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WO1996002661A1 - Procede permettant de produire une proteine dont le poids moleculaire peut etre reduit par une protease originaire d'une cellule hote - Google Patents

Procede permettant de produire une proteine dont le poids moleculaire peut etre reduit par une protease originaire d'une cellule hote Download PDF

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Publication number
WO1996002661A1
WO1996002661A1 PCT/JP1995/001399 JP9501399W WO9602661A1 WO 1996002661 A1 WO1996002661 A1 WO 1996002661A1 JP 9501399 W JP9501399 W JP 9501399W WO 9602661 A1 WO9602661 A1 WO 9602661A1
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Prior art keywords
protein
medium
host cell
molecular weight
column chromatography
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PCT/JP1995/001399
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English (en)
Japanese (ja)
Inventor
Muneo Tsujikawa
Ken Okabayashi
Masanori Morita
Toshizumi Tanabe
Koichi Yamanouchi
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The Green Cross Corporation
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Publication of WO1996002661A1 publication Critical patent/WO1996002661A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6456—Plasminogen activators
    • C12N9/6462—Plasminogen activators u-Plasminogen activator (3.4.21.73), i.e. urokinase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21073—Serine endopeptidases (3.4.21) u-Plasminogen activator (3.4.21.73), i.e. urokinase

Definitions

  • the present invention relates to a method for stably producing a large amount of an unstable protein which can be reduced in molecular weight by a protease derived from a host cell.
  • host cells that produce and secrete the protein obtained by genetic manipulation are cultured in a medium containing a specific substance to enhance the growth of the host cells and promote the production and secretion of the protein
  • the present invention relates to a method for mass-producing a protein that can be reduced in molecular weight by a host cell-derived lipase by suppressing the reduction in molecular weight.
  • tissue t-PA produced by vascular endothelial cells
  • perokinase UK
  • perokinase when used in large quantities, has the drawback of inducing the degradation and activation of coagulation and fibrinolytic factors and inducing a tendency to bleed.
  • blow mouth kinase an inactive precursor of human ostium kinase produced by human cells [hereinafter referred to as blow mouth kinase.
  • Japanese Unexamined Patent Publication (Kokai) No. 60-62981 (EP-B-1 394 47), J. Biol. Chem .. 260, 12 377 (1985)] is a fiber constituting a thrombus. It has a specific affinity for fibrin, has thrombolytic properties such as the selective degradation of fibrin, and more preferably, the blow mouth kinase, unlike perokinase, causes a bleeding tendency. Cell Struc. Func .. 10, 151 (1985)] found no defect.
  • blow mouth kinase having such excellent properties is expected to be widely used clinically as a fibrinolytic enzyme.
  • Pichia pastoris which has an effect of increasing the secretion amount in the case of human serum albumin or invertase, as a host cell (Bio / Technology, 5, 1305-1308. 1985). Although the oral kinase was examined in the same manner, the secreted amount of the protein into the medium was lower than that of human serum albumin and the like, and considerable brourokinase remained in the cells without being secreted.
  • the present inventors have found that when the Pichia yeast is cultured in a YPM medium, there are many degradants of about 47 kDa and about 30 kDa in addition to those of 5 O kDa which are considered to be intact proteins in the culture. I noticed that This 47 kDa protein was determined to be a deletion of the intact blow-mouth kinase N-terminus from the results of ⁇ -esten blotting with a monoclonal antibody recognizing the EGF domain (Evider-Malgro-factor). However, it was considered that the Blow-mouth kinase was reduced in molecular weight by proteases derived from host cells and the like present in the culture supernatant.
  • the present invention relates to the production of a protein that can be reduced in molecular weight by a protease derived from a host cell by an enzymatic or genetic engineering technique, wherein the protein is reduced in molecular weight. It is an object of the present invention to provide a method for mass production without mass production. Specifically, a host cell-derived protein that can be degraded by a host cell-derived protease is added to the host cell. It is an object of the present invention to provide a method for producing a large S protein by stably producing and secreting a large amount of white matter and suppressing the low molecular weight after secretion.
  • the recombinant host cell is at least one selected from acidic amino acids, basic amino acids, phosphates, ammonium salts and nonionic surfactants.
  • the present invention relates to a host cell that secretes and produces a protein that can be reduced in molecular weight by a protease derived from a host cell, and more particularly, to a method for transforming Pichia yeast into an acidic amino acid, a basic amino acid, a phosphate, an ammonium salt, and a nonionic interface. Culturing in a medium containing at least one selected from the group consisting of activators, and collecting the protein from the resulting culture supernatant. How to make protein
  • the present invention provides a method for collecting a protein which can be degraded by a protease derived from a host cell from a culture supernatant in the production method, the method comprising the steps of: using a buffer solution containing ammonium salt for column chromatography on a culture supernatant; A method for producing a protein that can be reduced in molecular weight with a host cell-derived protease, which comprises treating with a hydrophobic column chromatography and a cation exchange column chromatography, preferably.
