WO2001027310A1 - Procede de production d'un precurseur d'adrenomeduline - Google Patents

Procede de production d'un precurseur d'adrenomeduline Download PDF

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WO2001027310A1
WO2001027310A1 PCT/JP2000/007023 JP0007023W WO0127310A1 WO 2001027310 A1 WO2001027310 A1 WO 2001027310A1 JP 0007023 W JP0007023 W JP 0007023W WO 0127310 A1 WO0127310 A1 WO 0127310A1
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seq
precursor
fusion protein
adrenomedullin
gly
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PCT/JP2000/007023
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English (en)
Japanese (ja)
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Akio Takimoto
Yuuichi Mitsuda
Toshimasa Nakayama
Kenji Mitsushima
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Shionogi & Co., Ltd.
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Priority to JP2001530513A priority Critical patent/JP4332599B2/ja
Priority to AU75595/00A priority patent/AU7559500A/en
Publication of WO2001027310A1 publication Critical patent/WO2001027310A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to a method for producing an adrenomedullin precursor via a fusion protein.
  • Adrenomedullin is a peptide isolated from human pheochromocytoma tissue and is a 52-residue amino acid in which the C-terminal amino acid has been amidated (SEQ ID NO: 2). (Kitamura et al., Biochem. Biophys. Res. Commun., 192, 553, (1993)). This peptide has a vasodilator effect and is expected to be useful as a drug for treating hypertension, asthma, and infertility or as a diagnostic tool for the circulatory system. At present, a method of purifying from human tissue or producing with a peptide synthesizer is used, but industrial mass production of AM cannot be expected by a method of purifying from tissue or using a synthesizer.
  • AM is a peptide having a relatively small molecular weight
  • a secretory system such as yeast, insect cells, Bacillus subtilis, or a non-secretory system such as Escherichia coli can be considered. If the latter is used, it is susceptible to degradation in microorganisms. Therefore, it is difficult to produce only AM by direct genetic recombination technology.
  • a method using a fusion protein comprising a protective protein or an additional protein and a target protein to obtain the target protein is known.
  • the target protein can be obtained by subjecting the fusion protein to limited degradation using protease. In this case, chromatography has been used to purify the target protein (Japanese Patent Application Laid-Open No. 62-259595). Example 6 and Japanese Patent Application Laid-Open No. 1-10999 Example 7).
  • the target protein -type human atrial sodium diuretic peptide (ahANP) is obtained by the following steps.
  • the target protein is purified by chromatography at the stage of the limited digestion, the chromatographic mixture is still mixed with contaminants other than the target protein (especially a large number of unspecified peptide fragments derived from the limited digestion). Purification by chromatography has limitations in terms of throughput and recovery, and efficient recovery of the target protein could not be expected. Therefore, the conventional technology has difficulty in recovering the target protein on an industrial scale, and is not suitable for producing the target protein. Disclosure of the invention
  • the present inventors have conducted research for the purpose of efficient mass production of AM, and as a result, have found the present invention and completed it. That is, the present invention
  • a method for producing adrenomedullin which comprises producing a fusion protein containing an adrenomedullin precursor using a recombinant.
  • (2) a method for producing adrenomedullin comprising the following steps: (A) a step of producing a fusion protein containing an additional protein and an adrenomedullin precursor using a recombinant, and (B) a step of subjecting the fusion protein to limited digestion with protease to obtain a precipitate containing the adrenomedullin precursor;
  • a method for producing adrenomedullin which comprises the following steps: (A) a step of producing a fusion protein containing an additional protein and an adrenomedullin precursor using a recombinant,
  • step (C) a step of dissolving the precipitate obtained in the step (B) in a dissolving solution to extract an adrenomedullin precursor without containing contaminants;
  • step (A) comprises an additional protein, a linker peptide and an adrenomedullin precursor;
  • step (B) is a protease that specifically cleaves an amino acid or an amino acid sequence having no cleavage point within the adrenomedullin precursor.
  • step (B) The production method according to any one of (2) to (5), wherein the protease in step (B) is a glutamic acid residue-specific protease.
