WO1996003503A1 - Procede de production d'un inhibiteur de la trypsine urinaire et des domaines de celui-ci, nouveau polypeptide associe et procede de production de ce polypeptide - Google Patents

Procede de production d'un inhibiteur de la trypsine urinaire et des domaines de celui-ci, nouveau polypeptide associe et procede de production de ce polypeptide Download PDF

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WO1996003503A1
WO1996003503A1 PCT/JP1995/001449 JP9501449W WO9603503A1 WO 1996003503 A1 WO1996003503 A1 WO 1996003503A1 JP 9501449 W JP9501449 W JP 9501449W WO 9603503 A1 WO9603503 A1 WO 9603503A1
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PCT/JP1995/001449
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Japanese (ja)
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Shoji Ideno
Takashi Goto
Hajime Horii
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The Green Cross Corporation
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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
    • C07K14/81—Protease inhibitors
    • C07K14/8103—Exopeptidase (E.C. 3.4.11-19) inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a method for producing a urinary trypsin inhibitor (hereinafter, simply referred to as UTI) and a Kunitz-type domain thereof by a genetic engineering technique using Pichia yeast as a host cell. Furthermore, the present invention provides a novel polypeptide having excellent protease inhibitory activity, a DNA encoding the polypeptide, a vector having the DNA, a transformant transformed with the vector, and culturing the transformant. And a method for producing the novel polypeptide.
  • UTI urinary trypsin inhibitor
  • DIC disseminated intravascular coagulation
  • MOF multiple organ failure
  • the characteristic features of the transition from the occurrence of local inflammation to systemic disease states such as MOF and DIC are (1) the progression of the disease state from the local to the whole body rapidly progresses in a short time, (2) the final There are two points that involve the involvement of proteases such as elastase (HNE) released from activated neutrophils and elastase (HPE) released from skeletal cells. Therefore, in the treatment of otitis, etc., two approaches are considered: suppressing the progression in the early stage of inflammation and inhibiting the protease. However, in the former approach, it is difficult to develop an effective drug because of the involvement of multiple factors and the progress is rapid.At present, the latter approach is considered effective, including new development. I have.
  • HNE elastase
  • HPE elastase
  • those having elastase inhibitory activity include ⁇ -antitrypsin (1- ⁇ ) and UTI, all of which mainly inhibit neutrophil elastase.
  • al-AT is a serine protease inhibitor present in a large amount in blood and has a strong inhibitory activity on neutrophil elastase, but is known to be easily inactivated by active oxygen.
  • An object of the present invention is to provide a new method for producing UTI and its respective domains by genetic engineering techniques, especially using Pichia yeast as a host cell.
  • Another object of the present invention is to provide a useful novel polypeptide having a strong neutrophil elastase inhibitory activity and preferably capable of being produced in a large amount by a genetic engineering technique, and a method for producing the same by the genetic engineering technique.
  • a novel borohydride having improved neutrophil elastase inhibitory activity which is a disadvantage of natural UTI, having a stronger neutrophil elastase inhibitory activity, and preferably also having a trypsin inhibitory activity.
  • an expression vector for a yeast belonging to the genus Pichia containing DNA having a nucleotide sequence encoding the amino acid sequence of Kunitz type domain 1 of UTI, having a nucleotide sequence encoding the amino acid sequence of Kunitz type domain 2 of UTI An expression vector for Pichia yeast containing DNA, and an expression vector for Pichia yeast containing DNA having a base sequence encoding the amino acid sequence of UTI.
  • a method for producing a protein having at least one kind of Kunitz-type domain amino acid sequence of UTI characterized by comprising:
  • novel polypeptide may have an amino acid sequence represented by the formula:
  • a polipeptide having an amino acid sequence of the following formula IX is preferably used.
  • polypeptides may have an amino acid sequence represented by Formula I [N-terminal].
  • ys is preferably a novel polypeptide having an amino acid sequence represented by Formula IV. Ala-Val-Leu-Pro-Gln-Glu-Glu-Glu-Gly-Ser-
  • a vector comprising the DNA according to (7).
  • (10) A method of culturing the transformant of (9) to produce the novel polypeptide of (4) to (6), and collecting the novel polypeptide from the obtained culture. (4) The method for producing a novel polypeptide according to (6).
  • FIG. 1 shows the primary structure of UTI isolated from urine (Watcher. E and Hochstrasser. K., Hoppe-Seyler's ⁇ . Physiol. Chem., 362, 1351, 1981) ⁇
  • FIG. 2 is a diagram showing the primary structure of the Kunit ⁇ type domain 1 and the Kunitz type domain 2 of UTI.