  • the present invention provides a transformant (Bikia yeast) that produces and secretes blow mouth kinase.
  • FIG. 1 is a diagram showing a method of constructing the plasmids p K O 110, ⁇ ⁇ 1 113, and ⁇ ⁇ ⁇ 114.
  • FIG. 2 shows a method for constructing plasmids pKO116 and pKO117. 0
  • FIG. 3 is a diagram showing a method for constructing brassmid pKO122 and a blow-mouth kinase secretion expression vector ⁇ 126.
  • FIG. 4 is a diagram showing a restriction enzyme map of the blow mouth kinase secretion expression vector pKO126.
  • FIG. 5 is a graph showing the growth of Pichia yeast SC06 strain cultured in five types of methanol media in Experimental Example 1 over time.
  • ⁇ Medium 1
  • medium Medium 2
  • ⁇ Medium 3
  • X Medium 4
  • ⁇ Medium 5
  • FIG. 6 is a diagram showing the time course of the ⁇ activity in the culture supernatant obtained by culturing the Bikia yeast SC06 strain in five types of methanol media in Experimental Example 1.
  • ⁇ Medium 1
  • Medium 2
  • ⁇ Medium 3
  • X Medium 4
  • ⁇ Medium 5
  • FIG. 7 shows the results of a Western plot (electrophoresis) performed in Experimental Example 4.
  • Lane 1 Using ⁇ medium
  • Lane 2 YP 2 + 23 ⁇ 4NH 4 H 2 P0 4 + 23 ⁇ 4 (NH *) 2 HP0 4
  • Lanes 3 and 4 ⁇ + 0.013 ⁇ 4 Tri-ton X-100 + 0.1M arginine lanes 5 and 6: ⁇ 2 ten 0.013 ⁇ 4 Tri-ton X-100 + 0.1M arginine
  • host cell-derived protease refers to a protease encoded by a chromosome gene of a host cell.
  • proteases are presently found in yeast in about 30 or more species, and are present in vacuoles (yscA, yscB, yscY), mitochondria (yscMpI), periplasm (ysdl) and the like.
  • yscA, yscB, yscY mitochondria
  • mitochondria yscMpI
  • periplasm periplasm
  • the protein targeted by the production method of the present invention is not particularly limited as long as it can be reduced in molecular weight by the action of the above-described host cell-derived protease.
  • plow-mouth kinase human tissue plasminogen activator, blood coagulation factor IX, blood coagulation factor X, protein c, interferon, epidermal grosspha Kuta-1 (hereinafter referred to as EGF) and ⁇ -dolphin are exemplified.
  • EGF epidermal grosspha Kuta-1
  • ⁇ -dolphin is particularly preferred.
  • the protein targeted by the present invention is not limited to a naturally-derived protein, and may be a mutant obtained by genetically mutating a natural protein or a derivative obtained by artificially modifying the natural protein.
  • a mutant obtained by genetically mutating a natural protein or a derivative obtained by artificially modifying the natural protein For example, in the case of blow-mouth kinase, the entire region or part of the evidermal macroglobulin factor domain of the natural blow-mouth kinase is deleted, or the entire region or part thereof is S-replaced with another amino acid residue.
  • blow-mouth kinase derivative in which the asparagine residue at position 302 from the N-terminus of the natural blow-mouth kinase is substituted with a glutamic acid residue, and an annexin having thrombus affinity at the C-terminal side of the natural blow-mouth kinase And a fusion protein to which V protein is bound (PCT International Publication No. WO 92/19279).
  • These derivatives are sugar-free blow-mouth kinases to which no asparagine-linked sugar chain is added, and are expected to be applied as thrombolytic agents due to their high thrombus affinity.
  • the host cell used in the present invention is not particularly limited as long as it can secrete and produce a protein that can be reduced in molecular weight by the above-mentioned protease, and it does not matter whether the host cell is naturally derived or obtained by gene manipulation. Absent. In addition to those already described in publicly known documents, those that will be developed in the future can be used as appropriate. Specifically, a bacterium having a function of expressing a gene encoding a protein that can be degraded by a host cell-derived protease in a cell, producing the protein, and secreting the protein outside the cell. , Escherichia coli, yeast, mold and Bacillus subtilis.
  • Pichia pastoris Piichi na /) a5toris
  • GTS 1 15 his 4
  • Etc. NRRL accession number Y—1 585 1 Etc.
  • a method for preparing a host cell capable of secreting and producing a protein that can be reduced in molecular weight by the above protease by a gene recombination operation can be carried out by employing a known method or a method analogous thereto.