  • step (9) The production method as described in (8) above, wherein the solution in step (C) does not contain a denaturant;
  • step (C) is a sodium acetate solution
  • the additional protein in the step (A) is thioredoxin, a partial sequence of cephalosporin-C deacetylase, a partial sequence of methionine aliase, a maltose binding protein, glutathione-S transferase, (1)
  • a method for producing adrenomedullin comprising the following steps:
  • step (b) disrupting the host cultured in step (a), and obtaining an insoluble fraction containing the fusion protein by centrifugation;
  • step (c) extracting the fusion protein from the insoluble fraction obtained in step (b) with a solution containing a solubilizing agent
  • step (d) limitedly decomposing the fusion protein with BLase to obtain a precipitate containing an adrenomedullin precursor; and (e) The precipitate obtained in step (d) is dissolved in a dissolving solution containing no denaturant and having a pH of 3.5 to 4.5 by heating at 60 ° (: to 90 ° C) to remove contaminants. Extracting adrenomedullin precursor without containing it;
  • a fusion protein comprising a linker peptide and an adrenomedullin precursor having the amino acid sequence of SEQ ID NO: 8, and
  • a gene encoding a fusion protein comprising a nucleic acid sequence encoding the linker peptide and adrenomedullin precursor according to (20).
  • adrenomedullin means adrenomedullin derived from human. Adrenomedullin consists of 52 amino acid residues (SEQ ID NO: 2), the C-terminal tyrosine residue of which is an amidated peptide.
  • adrenomedullin precursor a peptide (AM-gly) consisting of a 53 amino acid residue (SEQ ID NO: 4), which is obtained by adding a glycine residue to the C-terminal tyrosine residue of adrenomedullin, and Is exemplified.
  • a peptide having one amino acid residue added to the C-terminal of Gly at position 51 from Tyr at position 1 of the amino acid sequence described in SEQ ID NO: 2 (AM (1-51) -AA: AA Is an arbitrary amino acid residue) is also included in the “adreno-medulin precursor” of the present invention.
  • a physiologically active AM can be obtained by treating it with an amidating enzyme.
  • a bioactive AM can be obtained by substituting AA with an amidated tyrosine residue.
  • fusion protein means a polypeptide containing an additional protein and an AM precursor.
  • additional protein, linker peptide and AM A polypeptide consisting of a precursor is meant.
  • the additional protein is located N-terminal to the fusion protein.
  • the AM precursor is located C-terminal to the fusion protein.
  • the linker peptide is located between the additional protein and the AM precursor.
  • Additional proteins are used to protect relatively low molecular weight AM from degradation. Therefore, any peptide can be used. However, from the viewpoint of productivity, peptides derived from Escherichia coli and peptides having 50 or more amino acids and 200 or less residues from the N-terminus are preferable. Additional proteins include thioredoxin (Bio / Technology, 11, 187-193 (1993)) and cephalosporin-C deacetylase (Appl.Environ.Microbiol., 2224-2229 (1995)). Sequence, partial sequence of methionine lyase (Anal. Biochem.
  • Partial sequence of cephalosporin-C deacetylase, partial sequence of methionine enzyme means a peptide consisting of the N-terminal partial sequence of each molecule.
  • the “linker peptide” is a peptide having an amino acid or an amino acid sequence recognized by a protease to be used at the C-terminus. Preferably, it means a peptide having a glutamate residue at the C-terminal.
  • As the linker peptide a peptide having 4 to 20 amino acid residues is preferable. Examples are the peptides consisting of the amino acid sequence described in SEQ ID NOs: 40 to 46.
  • the amino acid sequence described in SEQ ID NO: 44 is exemplified.
  • the “linker peptide and adrenomedullin precursor” preferably consists of the amino acid sequence of SEQ ID NO: 8.
  • the amino acid sequence described in SEQ ID NO: 8 consists of a sequence obtained by adding the AM precursor described in SEQ ID NO: 4 or SEQ ID NO: 6 to the C-terminal of the linker peptide described in SEQ ID NO: 44.
  • the “gene encoding a linker peptide and an adrenomedullin precursor” preferably comprises the nucleic acid sequence of SEQ ID NO: 7 encoding the amino acid sequence of SEQ ID NO: 8.
  • Examples of the fusion protein include thioredoxin as an additional protein and a peptide consisting of the amino acid sequence of SEQ ID NO: 44 and an AM precursor as a linker peptide.
  • the preferred amino acid sequence of the fusion protein is shown in SEQ ID NO: 10.
  • the amino acid sequence described in SEQ ID NO: 10 consists of the amino acid sequence described in SEQ ID NO: 8 with thioredoxin added to the N-terminus.
  • the nucleic acid sequence encoding the amino acid sequence described in SEQ ID NO: 10 is shown in SEQ ID NO: 9.