  • FIG. 3 is a diagram showing the nucleotide sequence (120 to 240) of the cloned UTI cDNA. The base number was in accordance with the report of J. F. Kaumeyer et al., Nucl. Acads Res. 14 (20), 7839-7850 (1986).
  • FIG. 4 is a diagram showing the nucleotide sequence (241 to 540) of the cloned UT1 cDNA.
  • the base number was determined according to the report of J. F. Kaumeyer et al., Nucl. Acads Res. 14 (20), 7839-7850 (1986). Note that the restriction enzyme sites in the figure are those used in each Example.
  • FIG. 5 is a diagram showing the base sequence (541-780) of the cloned UTI cDNA.
  • the base number was in accordance with the report of J.F. Kaumeyer et al., Nucl. Acads Res. 14 (20), 7839-7850 (1986).
  • the amino acid sequence determined from UTI cDNA is shown below.
  • the restriction enzyme sites shown in the figures are those used in each Example.
  • FIG. 6 shows the nucleotide sequence of the cloned UTI cDNA (781-1020).
  • the base number was in accordance with the report of F. Kaumeyer et al., Nucl. Acads Res. 14 (20), 7839-7850 (1986).
  • the amino acid sequence determined from the UTI cDNA is shown below.
  • the restriction enzyme sites shown in the figures are those used in each Example.
  • FIG. 7 is a view showing the base sequence (1021-1235) of the cloned UTI cDNA.
  • the base number was in accordance with the report of JF Kaumeyer et al., Nucl. Acads Res. 14 (20). 7839-9850 (1986).
  • the amino acid sequence determined from the cDNA of UTI is shown below.
  • the restriction enzyme sites in the figures are those used in each example.
  • FIG. 8 is a view showing a modified base sequence of a region encoding the N-terminus and a region encoding the
  • FIG. 9 is a diagram showing a construction process of # 7_BbeI—ERI.
  • FIG. 10 is a diagram showing the process of constructing pUT I-N from Bbe I—ER I # 7.
  • Fig. 11 is a diagram showing the process of constructing pUT I-C from # 7ZBb e I-ER I.
  • FIG. 12 is a diagram showing a construction process of pUTINC.
  • FIG. 13 is a diagram showing the steps of constructing pUTIN-CZERI.
  • FIG. 14 is a diagram showing a restriction map of pUTIN-CZERI and pA ⁇ 807N, and a process for constructing pHH310.
  • FIG. 15 shows the nucleotide sequence of the SUC2 signal.
  • FIG. 16 is a diagram showing the nucleotide sequence of a synthetic DNA that complements the region encoding the C-terminus of Kunitz type domain 1 of UTI.
  • FIG. 17 is a diagram showing the nucleotide sequence of a synthetic DNA that complements a region encoding the N-terminus of Kunitz type domain 2 of UTI.
  • FIG. 18 is a diagram showing the gene unit of the N-terminal butyl-added K unitz type domain 1 and the Kunitz type domain 2 of UTI. In the figure, the domain of each domain is indicated by the amino acid number of UTI.
  • FIG. 19 is a diagram showing a construction process of pHH305 and a restriction enzyme map thereof.
  • FIG. 20 is a diagram showing a restriction enzyme map of pHH313.
  • FIG. 21 is a diagram showing a construction step of pHH306 and a restriction enzyme map thereof.
  • FIG. 22 is a diagram showing a restriction enzyme map of pHH314.
  • FIG. 23 is a view showing a profile of HPLC-GPC of purified Intact-rUTI.
  • FIG. 24 is a view showing a profile of HP LC-GPC of purified rD2.
  • FIG. 25 is a diagram showing a region used for mutagenic primer.
  • FIG. 26 is a diagram showing a construction step of pHH334.
  • FIG. 27 shows the effect of chloramine T oxidation of Ep1-UTI and native UTI on neutrophil elastase inhibition.
  • Figure 28 shows the synthetic DNA corresponding to the region from the 5 'end to the Ec052 I site that encodes the 21 amino acid region added to the N-terminal side of the Kunitz type domain 1 of UTI. It is a figure which shows a base sequence. In the figure, the bases enclosed in squares are bases that have been replaced due to disappearance of the Pvull site.
  • FIG. 29 is a diagram showing a construction process of pHH336 and a restriction map thereof.
  • FIG. 30 is a diagram showing a construction step of pHH339 and a restriction enzyme map thereof.