  • a method for preparing a host capable of producing and secreting blow mouth kinase a method described in JP-A-60-180591 (EP-B-154272) (Saccharomyces ⁇ ), a human tissue plasminogen activator
  • JP-A-60-180591 EP-B-154272
  • Sacharomyces ⁇ a human tissue plasminogen activator
  • Examples of the method for producing a host that produces and secrete include the method described in Transfusion Medicine, p303-313. (1986).
  • Pichia yeast When Pichia yeast is used as a host cell, it can be prepared by contacting the S gene of the target protein with a promoter of the AOX gene that is strongly induced by methanol in the medium according to a conventional method.
  • both the autonomous propagation type and the chromosome integration type are used as the expression vector, but the expression vector is preferably the chromosome integration type. More preferably, the expression vector has the 5 'and 3' untranslated regions of the AOX gene at both ends, and is HIS4 which is one of the best candidates in addition to the AOX promoter, foreign gene and AOX terminator. It is a straight-type vector with a distant element.
  • Transformation was performed by the spheroblast method (Cregg, JM et al., Mol. Cell. Biol .. 5.3376-3385, 1983), the alkali cation method (Ito, H .. et al., J. Bacterid .. 153, (1) 163). -168. 1983) can be used.
  • a strain having histidine auxotrophy (his-) is preferably used as a host cell.
  • his- histidine auxotrophy
  • the production method of the present invention relates to a protein that can be reduced in molecular weight by a host cell-derived protease.
  • Host cells capable of secreting and producing white matter are cultured in a medium containing at least one selected from the group consisting of acidic amino acids, basic amino acids, phosphates, ammonium salts, and nonionic surfactants.
  • This makes it possible to obtain a large amount of a protein which can be reduced in molecular weight by a host cell-derived porcine protease from the culture supernatant in an intact state. That is, the culture method is considered to contribute to expression of the target protein in host cells, improvement of production efficiency, promotion of extracellular secretion, or suppression of low molecular weight by protease.
  • Examples of the acidic amino acid used in the present invention include aspartic acid and glutamic acid, and examples of the basic amino acid include arginine and lysine. Preferably, it is arginine or glutamic acid.
  • Acidic amino acids and basic amino acids may be used in the form of a salt. Examples include sodium aspartate, potassium aspartate, sodium glutamate, potassium glutamate, alginine hydrochloride, and lysine hydrochloride.
  • amino acid content fi in the medium is usually about 0.02 to 0.5M, preferably about 0.05 to 0.3M, and more preferably about 0.1M.
  • the phosphate used in the present invention include ammonium phosphate I NHJ HPO * or NH 4 H 2 PO «] and sodium phosphate [Na 2 HPO4 or
  • the content of phosphate in the medium is about 2 to 5 w / v%, preferably about 4 w / v%.
  • Anmoniumu salt used in the present invention phosphoric acid Anmoniu ⁇ [(NH) 2 HPO * or NH «H 2 PO4], Anmoniumu carbonate [(NH 4) 2 C0 3 or NH 4 HCO3], Anmoniumu chloride [ NH 4 Cl] and the like.
  • the content of ammonium salt in the medium is about 2 to 5 wZv%, preferably about 4 wZv%.
  • the phosphate or ammonium salt is preferably in the form of ammonium phosphate, more preferably in the form of a combination of (NH 4 ) 2 HPO * and NH 4 H 2 PO 4 .
  • (NH *) 2 ⁇ 5w / v% is a 2 HPO * and NH 4 H 2 PO * content in total, and preferably about 4wZv 3 ⁇ 4.
  • Li down ⁇ or Anmoniumu salts especially (NH 4) 2 HPO4 and ⁇ * ⁇ 2 ⁇ 0 by ⁇ the host cell of the present invention 4 in a medium containing, Protea is ⁇ secreted from the host cell Ichize
  • nonionic surfactant used in the present invention examples include polyoxyethylene mono ⁇ -tert-butyl tert-butyl ether and polyoxyethylene sorbin fatty acid ester.
  • the content of the nonionic surfactant in the medium is about 0.005 to 0.03 wZv%, preferably 0.01%.
  • the medium used in the present invention may contain at least one selected from the group consisting of the above acidic amino acids, basic amino acids, phosphates, ammonium salts and nonionic surfactants.
  • Arginine in Naka preferably contains Anmoniumu phosphate [(NHJaHPO * and NH4H 2 PO «] and 3-ton X- 1 00.
  • the other components are not particularly limited as long as the medium used in the present invention fulfills the above conditions and can grow and proliferate the host cells.
  • Other combinations Examples of the component include components contained in a known medium commonly used in this field.