  • examples of the fusion protein include UspA as an additional protein, and a peptide consisting of the amino acid sequence of SEQ ID NO: 44 and an AM precursor as a linker peptide.
  • Preferred amino acid sequences of the fusion protein are shown in SEQ ID NOs: 72, 74, and 76.
  • the amino acid sequence described in SEQ ID NO: 72, 74, 76 comprises a sequence obtained by adding a part or all of UspA to the N-terminal of the amino acid sequence described in SEQ ID NO: 8.
  • SEQ ID NO: ⁇ The nucleic acid sequence encoding the amino acid sequence described in 2 is shown in SEQ ID NO: 71, and the nucleic acid sequence encoding the amino acid sequence described in SEQ ID NO: 74 is shown in SEQ ID NO: 73, SEQ ID NO: 73.
  • the nucleic acid sequence encoding the amino acid sequence of 76 is shown in SEQ ID NO: 75.
  • the term "protease that restricts the fusion protein to a limited extent” means a protease capable of releasing an AM precursor by enzymatic cleavage of the fusion protein.
  • a "protease that specifically cleaves an amino acid sequence having no cleavage point inside the adrenomedullin precursor” is exemplified, and more preferably, "a glutamate residue-specific protease” is exemplified. You.
  • Glutamate-specific protease is a protease that specifically cleaves the C-terminus of glutamate residues.
  • proteases include BLase (Eur. J. Biochem., 204, 165-171 (1992)), V8 protease (J. Biol. Chem., 247, 6720-6726 (1972)), a protease derived from Si society ⁇ ios ⁇ ces fWseus (J. Biochem. , 104, 451-456 (1988)).
  • Examples of the “denaturing agent” include urea, guanidine hydrochloride, SDS, acetic acid and the like.
  • a sodium acetate solution can be used, but other solutions such as glycine monohydrochloride, sodium hydrogen phosphate monocitrate, sodium citrate monosodium citrate, etc. Can be exemplified.
  • the “solubilizing agent” examples include reagents such as SDS and guanidine hydrochloride in addition to the urea solution.
  • FIG. 1 shows the structure of PRS5402.
  • FIG. 2 shows the structure of pAME1102.
  • FIG. 3 shows the structure of pAME3201.
  • FIG. 4 shows a reverse phase HPLC elution pattern of AM-gly prepared in Example 5.
  • FIG. 5 shows the structure of pAME3202.
  • FIG. 6 shows the structure of pAMEllll.
  • FIG. 7 shows the structure of pAME3105.
  • FIG. 8 shows the structure of pAME1201.
  • FIG. 9 shows the change in the recovery rate due to insolubilization of AM-gly under various pH conditions.
  • FIG. 10 shows the extraction efficiency of AM-gly from the AM-gly precipitate under various pH conditions.
  • FIG. 11 shows a reversed-phase HPLC elution pattern of the AM_gly extract.
  • AM can be manufactured by the following steps. 1) Culture and disruption of transformants
  • An expression vector for expressing the fusion protein is prepared by incorporating the gene encoding the fusion protein containing the AM precursor, which is the target protein of the present invention, into an appropriate vector.
  • the gene encoding the fusion protein is composed of a gene encoding an additional protein, a gene encoding a linker peptide, and a gene encoding an AM precursor.
  • a source of the gene encoding the AM precursor for example, pHAM-3 (Biochem. Biophys. Res. Commun., 194, 720-725, (1993)
  • human cMA can be used as a source of the gene encoding the AM precursor.
  • an AM precursor synthesis gene that does not change the amino acid sequence of the AM precursor can be used.
  • the nucleic acid sequence described in SEQ ID NO: 5 encoding AM-gly can also be used.
  • the added protein is cephalosporin-C deacetylase
  • PCAH431 or Bacillus subtilis SHS 0133 strain chromosome MA as the source of the gene encoding cephalosporin-C deacetylase.
  • the additional protein is thioredoxin
  • a source of a gene encoding thioredoxin for example, pTrxFus or pThioHis (both manufactured by Invitrogen) or chromosomal DNA of E.co JM109 strain is used. can do.
  • chromosomal DNA of E.co JM109 strain can be used.
  • Synthetic DNA can be used as the gene encoding the linker peptide.
  • a phage or a plasmid is used as the vector.
  • a transformant is prepared by introducing this expression vector into a host.
  • Bacteria, yeast, insect cells and animal cells are used as hosts.
  • the host is preferably a bacterium, and more preferably Escherichia coli.