  • UTI was initially isolated from human urine according to the method of Proksch (Proksch G. J., et al., Lab. Clin. Med., 79, 491-499, 1972). It is a proteinase inhibitor that has been studied (Sumi et al., Japanese Journal of Physiology, Vol. 39, pp. 53-58, 1977) and has the primary structure shown in Fig. 1 by studies by Watcher, E. Known (Watcher, E and Hochstrasser, K., Hoppe-Seyler's
  • the primary structure of the protein shown in FIG. 1 is the amino acid sequence directly obtained from the purified protein.
  • the UTI is continuous from two structurally related Kunit I type domains.
  • one Knitz-type domain 1 is located in the N-terminal region of UTI and is defined as amino acid residues 22 to 77 counted from the N-terminal
  • the other Knitzitz domain 1 Type domain 2 is located in the C-terminal region of UTI, and is defined as amino acid residues 78 to 1443 counted from the N-terminus (Hochstrasser, et al., Hoppe-Seyler's Z.
  • the Kunitz-type domain 1 and Kunitz-type domain 2 of UTI referred to in the present invention are each. And has been reported to have the amino acid sequence shown in Fig. 2 [JF Kaum eyer et al., Nucl. Acids Res. 14 (20), p7844-7845. 1986: Amino acid numbers 86, 87 and 138 are from proteins. This differs from the directly determined amino acid sequence (Fig. 1).] Kunitz-type domain 1 and Kunitz-type domain referred to in the present invention.
  • the term “in 2” substantially means each of these domains, and according to this DNA recombination method, the UTI structural gene is composed of 441 bases and encodes 147 amino acids. No sequence with a terminal amino acid at position 147 in UTI has been reported. (Hoc strasser, K. et al., Hoppe-Seyler's Z. Physiol. Chem., 362, 1351, 1981). Therefore, when expressing UTI by genetic engineering, it is considered preferable to use a sequence found in nature.In the present invention, the amino acid at position 145 is replaced with the C-terminal amino acid of K unitz type domain 2. And
  • K unitz type domain 1 and K unitz type domain 2 have different specific protease inhibitory activities, respectively, edited by CGebhard & Hochstraber, Proteinase Inhibitors, Barrett & Salvesen, Elsevier ⁇ 1986, p. 375)
  • the present invention relates to an expression vector containing a DNA having a base sequence encoding the amino acid sequence of the Kunitz-type domain 1 of UTI, a nucleotide sequence encoding the amino acid sequence of the Kunitz-type domain 2 of UTI. And an expression vector containing a DNA having a base sequence encoding the amino acid sequence of UTI.
  • amino acid sequence of UTI specifically refers to an amino acid sequence represented by the following formula V:
  • amino acid sequence of nitz-type domain 1 specifically refers to the amino acid sequence represented by the following formula VI
  • amino acid sequence of K unitz-type domain 2 specifically corresponds to the following formula: ⁇ ⁇ ⁇ ⁇ Refers to the amino acid sequence represented by ⁇ .
  • the DNA encoding the amino acid sequence of UTI is defined as the Pichia yeast transformed by introducing the DNA into Pichia yeast by an appropriate method and transforming the Pichia yeast.
  • any DNA having any base sequence may be used as long as it can express a protein having the amino acid sequence of UTI.
  • DNA encoding the amino acid sequence of UTI preferably a DNA having a base sequence represented by base numbers 661-1095 among the base sequences shown in FIGS. 3 to 7 as a part of the base sequence.
  • the DNA encoding the amino acid sequence of Knitz-type domain 1 is preferably a nucleotide sequence represented by nucleotide numbers 724 to 891 in the nucleotide sequences shown in FIGS. 3 to 7 as a part of the nucleotide sequence. Is a DNA having
  • the DNA encoding the amino acid sequence of Knitz-type domain 2 is preferably represented by base numbers 892-2010 of the base sequences shown in FIGS. 3 to 7 as a part of the base sequence. It is a DNA having a base sequence.
  • each of the DNAs according to the present invention may be one or more of the base sequences shown in FIGS. 3 to 7 as long as it can encode the amino acid sequence of UTI. May be replaced by another base.
  • the origin and the like of the expression vector of the present invention are not particularly limited as long as it contains the above DNA and can express the DNA in Pichia yeast.
  • the expression vector preferably has, in addition to the above DNA, a promoter, a terminator, a homologous region, a marker gene, and the like necessary for expressing the DNA in Pichio yeast. It is preferable that the vector has a base sequence encoding a signal peptide. In addition, it may carry an autonomous replication sequence or the like that can be replicated in a host.
  • the base sequence encoding the promoter and signal peptide may be any sequence that functions in Pichia yeast.
  • the promoter examples include the A ⁇ X1 promoter [Japanese Patent Laid-Open No. 63-39484 (EP-A-244584)], the DAS promoter, and the mutant AOX2 promoter. No. 1 (Japanese Unexamined Patent Publication No. Hei 6-99068 (EP-A-56040)) and the like can be used.