  • various sugars (glucose, glycerol, etc.) are generally used as carbon sources, urea and usate are used as nitrogen sources, various vitamins and nucleotides are used as sss nutrients, and inorganic salts such as Mg, Ca. Fe, Na, K , Mn, Co, Cu salts and the like.
  • YNB liquid medium [0.7% amino acid-free yeast nitrogen base (manufactured by Difco), 2% glucose], YPD liquid medium [1 ⁇ yeast extract (manufactured by Diico), 2% bactopeptone (Difco) Co., Ltd., 2% glucose], etc.
  • a methanol-containing medium can be used. Is about 0.01 to 5%, and specifically, a YPM medium [1% yeast extract, 2 pact peptone, 0.01 to 5% methanol] is exemplified.
  • the medium used in the present invention is prepared by adding at least one selected from the above-mentioned acidic amino acids, basic amino acids, phosphates, ammonium salts and nonionic surfactants to a conventionally known medium. Can be easily prepared.
  • the pH of the medium may be neutral, weakly basic or weakly acidic. It is preferably pH 6-8.
  • the cultivation can be carried out by using the above-mentioned medium and appropriately selecting conditions under which the cells produce the maximum amount of the target protein and efficiently secrete it in a conventional manner.
  • the culturing temperature is usually 15 to 43'C, preferably 20 to 3O'C, and the culturing time is usually about 1 to 1,000 hours. If necessary, it can be cultured under aeration.
  • preculture Prior to the main culture, preculture can be performed under the same conditions.
  • the target protein secreted into the culture supernatant can be collected from the culture by known separation and purification means.
  • the culture is subjected to ultrafiltration or centrifugation to obtain a culture supernatant, and the culture supernatant is subjected to salt precipitation such as ammonium sulfate precipitation, gel-based treatment, treatment with an anion exchanger, cation Purification of the target protein by subjecting it to various treatments such as treatment with an exchanger and treatment with hydrophobic chromatography Can be.
  • the target protein of the present invention is easily degraded by a protease derived from a host cell, it is preferable to purify the target protein from the culture by the following method.
  • a culture solution containing ammonium salt is used in the initial stage of purification by a conventional method such as a centrifugal separation method or an ultrafiltration method. This is a method of treating by column chromatography under the condition of low protein S.
  • the buffer used here is not particularly limited as long as it is a buffer containing an ammonium salt.
  • it is a buffer having a buffering capacity in a neutral region (pH 6 to 8, preferably about pH 7), and the concentration of the contained ammonium salt is preferably 0.05 to 5 M.
  • concentration of the contained ammonium salt is preferably 0.05 to 5 M.
  • the present invention can effectively suppress the reduction of the molecular weight of the target protein by a protease, and can purify and obtain an intact protein in a high yield.
  • Column chromatography includes ordinary protein purification such as anion exchange column chromatography, cation exchange column chromatography, gel permeation column chromatography, hydrophobic column chromatography, chelate resin column chromatography, etc.
  • Column chromatography used for purification of proteins produced by gene engineering techniques is exemplified.
  • a preferred embodiment of the present invention includes a combination of hydrophobic force column chromatography and positive ion exchange column chromatography.
  • aqueous column chromatography and cations are preferably carried out using a buffer solution containing ammonium salt, more preferably using an ammonium phosphate buffer solution (pH 6 to 8).
  • a method of performing exchange column chromatography By such a method, the target protein having high specific activity can be isolated at a high yield with a small amount of intact with a small amount of degraded product.
  • the treatment by the hydrophobic chromatography used in the present invention can be performed according to a conventional method.
  • hydrophobic chromatography carrier examples include an insoluble carrier having an alkyl group having 4 to 18 carbon atoms (such as a butyl group, an octyl group, and an octyldecyl group) or a phenyl group.
  • Preferable examples include a fuunil base type, and specific examples include phenyl cellulose (trade name: phenyl cell mouth fine, manufactured by Seikagaku Corporation) and phenyl agarose (trade name: phenyl sepharose, manufactured by Pharmacia). Is exemplified. Adsorption and elution conditions can be appropriately selected according to the target protein to be purified.
  • the contact conditions include pH 6 to about 8, preferably pH 7 and a salt concentration of about 0.5 to 1.5M, preferably about 0.5 to 0.8M.
  • the elution conditions are about pH 6 to 8, preferably about pH 7, a salt concentration of about 0.05 to 0.7 M, preferably about 0.1 to 0.5 M, and more preferably about 0.2 M.
  • the treatment by cation exchange chromatography can also be performed according to a conventional method.
  • any insoluble carrier having an intestinal ion exchange group can be used.