  • a fusion protein of interest When the host is Escherichia coli, in order to efficiently express a fusion protein of interest has a suitable promoter lac that functions in the host, tac, trc, trp, the P L etc.) and Shine- Dalgarno (SD) sequence Insert the gene fragment containing the fusion protein of interest into an expression vector (such as pKK223-3, pBS, pDR540, or pP lambda) or an ATG vector (such as pTrc99A) that has a translation initiation codon ATG or a regulatory sequence. Good.
  • an expression vector such as pKK223-3, pBS, pDR540, or pP lambda
  • ATG vector such as pTrc99A
  • a transformant can be obtained by introducing the expression vector into an appropriate host cell (for example, E. coli iJM103, JM109, JM110, HB101, C600, BL21 strain, etc.).
  • the desired transformant can be obtained by culturing the transformant under appropriate conditions and disrupting the transformant by a conventional method.
  • the fraction containing the fusion protein having an inclusion body formed in the cells can be collected by centrifugation. Centrifugation can be performed according to a conventional method. Alternatively, it is also possible to recover using a membrane. When an inclusion body is not formed, it can be purified by isoelectric precipitation or a method using an ion exchange resin. In this case, operation 3) below is not required.
  • the fusion protein in the insoluble fraction can be extracted with a solubilizing agent.
  • solubilizers include urea solutions of 4 to 10 mol / l, and reagents such as SDS and guanidine hydrochloride. You. However, in order to perform limited decomposition in the extract, it is preferable to extract with a urea solution that does not lose the activity of protease.
  • the fusion protein is cleaved with a glutamate residue-specific protease according to a conventional method to obtain an AM precursor as a target protein.
  • glutamate residue-specific protease for performing the limited degradation include Bacillus licheniformis Glu-specific endopeptidase (BLase) and V8 protease.
  • the limited decomposition is preferably performed after adjusting the urea concentration of the above-mentioned extract by dialysis or dilution. A preferred urea concentration is 0 mol / L to 3 mol / L.
  • the solution treated with protease contains not only AM precursors but also an unspecified number of peptide fragments and various other contaminants caused by limited degradation. Therefore, removal of contaminants is indispensable for efficient production of AM.
  • purification by chromatography was performed with insufficient removal of contaminants, so the burden on chromatography was high, and it was difficult to efficiently obtain large amounts of the target protein with high purity. .
  • the contaminant was successfully removed by performing the limited decomposition in a pH-adjusted solution to precipitate the AM precursor while the contaminant was dissolved in the supernatant.
  • the isoelectric point of AM-gly which is an AM precursor, is 10.0 as calculated by computer.
  • AM-gly was able to precipitate efficiently at around pH 8.0.
  • no additional protein was precipitated under these conditions.
  • disconnection occurs inside AM_gly.
  • Below pH 7.0 the yield of AM-gly decreases. Therefore, in order to separate AM-gly from contaminants by precipitation, ⁇ 7.0 AM-gly can be separated efficiently by adjusting the pH to a range of, preferably, PH8.0.
  • AM (1-5U-AA) which is an AM precursor, can be similarly separated by ⁇ adjustment.
  • precipitation can be performed by ammonium sulfate precipitation, ethanol precipitation, or precipitation using an organic polymer.
  • the precipitation of AM precursor can be assisted by adding 20-30% saturated ammonium sulfate. 6) Extraction of AM precursor with solution and removal of contaminants
  • the above-mentioned precipitation of AM precursor may not completely remove contaminants in some cases.
  • contaminants can be removed as a precipitate by specifically dissolving the AM precursor in a dissolution solution adjusted to about pH 4.0.
  • the removal of the contaminants can be performed efficiently even in the absence of a denaturing agent.
  • a sodium acetate solution can be used, and other examples of the dissolving solution include a solution of glycine monohydrochloride, sodium hydrogen phosphate, sodium citrate and the like.
  • the extraction is preferably performed by heat treatment.
  • the temperature at this time is preferably from 60 ° C to 90 ° C. 7) C-terminal amidation of AM precursor
  • the C-terminus of AM-gly which is an AM precursor, is substituted with peptidylglycine alpha-amidase (Eur. J. Biochem., 201., 551-559 (1991)).
  • carboxypeptidase to replace the C-terminal amino acid residue of ⁇ (1-51) - ⁇ ( ⁇ is a suitable amino acid residue) with Tyr-NH ⁇ I can do it.