  • Examples of the signal peptide include yeast invertase (SUC 2) [Japanese Patent Application Laid-Open No. 60-41888 (EP-A-127304)], ⁇ -factor [Japanese Patent Application Laid-Open No. 1 1 ⁇ ⁇ 1 ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ HS ⁇ ⁇ ⁇ ⁇ , A ⁇ ⁇ ⁇ HS HS HS No. 5 (EP—A—3 196 4 1)], an artificially created signal sequence [JP-A 1-240191 (EP—A—3 291)] Etc. can be used.
  • AOX 1 evening light and evening Japanese Patent Application Laid-Open No. 63-39584 (EP-A-24444598)
  • Examples of the homologous region include HIS4 (Japanese Patent Application Laid-Open No. 61-108391 (EP-A-1808999)), URA3, LEU2, and ARG4.
  • an antibiotic resistance gene As the marker gene, an antibiotic resistance gene, an auxotrophic complement gene, or the like is used.
  • antibiotics include cycloheximide, G-418, chloramphenicol, bleomycin, hygromycin and the like.
  • the auxotrophic complement gene includes HIS 4, URA 3, LEU 2, ARG 4, and the like.
  • the expression vector of the present invention is simply a plasmid vector or a plasmid vector having in advance a nucleotide sequence necessary for expression, production and secretion of UTI such as a promoter, a signal peptide-encoding nucleotide sequence or each Knitz-type domain thereof.
  • a phage vector is prepared by introducing the above DNA into a first-class phage vector according to a conventional method (eg, Molecular Cloning, a laboratory manua 1, Second edition, edited by T. Maniatis, Cold Spring Harbor Laboratory, 1989). Can be.
  • a nucleotide sequence required for expression can be chemically synthesized and introduced into a plasmid vector or the like together with the above DNA.
  • the present invention also relates to a Pichia yeast transformed with the above-described expression vector of the present invention.
  • the transformant was prepared by the spout plast method (Cregg T. et al., Mol. Cell. Biol. 5.3 pp. 376-3385, 1985), the alkali cation method Utoh H. et al., J. Bacteriol. 153, 163- 168), by transforming a yeast belonging to the genus Pichia with the vector of the present invention.
  • Pichia yeast to be transformed examples include Pichia pastoris GTS115 strain (NRRL accession number: Y-15851).
  • the vector to be introduced into the transformant encodes those proteins.
  • those having a base sequence encoding the aforementioned signal peptide in addition to the above DNA are preferable.
  • the Pichia yeast which is the transformant of the present invention, more preferably has the property of producing a protein having the amino acid sequence of UTI or each domain thereof and secreting the protein out of the transformant.
  • the present invention also provides a method for producing a protein having an amino acid sequence of at least one Knitz type domain of U UI by culturing the above-mentioned transformant (Pichia yeast), and transforming the protein from the culture. Collecting a protein having an amino acid sequence of at least one Knitz-type domain of UTI. W / 0 03
  • the manufacturing method is characterized by performing the following steps (a) to (c).
  • Pichia yeast is transformed with the expression vector containing the DNA obtained in (a) to obtain a transformant.
  • a protein having an amino acid sequence of at least one kind of Kunitz-type domain of UTI refers to a protein having at least the amino acid sequence of either Kunitz-type domain 1 or Kunitz-type domain 2 of UTI. Means therefore, if it has the amino acid sequence of Kunitz-type domain 1 or Kunitz-type domain 2, it may have another amino acid sequence at its N-terminal and / or C-terminal. A protein having an amino acid sequence is also included.
  • the Pichia yeast which is a transformant, may be cultured according to a general method used for culturing Pichia yeast, for example, a method described in JP-A-6-22784.
  • the medium may be a 0.01% to 8% methanol-containing YNB liquid medium [0.7% Yeast Nitrogen Base w / o Amino Acids (Difco)] and a 0.01% to 8% methanol-containing medium.
  • YP medium 1% Yeast Extract (Difco), 2% Peptone (Difco)] and the like are exemplified.
  • the cultivation is usually carried out at 15 to 43 (preferably about 20 to 30 ° C.) for about 20 to 360 hours, and if necessary, aeration and stirring can be applied.
  • the target protein produced by the yeast of the genus Pichia is collected from the culture of the yeast of the genus Pichia.
  • the produced protein is not secreted outside the Pichia yeast, it is preferably isolated from the yeast cells of the genus Pichia, and when secreted, it is preferably isolated from the culture supernatant.