  • 3 ⁇ 4Examples of the ion exchange group include a carboxymethyl (CM) -based type and a sulfopropyl (SP) -based type. It is preferably an SP type, and specific examples include SP-agarose (trade name: S-Sepharose, manufactured by Pharmacia), SP-dextran (trade name: SP-Sephadex, manufactured by Pharmacia) and the like. You. Preferred conditions for adsorption and elution can be appropriately selected according to the target protein to be purified.
  • the contact conditions include pH 6 to about 8, preferably pH 7 and salt odor of about 0.01 to 0.2M.
  • the elution conditions include about pH 6 to 8, preferably about pH 7, and a salt concentration of about 1 to 1M.
  • a protein that can be reduced in molecular weight by a protease produced by a genetic engineering technique or an enzymatic method can be produced in large form in large size in a complete state. That is, by culturing a host cell that secretes and expresses the protein in the medium referred to in the present invention, it enhances the expression of the host cell, improves the production S of the protein, and secretes the protein from the host cell. Is promoted. Further, according to the present invention, it is possible to effectively suppress the low molecular weight of the protein secreted into the culture medium.
  • the nonionic surfactant used in the present invention does not directly contribute to the growth of host cells or the suppression of protein depolymerization, but has an effect of suppressing the disappearance of proteins by adsorption to instruments and the like.
  • Amino acids especially arginine and glutamic acid, have the effect of increasing the growth of host cells and inhibiting the production of low-molecular-weight proteins.
  • ammonium phosphate effectively suppresses the reduction of protein molecular weight.
  • the growth of host cells and the production and secretion of proteins are synergistically improved.
  • the unification method of the present invention it is possible to effectively suppress the decomposition of proteins that are liable to be reduced in molecular weight at the stage of production, and to efficiently purify and intact intact proteins in high yields. Further, the present invention can provide, for the first time, a Pichia yeast (transformant) capable of secretory production of a blow mouth kinase.
  • NH 4 -PO * refers to ammonium phosphate buffer containing (H *) 2 HPC and thigh 4 H 2 PO4, and Na—P0 * refers to Na 2 HPO «and NaH 2 P 0 «. Means the contained sodium phosphate ⁇ city liquid.
  • Plasmid JUKI (Hiramatsu et al., Gene. 99. 235-), an expression vector for Sacch ronyces cerevisiae containing a gene in which a signal sequence derived from the gene and the Blow Mouth Kinase cDNA are connected in frame. 241. 1991, JP-A-3-240493 and JP-A-4-166088) were partially digested with B1II and then digested with PstI to obtain a part of the signal sequence and A 330 bp fragment containing a portion of the oral kinase cDNA was isolated.
  • T4 polynucleotide kinase and ATP are added to 5 jiig linkers a and b (Table 1) purified using an OPC column (Abride 'Biosystems Japan) and incubated at 37'C for 30 minutes. To phosphorylate the 5 'end.
  • Linker a 5 ⁇ -TCGAGATGTTGTTCTCTAA-3 '
  • Linker b 3'-CTACAACAAGAGATTCTAG-5 '
  • linkers a and b were mixed and heated at 70 for 20 minutes, and then allowed to cool to room temperature.
  • the annealed linker and 330 bp PstI-Bg1II fragment prepared from Brasmid JUK1 were mixed and ligated, followed by ethanol precipitation. ⁇ was performed to recover DNA.
  • the precipitate was dissolved in TEClOmM Tris-HC1 (pH8.0), 1 mM EDTA], mixed with a PstI-Bg1II fragment, ligated, and ethanol precipitated S to recover DNA. .
  • the precipitate was dissolved in TE, and digested with PstI-XhoI to isolate a 36-Obp fragment.
  • This fragment was ligated with a 2.7 kb Pstl-XhoI fragment prepared from pKO110 and then introduced into Escherichia coli for transformation.
  • Brasmid was isolated from 39 clones of ampicillinous colonies.
  • Primary screening by restriction enzyme digestion was carried out to select 6 clones of plasmid, and the nucleotide sequence in the vicinity of the introduced linker was examined with a liquid phase sequencer Quensa-1 (Pharmacia). The sequence was as expected (sequence listing, SEQ ID NO: 2).
  • Two clones were selected and named pKO I13 and pKO114 (3.05 kb) (Fig. 1). Mucor.
  • Brasmid N302Q is aspartic acid, the 302th amino acid from the N-terminus of the brass-mouth kinase cDNA of Brasmid JUKI. Codon AAT of glutamine to CAG of glutamine, site-specific mutagenesis method (Molecular Cloning, A Laboratory Manual. Second edition. Sambrook. J. et al., 1989, modified by Cold Spring Harbor Laboratory, New York), in which the asparagine-linked glycosylation site has disappeared.
  • pKO116 was digested with BamHI and ⁇ I to separate a 3.95 kb DNA fragment.