  • the amidation reaction can be performed using the AM-gly solution obtained by the above-mentioned extraction step.
  • the AM-gly amidation reaction requires the use of a commercially available amidating enzyme. Can be.
  • amidating enzymes include peptidylglycine alpha-amidating enzyme (Wako Pure Chemical Industries). Even if the amount of enzyme required for the amidation reaction is 1/1200 or more (W / W) with respect to the AM precursor, the reaction can be performed without generating reaction by-products.
  • the substitution reaction of AM (1-51) -AA was carried out by using carboxypeptidase-Y (Carlsberg Res.Commun., 46, 121-128 (1981)) and carboxypeptidase-WI I (Carlsberg Res. Commun., 50, 309-323 (1985)), Carboxypeptidase-MI (Carlsberg Res. Commun., 48, 217-230 (1983)), Carboxypeptidase-SI (Carlsberg Res. Commun) , 53, 309-320 (1988)). 8) AM purification
  • the amidated AM can be purified by various types of chromatography. Purified AM can be stored for long periods of time by freeze-drying.
  • AM-gly Glycine extended human adrenomedul 1 in (hereinafter abbreviated as AM-gly (SEQ ID NO: 4)) into the medium.
  • the AM-gly structural gene portion (SEQ ID NO: 3) was prepared as follows. First, pHAM-3 [Biochem. Biophys. Res. Commun., 194. 720-725 (1993)] was synthesized by polymerase-chain-reaction (PCR) using TaKaRa EX Taq (Takara Shuzo). A Snal-Sall fragment containing the AM-gly gene as type ⁇ ⁇ was amplified (using AMPCR2 and AMSalR as primers).
  • This DNA fragment was cloned into the Asal site of pUC18 to obtain pUC18 / AM-gly.
  • the synthesized DNA primer was synthesized using a DNA synthesizer (Gene assembler Plus) manufactured by Amersham-Pharmacia.
  • AMPCR2 5'-GGGGTATACCGCCAGAGCATGAAC-3 '(SEQ ID NO: 11)
  • pUC18 / AM-gly was digested to obtain an AM-gly fragment, which was inserted and fused downstream of the mating factor a 1 (hereinafter abbreviated as MFa1) signal sequence cloned on pUC18. .
  • MFa1 mating factor a 1
  • S. cerevisiae BJ1991, SH2676, SH2779 strains were transformed with pAMG2A01 by the lithium acetate method, and YNB (-Leu) plate medium (yeast nitrogen base 0.673 ⁇ 4, glucose 2%, glucose 2%, amino acids 0.0053 ⁇ 4 each (without leucine), adenine 0.0025 I, uracil 0.0025%, pH 6.0, 2% agar) to give a transformant.
  • SH2676 and SH2779 shares were transferred from Prof. Shun Harashima of Osaka University. These strains were cultured in flasks and examined for productivity.
  • the Pichia pasio / sKMYl strain (manufactured by invitrogen) was transformed with pAMGPiOl cut with Stul or with Electroporion, and the resulting cells were plated on an MD plate medium (1.34% yeast nitrogen base, 4x10 " s % biotin, 1% dextrose, 2% agar)
  • the resulting transformant was cultured using a 24-well plate, and AM-gly in the culture supernatant was purified using egret anti-AM antiserum (Peptide Laboratories).
  • a plasmid was constructed that fused and expressed AM-gly with the whole or partial sequence of the heterologous protein.
  • a method for limited degradation of the fusion protein a method using glutamate residue-specific proteases such as V8 protease and BLase was assumed and linked via Glu.
  • the following oligonucleotides for PCR used in the construction were synthesized.
  • PCRTRXA1 5'-GCCGGCCAGGTTAGCGTCGAGGAACTC-3 '(SEQ ID NO: 21)
  • the Hpa ⁇ _Nsp fragment containing the gene encoding the partial sequence from the N-terminus of the CAH of four different lengths was amplified by PCR using TaKaRa EX Taq (manufactured by Takara Shuzo) using pCAH4312 as type II. (Use HPAI-CAH and any of E41REV, E98REV, E162REV, E307REV as primers).
  • the Nsp-Xba ⁇ fragment containing the AM-gly gene was similarly amplified by PCR using pHAM-3 as type III (NSPV-AM and AM-XBAIR were used as primers).
  • chromosomal DNA is extracted and purified from E.coi JM109 strain (Takara Shuzo), and purified by PCR using TaKaRa EX Taq (Takara Shuzo) to obtain the thioredoxin gene.