  • the method of collecting the target protein from the culture can be performed by a method usually used for protein purification, for example, “Biochemical Experiment Lecture 1, Protein Chemistry” (edited by the Biochemical Society of Japan, 1979, The method can be carried out with reference to methods described in many documents such as Tokyo Chemical Dojin.
  • the obtained protein can be further purified if necessary.
  • Purification methods include salting-out, ultrafiltration, isoelectric precipitation, gel filtration, ion-exchange chromatography, hydrophobic chromatography, affinity chromatography, chromatofocusing, and adsorption.
  • Chromatography and reversed-phase chromatography Examples include various types of chromatography.
  • the present invention also relates to novel polypeptides having useful protease inhibitory activity.
  • the novel polypeptide of the present invention is characterized in that it has protease inhibitory activity. Specifically, at least one enzyme among eye enzymes such as tribsine and elastase, inflammation-related enzymes such as neutrophil elastase, closin and fibrinolytic enzymes such as plasmin, factor-1Xa, and kallikrein. It shows inhibitory activity.
  • the novel polybeptide of the present invention preferably has a neutrophil elastase inhibitory activity that is stronger than the neutrophil elastase inhibitory activity of natural UTI derived from urine, and more preferably It also has an inhibitory activity on tribsine.
  • novel polybeptide of the present invention may be any amino acid sequence as long as it has the above-mentioned characteristics as long as it contains the amino acid sequence of the following formula I as at least a part of the amino acid sequence defining its primary structure. May also be used.
  • polypeptide of the present invention may be a polypeptide defined by the amino acid sequence itself represented by Formula I, or an arbitrary amino acid sequence at the N-terminal and / or C-terminal of the amino acid sequence of Formula I. It may be a polypeptide to which two or more amino acids have been added.
  • novel polypeptide of the present invention may have an amino acid sequence represented by the following formula VII, formula II or formula VII on the N-terminal side thereof.
  • a polypeptide having an amino acid sequence represented by the formula II on the N-terminal side is converted into a polypeptide having no amino acid sequence due to having the amino acid sequence.
  • the expression level is remarkably large in comparison, and it is useful in that it can be produced in large quantities by recombinant DNA technology.
  • novel polypeptide of the present invention may have an amino acid sequence represented by the following formula II on the C-terminal side of the amino acid sequence.
  • Such a novel polybeptide is useful in that it has not only neutrophil elastase inhibitory activity but also tribcine inhibitory activity.
  • Examples of such a polypeptide include a polypeptide represented by the following formula (IX).
  • polypeptide having an amino acid sequence represented by the following formula IV is a polypeptide having an amino acid sequence represented by the following formula IV.
  • the polypeptide of the present invention includes not only the amino acid sequence represented by Formula IV or Formula IX but also the polypeptide defined by itself, and the N-terminal and Z or C of the amino acid sequence represented by Formula [V or Formula. It also includes a polypeptide having one or more optional amino acids added to the terminal. More preferably, it is a peptide having an amino acid sequence represented by Formula IV or Formula IX, and having a trypsin inhibitory activity and a neutrophil elastase inhibitory activity, wherein the neutrophil elastase inhibitory activity is a natural UTI derived from urine. It is significantly enhanced as compared to the activity.
  • polypeptides are exemplified Novel polypeptides have extremely strong neutrophil elastase inhibitory activity And can be produced in large quantities using recombinant DNA technology.
  • polypeptide having an amino acid sequence represented by the formula II on the N-terminal side is useful in that the expression level is extremely high.
  • novel polypeptide of the present invention is expected to have an amino acid sequence partially common to UTI, to be high in tissue permeability due to being a small molecule, and not to exhibit antigenicity to humans. You. Further, it is useful as a material for producing a fusion protein, for example, by fusing another polypeptide having another function to the N-terminus and / or the C-terminus as necessary.
  • the novel polypeptide of the present invention may or may not have a sugar chain.
  • the origin of the acquisition is not particularly limited.
  • it may be chemically synthesized using a peptide synthesizer or the like.
  • it is a polypeptide produced using recombinant DNA technology.
  • it is a polypeptide produced by recombinant DNA technology using yeast or Pichia yeast or Escherichia coli as a host.
  • the polypeptide may be chemically modified (for example, sugar addition, alkylation, etc.) as long as the specific activity is not substantially reduced.
  • a substance obtained by forming a complex with a pharmacologically acceptable acid or base or a polymer such as polyethylene glycol is also included in the form of the novel polypeptide of the present invention.
  • the present invention also relates to DNA having a nucleotide sequence encoding the novel polypeptide.