  • p SV—G 1 -UK which is an expression vector for mammalian cells containing blow mouth kinase cDNA (see Japanese Patent Application Laid-Open No. 63-105675 (EP-A-2656784)).
  • a 320 bp DNA fragment was obtained by digestion with BamHI and KpnI, ligated to a PKO116 DNA fragment (3.95 kb), and excised pKO 118 was obtained.
  • ⁇ 118 was digested with ⁇ I, blunt-ended with ⁇ 4DN ⁇ volimerase, and then added with pXhoI linker (Takara Shuzo).
  • the 1.57 kb fragment was separated by XhoI digestion and cloned into the XhoI site of pKO110 to obtain the plasmid pK0121.
  • PK0121 was digested with XhoI to isolate a 1.57 kb DNA fragment, which was cloned into the XhoI site of pA0807NX.
  • PKO126 was isolated with the insert inserted in the forward direction ( Figures 3 and 4).
  • ⁇ pAO807Nx is a cloning site for plasmid pHIL-D2 (Invitrogen) Ec This is a plasmid in which the oR I recognition site has been converted to an Xhol recognition site (Esco R I digestion of the plasmid pH IL-D2, fill-in with Klenow fragment, addition of pXho I linker, and then Xho I digested fragments were ligated and ligation reaction was performed to prepare pAO80707NX).
  • PKO126 (1Oiig) obtained in Example 1 was digested with zymorylacese, and treated with calcium chloride to make it a competent Pichia yeast iPichia pastoris GTS 115: NRRL deposit number Y—15851)
  • HI S + transformant was selected by introducing into suspension S 100 / z1. The transformant was stroked on a plate containing SD CO. 67% non-containing amino acid YNB, 2% dextrose] to starve a single colony. Thirty clones were isolated as transformants into which pKO126 was introduced, and were named SC01 to SC30.
  • SC 02, 06, 08, 1 0, 1 2, 1 3, 1 8, 1 9, 20, 22, 24, 25, 28 and 14 strains of SC 29 are plated on YPD plates and incubated at 30'C for 2 days Cultured. Inoculate a single colony with 10 clones into HI S + selective plate [Noble agar 15g, sorbitol 91g, glucose 10g, YNB (w / o) 3.35g, biotin 0.2fflg / 500ml], and incubate at 30'C for 2 days Cultured. Proliferation of all colonies was observed. It was also thought that HIS 4 gene stably maintained.
  • the cells were cultured in at 30 e C 1 above the 4 strains 3 Om 1 test tube 5 m 1 Y medium (1% methanol-containing YPM medium), 46, 70, 9 of 9 hours after blowing port kinase activity CPA activity (Plasminogen activator activity) as an index], RPHA
  • SCO 2, 06, 13 and SC 28 were cultured for 2 days in YPM, medium (YPM medium containing 1% methanol) and YPN! * Medium (YPM medium containing 4% methanol), respectively.
  • a stamp lot of the culture was performed. Band was observed in YP M 4 medium about 50 kD a in culture cleansed of its bands are bought ⁇ especially in SC O 6.
  • DNA was extracted from SCO 2.06, 10, 12, 19, 22, 29 and Pichia yeast GTS-5, respectively, and the presence of the expression cassette on the chromosome was examined by Southern method.
  • the probe consisted of a 0.9 kb fragment of PHIL-D2 (manufactured by Invitrogen) corresponding to the AOX1 promoter digested with NotI-EcoRI and pMTO15 (corresponding to the structural gene for perkinase).
  • XUCl-digested 1.2 kb fragment of pUC18 into which Blow-mouth kinase cDNA was incorporated) was hybridized with a DNA fragment digested with KpnI, PstI, and NcoI. Isezio Performed.
  • Blow-mouth kinase secretion-expressing strain SC06 obtained in Examples 1-2 was pre-cultured in YPD medium for 30 days, subcultured twice for 2 days, and 0.01% Triton was added to YPM 2 medium containing 2% methanol.
  • step (2) the area that does not contain the 52 kDa protein but has a high content of the 50 kDa protein is bulged, and is subjected to S-condensation using an ultrafiltration membrane (FILTRON STRRED CELLS: Filtron, Technology). did.
  • the PA activity of 1 Oml of the concentrated solution was 43000 IU / m1, and a recovery of 90% was observed.
  • PA activity of 105 IU / m1 was observed in 52 ml of the pass fraction.
  • the Km value was determined and compared with the Km value of natural blow-mouth kinase (hereinafter, referred to as thrombolyse) produced by human Xu cells.
  • the Km value for the synthetic substrate S-2444 color-forming synthetic reagent, manufactured by Kabi-Vitrium
  • Brasmin The change over time under the activity of blow mouth kinase was not significantly different from that of thrombolyse.
