease is thrombin, a production method in which Gly-Pro-Arg is linked to the N terminus of each target peptide, and the target peptide does not have an amino acid sequence represented by Gly-Pro-Arg.
[9" claim-type="Currently amended] The method according to any one of claims 1 to 3, wherein the desired peptide is a KiSS-1 peptide.
[10" claim-type="Currently amended] The preparation method according to any one of claims 1 to 3, wherein the peptide of interest is a GPR8 ligand.
[11" claim-type="Currently amended] The N-terminus of each GPR8 ligand in the precursor protein, which was repeatedly linked three times by adding an enterokinase cleavage sequence to the N-terminus of the GPR8 ligand and a cysteine residue at the C-terminus, was cut with enterokinase and the N-cysteine N A process for producing a GPR8 ligand or a salt thereof, characterized by treatment at the terminal side with a cleavage reaction.
[12" claim-type="Currently amended] The production method according to claim 10 or 11, wherein the GPR8 ligand is a polypeptide containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 44.
[13" claim-type="Currently amended] The method of claim 10 or 11, wherein the GPR8 ligand is represented by SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, or SEQ ID NO: 50. A method of making a polypeptide having an amino acid sequence.
[14" claim-type="Currently amended] The production method according to claim 10 or 11, wherein the GPR8 ligand is a polypeptide having an amino acid sequence represented by SEQ ID NO: 44.
[15" claim-type="Currently amended] A methionine residue or protease cleavage sequence at the N terminus of the target peptide, a cysteine residue or a cysteinyl peptide at the C terminus, provided that the peptide portion of the cysteinyl peptide differs from the target peptide and a methionine residue at the N terminus. The peptide moiety does not have a methionine residue when added).
[16" claim-type="Currently amended] A recombinant vector containing the DNA of claim 15.
[17" claim-type="Currently amended] The recombinant vector according to claim 16, wherein the transformant represented by FERM BP-8023 is retained in Escherichia coli MM294 (DE3) / pTCGPR3.
[18" claim-type="Currently amended] A transformant transformed with the recombinant vector of claim 16.
[19" claim-type="Currently amended] The transformant of claim 18, wherein the transformant is represented by FERM BP-8023. Escherichia coli MM294 (DE3) / pTCGPR3.
[20" claim-type="Currently amended] A methionine residue or protease cleavage sequence at the N terminus of the target peptide, a cysteine residue or a cysteinyl peptide at the C terminus, provided that the peptide portion of the cysteinyl peptide differs from the target peptide and a methionine residue at the N terminus. The peptide moiety does not have a methionine residue when added).
[21" claim-type="Currently amended] The method of claim 4, wherein the precursor protein is a recombinant precursor protein prepared by culturing the transformant of claim 18.

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同族专利:

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公开号 | 公开日

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US20040185525A1|2004-09-23|

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EP1391518A4|2006-06-28|

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JP2003070488A|2003-03-11|

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EP1391518A1|2004-02-25|

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WO2002092829A1|2002-11-21|

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KR100599419B1|2006-07-10|

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CN1509336A|2004-06-30|

引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
2001-05-17|Priority to JP2001147341

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2001-05-17|Priority to JPJP-P-2001-00147341

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2002-05-16|Application filed by 가부시키가이샤 시마즈세이사쿠쇼

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2002-05-16|Priority to PCT/JP2002/004735

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2004-03-31|Publication of KR20040026654A

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2006-07-10|Application granted

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2006-07-10|Publication of KR100599419B1

优先权:

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申请号 | 申请日 | 专利标题

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JP2001147341|2001-05-17|

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JPJP-P-2001-00147341|2001-05-17|

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PCT/JP2002/004735|WO2002092829A1|2001-05-17|2002-05-16|Process for producing peptide|