  • the ael fragment containing (r) was amplified (PCRTRXS1 and PCRTRXA1 were used as primers). Cloned on pMOS i / eT-vector.
  • a synthetic DNA linker (5'-CTAGAGGATCCGCCGGCTCTGGTTCTGGTGAA-3 ') (SEQ ID NO: 24)
  • trxA was included between al- ⁇ el.-The ael fragment was subcloned to thioredoxin- [GSGSGE] -AM-gly-. include).
  • a multicloning site (X-Mlul-Stul-Notl-Xhol) (5'-AATTCGGTACCACGCGTAGGCCTGCGGCCGCCTCGAGTGATC-3 ') (SEQ ID NO: 25) was inserted between fcoRI-Jal of PATG2131 to give PATG2131M.
  • AI-thioredoxin- [GSGSGE] -AM-gly-5aiI was inserted between fcoRI and XAol of this plasmid to obtain pAME3201 (Fig. 3).
  • the coRI and Jal sites were cleaved, smoothed with nmng bean nuclease (Takara Shuzo), and ligated.
  • the terminal sites of Sa7I and AoI sites are the same, so ligation is possible.
  • E. coli JM109 strains into which plasmids pAMEllOl, pAME1102, ⁇ 1103, ⁇ or ⁇ 3201 were introduced were cultured in test tubes using modified TB or CAH-A medium containing 10 ng / mL tetracitalin.
  • E. coli JM109 / PAME1102 was found to produce at least two analogous proteins on the high molecular weight side in addition to CAH-96]-[FE] -AM-gly.
  • E.co7i JM103, JM110, DH5, DH5, DH5a F'lQ, BL21 were used to solve the problem by changing the host Escherichia coli to produce analogs using the E.co'BL21 strain. The amount dropped.
  • the prediction of the secondary structure of mRNA was performed throughout the computer.As a result, it was revealed that there was an inverted repeat consisting of 9 bases centered on the stop codon, and the possibility of secondary structure formation was strong. .
  • the stop codon (TGA) was replaced with TAA or TAG by site-directed mutagenesis to destroy the secondary structure.
  • Glu (17,26,33,34) present in CAH [1-96] was replaced with Lys or Gin in order to simplify the purification procedure.
  • the mutant plasmid holding strain in which the stop codon was replaced with TAA did not produce the analog protein.
  • linker-peptides were inserted around a peptide having a sequence with different properties connected to the N-terminal of Asp-Ala-Phe-Glu to select a sequence suitable for the reaction.
  • the synthetic DNA linker was inserted into the spV site immediately before the AM-gly gene of pAME1102Q, and seven types of fusion protein production plasmids having different sequences immediately before the AM-gly were constructed (PAME1102Q-11 to 7).
  • the synthetic DNA used for each is as follows.
  • the PAME1102Q-11 ⁇ 7 transfected ⁇ coi strain JM109 was cultured in a test tube (37 ° C) in 10 mL of CAH-A medium (10 JUL g / mL tetracycline), and the collected bacterial cells were super- After sonication, the insoluble fraction was collected by centrifugation. Dissolve 8 mol / L urea, 2 mmol / L CaCl 2 , 50 mmol / L Tris-HCl, ⁇ 80 so that the protein concentration becomes 1 mg / mL, and add 2 ⁇ g / mL BLase. The mixture was added and reacted at 37 ° C for 1 hour in a test tube.
  • the human AM-gly gene has some rare codons in Escherichia coli and may have reduced translation efficiency [Current Opinion in Biotechnology, 6, 494 (1995)]. For this reason, plasmids were constructed and expressed using an AM-gly synthetic gene (SEQ ID NO: 5) in which these amino acid codons were replaced with frequently used codons.
  • the substituted codons are Leu 11 (CTC, 12.13 ⁇ 4 ⁇ CTG, 53.71), Arg 1!
  • CAH [1-96]-[FE] -AM-gly had low solubility and required the addition of a denaturant such as urea in the purification operation. For this reason, the added protein was CAH [1-96] (E17,26,33,34Q)-[GSGSGDAFE], and [GSGSGDAFE] was added to highly soluble thioredoxin.
  • an ad-gene containing the [GSGSGDAFE] -AM-gly synthetic gene (SEQ ID NO: 7) was prepared by PCR using the following four synthetic DNAs and cloned into PUC18 ([GSGSGDAFE] -AM -gly / pUC18).