  • N-terminal amino acid sequence of the obtained purified blow-mouth kinase was determined.
  • 2X BJ Blue Juice: lO OmM Tris HC 1 (H6.8), 43 ⁇ 4SDS, 0.2% bromophenol blue, 20 glycerol
  • Add 2X BJ Blue Juice: lO OmM Tris HC 1 (H6.8), 43 ⁇ 4SDS, 0.2% bromophenol blue, 20 glycerol
  • PVDF membrane At 7 OmA for 4 nights.
  • the blocking buffer used was 25 mM Tris, 192 mM glycine, and 20% methanol.
  • blow-mouth kinase secretion-expressing strain SC06 obtained in Examples 1 and 2 was cultured in YPD medium for 30 days, subcultured twice for 2 days, and placed in a 300-ml 1-volume flask.
  • the medium 1 to 5 was inoculated into 50 ml of a methanol medium so that the absorbance (A s ⁇ .
  • Fig. 5 shows the cell growth curve.
  • Medium 1 and Medium 2 showed almost the same curve, and A "was changed from 2" to 2 to 23, and then decreased.
  • Medium 3 increased to 41.4 on day 3 and then decreased.
  • the growth on day 1 was very slow in medium 4 and medium 5, but the growth rate then increased, increasing to 36.4 on day 4 in medium 4 and 42.1 in medium 5. Since then, it has decreased.
  • FIG. 6 shows the PA activity by the fibrin plate method.
  • medium 1 showed an upper bound of 71 IU / ml, and then decreased.
  • Medium 4 was lower than medium 2 and about twice as high.
  • the activity was remarkably increased, and was 867 IU / ml on the fifth day.
  • the 50 kDa band was predominant until day 3, but then decreased. However, the amount of degradation products from 29 kDa to 35 kDa as in the medium 3 was small. In the medium 5, up to the sixth day, the band of 50 kDa was mainly contained, and the amount of degradation products was small. From these results, it was found that the addition of 0.01 kappa Triton X-100 did not affect bacterial growth or suppress the molecular weight of prokinase, but the activity was increased by more than 3 times. e which is expected to have significantly suppressed adsorption to the flask mouth kinase
  • Blow-mouth kinase secretion-expressing strain SC06 prepared in Examples 1-2 was cultured overnight in YPD medium (1% yeast extract, 2% bactopeptone, 2% glucose) for 30 days. Then, inoculate 30 ml of each of the culture media listed in Table 4 in a 300-ml one-volume flask so that the "" becomes 0.1 degree of S. The culture solution was cultured at 30'C for 72 hours and 144 hours. A, «, and PA activity were measured (Table 4) In the following table, Arg indicates arginine hydrochloride, and Glutamate indicates sodium glutamate.
  • Table 6 shows that when 2% NH 4 H 2 PO * + 2% (NH 4 ) 2 HPO4 was added, the growth was promoted and the PA activity was higher than that of the Triton X-100 + arginine supplemented medium. It also rose 1.4 times. A stamp lot using the anti-UK antibody on this culture was performed. In YPM 2 medium was not 50 kDa band, but 5 0 kDa band was observed in 4% phosphate 3 ⁇ 4 Anmoniumu added. When Triton X-100 arginine was added, there were bands at 50 kDa and 47 kDa, and the band at 47 kDa was deeper, but when 4% ammonium phosphate was added, the band at 50 kDa was predominant. The 47 kDa band was few. From these results, it was considered that the addition of 4% ammonium phosphate suppressed the reduction of the protein from 50 kDa to 47 kDa (Fig. 7).
  • Arginine ben-glutamate; 7-midine, abrotinin, casein hydrolyzate, ammonia (PA activity Roh A S ")
  • the PA activity was increased 4.3 times with the addition of 0.1 IN! Glutamic acid compared to the basal medium, and was more effective than with the addition of 0.1 M arginine.
  • Addition of benzyl-midine at lmM or 1 OmM reduced PA activity. 10 IU / ml abrotinin did not affect PA activity.
  • 3% casamino acid of the casein hydrolyzate promotes cell growth, An increase in PA activity about 8 times that of this medium was observed.
  • 4 CNH 4 C 1 suppressed cell growth and production of blow-mouth kinase. 0. 25M Na-P0 4 at pH 7. 0 or less in addition ⁇ 3 ⁇ 4 were subjected to Western plot also by PA activity was low c anti UK antibody on these culture cleanse regardless're promoted.
  • ammonium salt has an effect of remarkably suppressing the reduction of the molecular weight of the 50 kDa protein.
  • the reaction solution (50 ⁇ ) was placed in a siliconized conical tube, incubated at 30 for 24 hours, and the degree of decomposition of Blow ⁇ -kinase was so-called “stan-blot”. Is shown in Table 9.