  • the DNA may be an amino acid sequence represented by any one of Formula I, Formula IV or Formula IX, or other amino acids or peptides at the N-terminal and Z- or C-terminals of the amino acid sequence thereof (for example, Formula I I. VI I, VI II or those having the amino acid sequence represented by Formula III), or any other nucleotide sequence within a range that does not cause a change in the amino acid sequence of the polypeptide. Good.
  • the nucleotide sequence represented by nucleotide numbers 736 to 888 from the 5 ′ side The base number of 766 to 780 is ATCGCTTTCT TTCCT
  • nucleotide sequence of the nucleotide sequence represented by nucleotide numbers 724 to 1095 from the 5 'side of the nucleotide sequence shown in FIGS. 766 to 780 bases are represented by nucleotide numbers 724 to 1095 from the 5 'side of the nucleotide sequence shown in FIGS. 766 to 780 bases.
  • DNA having a nucleotide sequence substituted by in addition, as the DNA encoding the polypeptide represented by the formula IV, in the base sequences shown in FIGS. 3 to 7, bases represented by base numbers 661 to 1095 from the 5 ′ side are used. Nucleotide number 766-780 of the sequence is
  • DNA encoding the polypeptide represented by Formula II a DNA having a base sequence represented by base numbers 61 to 723 from the 5 'side of the base sequences shown in FIGS. 3 to 7,
  • Examples of the DNA encoding the polypeptide shown in [1] include, among the nucleotide sequences shown in FIGS. 3 to 7, a DNA having a nucleotide sequence represented by base numbers 661 to 735 from the 5 ′ side.
  • the DNA encoding the polypeptide represented by the formula III is, for example, a base sequence represented by base numbers 892-2010 from the 5 'side of the base sequences shown in FIGS. 3 to 7. DNA having the following is exemplified.
  • the DNA of the present invention has, in the base sequence specified in FIGS. Also included are sequences having a base sequence in which one or more bases have been replaced with other bases.
  • the DNA of the present invention has a 5′-terminal in addition to the nucleotide sequence specified in the above figure. And one or more arbitrary bases added to the 3 ′ end.
  • the base to be added may be any base or base sequence as long as it does not cause a change in the amino acid sequence encoded by the base sequence to be added by being added to the above base sequence. Good.
  • the base sequence added to the 5 ′ end includes the base sequence ATG as a start codon
  • the base sequence added to the 3 ′ end includes the stop codon TAA, TAG or TGA and the like.
  • the DNA of the present invention is a nucleotide sequence encoding another amino acid or polypeptide at the 5 ′ end and Z or 3 ′ end of the nucleotide sequence encoding the amino acid sequence of the novel polypeptide of the present invention. May be provided.
  • the DNA of the present invention may be obtained by any method. For example, a method of chemically synthesizing with reference to the nucleotide sequences shown in FIGS. 3 to 7, a method using recombinant DNA, using an appropriate chromosomal DNA library or cDNA library as a material, etc. There is a way to get it.
  • the chemical synthesis of the DNA of the present invention is performed, for example, as follows.
  • a desired DNA having a nucleotide sequence encoding the novel polypeptide of the present invention is designed, and if necessary, the designed DNA is divided into appropriate fragments, and an oligomer corresponding to each fragment is converted into a fully automatic DNA synthesizer. (Eg, Type 381 A, manufactured by Applied Biosystems).
  • an appropriate cDNA library, a chromosomal DNA library, or an amino acid sequence represented by Formula I or Formula IV is encoded.
  • Site-directed mutagenesis using DNA or the like as material hereinafter referred to as Site-directed mutagenesis, Kramer. W, et al., Ucleic Acid Res., 12, 944, 9456, 1984 and Kunkel, TA, etc. Enzymology, Vol. 154, pp. 367-382, 1987), and a method of modifying a base sequence and amplifying DNA by a known method such as PCR.
  • the present invention provides a DNA having a nucleotide sequence encoding the novel polypeptide described above. 9
  • the origin of the vector is not limited as long as it contains DNA having a nucleotide sequence encoding the novel polypeptide of the present invention.
  • the DNA of the present invention was introduced into an appropriate position in various plasmid vectors such as pBR322, pUC18 and pUC19, and phage vectors such as ⁇ gt10 and ⁇ gt11. Things.
  • it is a vector that can be replicated in the host cell to be used or a vector that can be integrated into the host chromosome. More preferably, it is a vector that can be replicated in Escherichia coli or yeast, preferably yeast of the genus Pichia, or that can be integrated therein. K It is the evening.