  • the Cal site in the thioredoxin gene was deleted by introducing a point mutation without amino acid substitution.
  • the fragment was inserted between EcolV-Nael of ([GSGSGDAFE] -AM-gly / pUC18) to give thioredoxin- [GSGSGDAFE] -AM-gly / pUC18.
  • the Cpol-Xbal fragment of pAME3201 was replaced with the Cpol-Xbal fragment of thioredoxin_ [GSGSGDAFE] -AM-gly / pUC18 to obtain PAME3202 (Fig. 5).
  • AI fragment of pAMEl 102Q-15 was replaced with the A3 ⁇ 4pV-AM-gly- ⁇ 3 ⁇ 4al fragment of thioredoxin- [GSGSGDAFE] -AM-gly / pUC18 to obtain pAMEllll (Fig. 6). .
  • the C fragment containing the entire fusion protein gene of pAMEllll was exchanged for PAME3202 to obtain PAME3105 (FIG. 7) and pAME1201 (FIG. 8).
  • the sequence of the synthetic DNA used in the construction of the plasmid is as follows.
  • the underlined portions are sequence portions complementary to each other. 5'- AC
  • IPTG isopropyl-?-D-thiogalactopyranoside
  • E. coli BL21 / pAME3105 was cultured, collected, disrupted, and centrifuged to collect fractions containing inclusion bodies. Then after washing with buffer at neutral pH range, 6 mo 1 / L urea, 2 dishes 01 / L CaC 1 2, 50 mmol / L Tris-HCl, it was dissolved in pH 8.0. After the urea concentration was adjusted to 0.5 mol / L or 1 mol / L by dialysis, the protein concentration was adjusted to 1 mg / mL, and a limited degradation reaction with BLase was performed. As a result, a phenomenon in which the solution became cloudy with the progress of the reaction was observed. HPLC analysis revealed that AM-gly was specifically insolubilized and precipitated.
  • the protein concentration during the BLase reaction was increased to 10 mg / mL in the presence of 1 mol / L urea, and the reaction was allowed to stand at 5 ° C for 16 h to increase the yield to 87% (Table 4).
  • the urea concentration was adjusted to 1 mol / L by diluting with 2 mmol / L CaCl 50 mmol / L Tris-HC1, pH 8.0, and the protein was treated with a final protein concentration of 2 mg / mL. Even in this case, AM-gly could be recovered with the same efficiency.
  • the fusion partner protein, thioredoxin also has five glutamate residues in the molecule, but the cleavage rate at these sites is extremely slow under urea concentrations of less than mol / L. Addition of ammonium sulfate could help AM-gly precipitate formation. By adding 20-30% ammonium sulfate, more than 95% of AM-gly could be recovered (Table 4). The released thioredoxin can maintain solubility under these conditions. Wear.
  • the dissolution conditions of the precipitated AM-gly precipitate were examined. It is possible to dissolve efficiently at pH 2.5 or pH 4.0 even in the absence of a denaturant (Fig. 10). When dissolved at pH 4.0, a large amount of contaminants can be removed. did it. In addition, heat extraction (80 ° C, 30 min) increased the dissolution efficiency, which was effective in inactivating BLase and removing other contaminating proteins. Compared with the absorbance of lysates was dissolved in pH 2.
  • the relevant gene portion was amplified by PCR using the chromosomal DNA of E.co7i JM109 strain as type III, and the obtained fragment was placed on the PUC18 at the 5 'end of the [GSGSGDAFE] -AM-gly gene (Example 8).
  • cloned between the i "coRI- Was. ⁇ coRI-cut ends are blunt-ended with Mung Bean Nuclease, and those that require the 5 'side of the gene of each protein to be ligated are also blunt-treated with MungBean Nuclease or T4 DNA polymerase to obtain the wild-type 5' sequence.
  • An Ala residue was added to the 3'-side ligation of the additional protein gene for convenience using an ael cleavage sequence.
  • the sequence of the DNA used for PCR is shown below. Primer on sense side
  • HdeB Pstl 5'-CTGCAGGTTACAAAATGAATATTTCATCTCTCC-3 '(SEQ ID NO: 53) HdeB (-signal) Ehel 5'-GGCGCCAATGAATCCGCTAAAGATATGACCTGC ⁇ 3' (SEQ ID NO: 54) YfiD Sspl 5'-AATATTACAGGTATCCAGATTACTAAAGCTCTAGAGCTCTCT
  • E. coli BL21 strain carrying the constructed expression plasmid was cultured in a test tube (10 mL CAH-A medium (5 g / ml tetracycline), 37). SDS_PAGE and CBB staining of the total protein of the cultured cells were fused with [GSGSGDAFE] -AM-gly except when HdeB, which had a residual signal, was used. Evening protein production It could be confirmed. Above all, the expression level was high when UspA was used, and this plasmid was named PAME9101.