  • the buffer [5 OmM Tris-HCl (pH7.5), 0.5M NaC0 0.1 Triton X-100] and eluted with an eluent [0.2M glycine hydrochloride (pH 2.5), 0.5M NaCl, 0.01% Triton X-I 00]. .
  • the eluate with PA activity was concentrated approximately 100-fold with molcut L (fraction molecular weight 10,000, UFP2LGC24, manufactured by Miriboa) and Ultra-free C3-LGC (fraction molecular weight 10,000, manufactured by Millipore).
  • the band of 30 kDa was cut out by -PAGE, and the amino acid sequence of the 30 kDa protein obtained by extracting from the band was determined.
  • the amino acid sequence of 1 matches the sequence from the isoleucine at the 159th position from the N-terminal of the blow-mouth kinase, and the amino acid sequence of 2 is 1336 from the N-terminal of the blow-mouth kinase. It matched the sequence from the lysine of the eye. This suggests that the 30 kDa protein is a small double-stranded perovin kinase.
  • PA activity was measured according to the method of Levin. E. G .. et al. (Fibrin plate method, J. Cell Bio., 94, 631-636. 1982).
  • Sequence type nucleic acid
  • Sequence type nucleic acid

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Abstract

Procédé de production d'une protéine dont le poids moléculaire peut être réduit par une protéase originaire d'une cellule hôte, et consistant à cultiver une cellule hôte capable de produire et secréter ladite protéine dans au moins un milieu choisi dans le groupe comprenant des acides aminés acides, des acides aminés basiques, des sels d'ammonium, des phosphates et des tensioactifs non ioniques, puis à séparer la protéine cible du surnageant se trouvant dans le milieu. Il en existe un autre procédé de production de ladite protéine qui consiste à séparer la protéine cible par traitement du surnageant à l'aide d'un tampon contenant un sel d'ammonium dans un chromatographe à colonne. L'invention porte également sur une levure du genre Pichia sécrétant et exprimant de la prourokinase. Ce procédé permet de produire en masse sous forme complète une protéine dont le poids moléculaire peut être réduit par une protéase obtenue par des techniques de génie génétique ou par fermentation; il peut de plus accroître la croissance des cellules hôtes, et la production de la susdite protéine et accélérer la sécrétion de protéines à partir de la cellule hôte; il permet enfin de moduler la réduction du poids moléculaire de la protéine sécrétée dans le surnageant.
PCT/JP1995/001399 1994-07-14 1995-07-13 Procede permettant de produire une proteine dont le poids moleculaire peut etre reduit par une protease originaire d'une cellule hote WO1996002661A1 (fr)

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JP6186602A JPH0823995A (ja) 1994-07-14 1994-07-14 宿主細胞由来のプロテアーゼで低分子化され得る蛋白質の製造方法
JP6/186602 1994-07-14

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6309864B1 (en) 1997-04-03 2001-10-30 Yoshitomi Pharmaceutical Industries, Ltd. Process for producing foreign proteins
US10765245B2 (en) 2009-07-14 2020-09-08 Belgravia Wood Limited Power pole for artificial tree apparatus with axial electrical connectors

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP6411891B2 (ja) * 2012-10-03 2018-10-24 協和発酵キリン株式会社 培養液にアミノ酸を添加することによるポリペプチドの還元防止方法
JP7050528B2 (ja) * 2017-03-01 2022-04-08 三洋化成工業株式会社 有用物質の生産方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS63296689A (ja) * 1987-04-15 1988-12-02 ノバルティス　アクチェンゲゼルシャフト ウロキナーゼ型プラスミノーゲン活性化因子及びその製造方法
JPH02104292A (ja) * 1988-04-25 1990-04-17 Phillips Petroleum Co ヒト―インターロイキン―２のメチロトローフ酵母での発現
JPH05260986A (ja) * 1992-03-16 1993-10-12 Green Cross Corp:The ヒト血清アルブミンの着色抑制方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS63296689A (ja) * 1987-04-15 1988-12-02 ノバルティス　アクチェンゲゼルシャフト ウロキナーゼ型プラスミノーゲン活性化因子及びその製造方法
JPH02104292A (ja) * 1988-04-25 1990-04-17 Phillips Petroleum Co ヒト―インターロイキン―２のメチロトローフ酵母での発現
JPH05260986A (ja) * 1992-03-16 1993-10-12 Green Cross Corp:The ヒト血清アルブミンの着色抑制方法

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6309864B1 (en) 1997-04-03 2001-10-30 Yoshitomi Pharmaceutical Industries, Ltd. Process for producing foreign proteins
US10765245B2 (en) 2009-07-14 2020-09-08 Belgravia Wood Limited Power pole for artificial tree apparatus with axial electrical connectors

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