  • the vector of the present invention comprises, in addition to DNA encoding the novel polypeptide of the present invention, bases such as a promoter and an optional ribosome binding site necessary for expressing the novel polypeptide of the present invention in a host. It is preferable to have a sequence. Particularly, when the vector of the present invention is a vector for secretion expression, a vector further having a base sequence encoding a signal peptide and the like is preferable.
  • the above-mentioned promoter, the ribosome binding site, the nucleotide sequence encoding the signal peptide, and the like may be any sequence that functions in the host used. Examples of the promoter and signal peptide include those described above.
  • the vector of the present invention simply has in advance a nucleotide sequence necessary for the expression, production and secretion of the novel polypeptide of the present invention, such as a promoter, a ribosome binding site, and a nucleotide sequence encoding a signal peptide.
  • the DNA of the present invention is introduced into a brasmid vector or a phage vector according to a conventional method (eg, Molecular Cloning, a laboratory manual, Second edition T. Maniatis, etc., Cold Spring Harbor Laboratory, 1989). It can be manufactured by the following. Alternatively, it is also possible to chemically synthesize a nucleotide sequence required for expression and introduce it into a plasmid vector or a phage vector together with the DNA of the present invention.
  • the present invention also relates to a transformant transformed with a vector comprising a DNA having a nucleotide sequence encoding the novel polypeptide.
  • the transformant was prepared by the calcium chloride method, the rubidium chloride method, Hanahan. D, Techniques for Transformation of E. coli. In: DNA cloning vol 1, Gl over, DM (ed.) 109-136, IRL press, 1985) and by transforming a suitable host cell with the vector of the present invention.
  • yeast When yeast is used as a host cell, the blast method (Hinnen A. et al., Proc. Natl. Acad. Sci. USA 75, 1929-1933, 1978, Cregg JT et al., OLCell. Bio 1.5, p. 3376-3385 198) and the known method such as the alkali cation method (Itoh H. et al., J. Bacteriol. 153, pp. 163-168, 1983).
  • the transformant of the present invention more preferably has a property of producing the novel polypeptide of the present invention and secreting it outside the transformant.
  • the introduced vector may be a signal peptide in addition to the DNA of the present invention. Those having an encoding base sequence are preferred.
  • any eukaryotic cell typified by yeast or the like can be used as long as it is suitable for expression and production of the polypeptide of the present invention. It may be a biological cell. Preferably, Escherichia coli and yeast are used, and more preferably, Pichia yeast is used.
  • the present invention provides a novel polypeptide of the present invention, which comprises culturing the above-mentioned transformant, producing the novel polypeptide of the present invention, and collecting the polypeptide from the obtained culture. It is a manufacturing method.
  • the production method is a method for producing a novel polypeptide of the present invention, which comprises performing the following steps (a) to (c).
  • the novel voriveptide of the present invention produced by the transformant is collected from the culture of the transformant.
  • the produced polypeptide is not secreted outside the transformant, it is preferably isolated from the transformant, and when secreted, it is preferably isolated from the culture supernatant.
  • the collection and purification of the polypeptide can be carried out according to a conventional method as described above.
  • the present invention also relates to a method for enhancing expression of a polypeptide having a protease inhibitory activity in a transformant in a large amount.
  • the present inventors have found that a polypeptide having an amino acid sequence represented by the formula II on the N-terminal side is more effective than a polypeptide having no amino acid sequence represented by the formula ⁇ on the N-terminal side. It was found that the expression level in yeast was remarkably large. Therefore, by binding the amino acid sequence represented by the formula H to the N-terminal side of olibeptide having a protease inhibitory activity such as neutrophil elastase and trypsin, the polypeptide is added to the N-terminal side.
  • a large amount of a polypeptide useful as a protease can be obtained. That is, a vector is prepared in which a nucleotide sequence encoding an amino acid sequence represented by the formula I is linked to the 5 'end of a nucleotide sequence encoding an amino acid sequence containing a protease inhibitory activity site, and this vector is used. As a result, the expression of borobeptide useful as a protease inhibitor in a transformant is enhanced, and a large amount of protease inhibitor can be obtained.
  • One or more amino acids may be arranged between the amino acid sequence of the protease inhibitory active site and the amino acid sequence of the formula II.
  • polypeptide to which the amino acid sequence represented by the formula 11 can be added to the N-terminus at the N-terminus is not limited to the novel polypeptide of the present invention having the amino acid sequence represented by the formula I or IX described above.
  • Neutrophil elastase, trypsin and other proteases Since the method can be applied to other polypeptides having an inhibitory activity, the expression enhancing method of the present invention is extremely useful.
  • the present invention it is possible to provide a method for producing UTI and its respective K unitz-type domains by a genetic engineering technique using yeast of the genus Pichia as a host cell, and an expression system related thereto.