  • Example 12 AM-gly fusion expression using low molecular weight UspA
  • UspA- [A]-[GSGSGDAFE] -AM-gly expression plasmid A plasmid using the N-terminal partial sequence of UspA was prepared in the same manner as in pAME9101. If the molecular weight of the additional protein is reduced within a range where the expression level does not significantly decrease, the production efficiency can be increased.
  • UspA [l-84], UspA [l65], UspA [l-59], UspA [1-57] were used as partial sequences, and the sequences of the primers used for PCR in these regions were as follows. Shown in Sense primer (common)
  • AM can be efficiently mass-produced by gene recombination technology.

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Abstract

L'invention porte sur un procédé permettant de produire efficacement et en grande quantité de l'adrénoméduline. Ce procédé est caractérisé par plusieurs étapes consistant à: (A) produire une protéine de fusion composée d'une protéine d'addition, d'un peptide de lieur et d'un précurseur d'adrénoméduline au moyen d'un agent de recombinaison; (B) soumettre la protéine de fusion à une digestion restreinte avec une protéase afin d'obtenir un précipité contenant le précurseur d'adrénoméduline; et (C) dissoudre dans un solvant le précipité obtenu à l'étape (B) afin d'en extraire le précurseur d'adrénoméduline exempt de contaminants.
PCT/JP2000/007023 1999-10-15 2000-10-10 Procede de production d'un precurseur d'adrenomeduline WO2001027310A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
JP2001530513A JP4332599B2 (ja) 1999-10-15 2000-10-10 アドレノメデュリン前駆体の製造方法
AU75595/00A AU7559500A (en) 1999-10-15 2000-10-10 Process for producing adrenomedulin precursor

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP11/294147 1999-10-15
JP29414799 1999-10-15

Publications (1)

Publication Number Publication Date
WO2001027310A1 true WO2001027310A1 (fr) 2001-04-19

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PCT/JP2000/007023 WO2001027310A1 (fr) 1999-10-15 2000-10-10 Procede de production d'un precurseur d'adrenomeduline

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JP (1) JP4332599B2 (fr)
AU (1) AU7559500A (fr)
WO (1) WO2001027310A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005044846A1 (fr) * 2003-10-24 2005-05-19 Mochida Pharmaceutical Co., Ltd. Adrénomédulline 2 et utilisation

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS62259595A (ja) * 1986-05-02 1987-11-11 Suntory Ltd 生理活性ペプチドの製造方法
WO1999038984A1 (fr) * 1998-01-30 1999-08-05 Suntory Limited Procede de production de peptide au moyen d'un peptide accessoire

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS62259595A (ja) * 1986-05-02 1987-11-11 Suntory Ltd 生理活性ペプチドの製造方法
WO1999038984A1 (fr) * 1998-01-30 1999-08-05 Suntory Limited Procede de production de peptide au moyen d'un peptide accessoire

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
KAZUO KITAMURA ET AL.: "Adrenomedulin no kiso to sono Rinsho Ouyou Adrenomedulin oyobi Kanren Peptide no aratana Bunshigata to Sokuteijo no Mondaiten", MOLECULAR MEDICINE, vol. 35, no. 8, 1998, pages 952 - 960, XP002935847 *
KAZUO KITAMURA ET AL.: "Cloning and characterization of cDNA encoding a precursor for human adrenomedulin", BIOCHEMICAL AND BIOPHISICAL RESEARCH COMMUNICATIONS, vol. 194, no. 2, 1993, pages 720 - 725, XP002935845 *
KLAUS BREDDAM ET AL.: "Substrate preferences of glutamic-acid-specific endopeptitases assessed by synthetic Peptide Substrates based on intramolecular fluorescence quenching", EUR. J. BIOCHEM., vol. 206, no. 1, 1992, pages 103 - 107, XP002935846 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005044846A1 (fr) * 2003-10-24 2005-05-19 Mochida Pharmaceutical Co., Ltd. Adrénomédulline 2 et utilisation

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JP4332599B2 (ja) 2009-09-16

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