  • An expression vector comprising a DNA encoding the UTI of the present invention or a Kunitz-type domain thereof, an expression system comprising Pichia yeast transformed with the vector, and a UTI using the same and each of them.
  • the method for producing a K unitz-type domain enables simple and efficient mass production of UTI and its respective K unitz-type domain.
  • mutants or derivatives of UTI and its respective K unitz-type domains can be easily produced, so that new protease inhibitors, such as the development of useful protease inhibitors by studying structure-activity relationships, can be used. It can contribute to drug development.
  • the present invention provides a novel and useful polypeptide, which has one or more protease inhibitory activities and is superior in neutrophil elastase inhibitory activity to urine-derived UTI, and is preferably a superior neutrophil elastase. It is possible to provide a useful novel polypeptide having an inhibitory activity and having a high expression level in a genetic engineering technique and capable of being produced in large quantities, and a method for producing the polypeptide by the genetic engineering technique.
  • the method for enhancing the expression of a protease inhibitor of the present invention it is possible to remarkably increase the expression amount of a polypeptide having protease inhibitory activity in yeast.
  • the method is extremely useful in that it can be applied to polybeptides having a protease inhibitory activity other than the novel polypeptide of the present invention.
  • Probes 1 and 2 were designed to have a GC content of 50% or more. The area of each probe is shown below.
  • Oligonucleotides were synthesized on a DNA synthesizer (Model 392, manufactured by Applied Biosystems Japan) using a 0.2 M FOD column (manufactured by Ablied Biosystems Japan) based on this sequence.
  • the synthesized oligonucleotides are used in a 0PC cartridge After purification using Thames, Japan, the gel was electrophoresed with a 7M urea 10% acrylamide-modified gel. As a result, both probes showed a single band.
  • oligonucleotides 7 - 3 was end labeled with P ATP.
  • the dilution indicator bacterial solution was prepared as follows.
  • E. c01iC600hf1 strain was inoculated into a 5 ml 1L medium, and cultured at 37'C for 9 hours. This was inoculated in its entirety into 500 ml of TBIO medium dispensed into a 2 L kolben, and cultured at 37 ° C.
  • the culture was stopped when the blacks became visible, and stored at 4 ° C as a master plate.
  • Two membranes (colony / plaque screen, manufactured by DuPont) were prepared per master plate.
  • the resulting membrane was incubated on a TB10 bottom agar plate at 37 ° C- ⁇ to amplify phage.
  • These membranes were immersed in a 0.5N aqueous solution of sodium hydroxide for 5 minutes, and subjected to a denaturation treatment. After repeating the same operation, bacterial cell residues were removed with paper. Then, the plate was immersed in 1M Tris-HCl buffer (PH7.5) for neutralization, and rinsed with 2 ⁇ SSC buffer.
  • PH7.5 Tris-HCl buffer
  • the cells were incubated in a prehybridization solution (2 ⁇ SSC.2% SDS, 5 ⁇ Denhardt's soln., 500 Aig / ml yeast tRNA) at 60 ° C. for 4 hours.
  • a prehybridization solution (2 ⁇ SSC.2% SDS, 5 ⁇ Denhardt's soln., 500 Aig / ml yeast tRNA) at 60 ° C. for 4 hours.
  • the membrane was washed with 2 x SSC-23 ⁇ 4 SDS aqueous solution for 20 minutes at room temperature, 2xSSC-2% SDS aqueous solution at 52 ° C for 20 minutes, and further washed with 1 XSSC-2% SDS aqueous solution at 45 ° C for 40 minutes. (Registered trademark) and exposed to light at 180 ° C.
  • each membrane had 30 to 40 specific signals, and a total of about 400 signals were obtained. Most signals were consistent for probes 1 and 2.
  • the phage was not amplified on the membrane at 37 ° C for 6 hours and stored at 4.
  • the membrane was alkali-denatured, neutralized, and rinsed with 2 x SSC, and then incubated in a prehybridization solution (2XSS 2% SDS. 5x Denhardfs soln., 100 / g / ml yeast tRNA) for 60'C for 6 hours. .
  • prehybridization hybridization was performed using probe 2 in the same manner as in primary screening.
  • Each membrane was washed twice with 1 ⁇ SSC-2 SDS aqueous solution at room temperature for 10 minutes. Thereafter, each membrane was covered with Saran Wrap (registered trademark) and exposed at room temperature for 6 hours.
  • the plaque corresponding to the signal from each master plate used for secondary screening was mixed with a toothpick using indicator bacterial plate (100/1 diluted indicator bacterial solution and 3 ml TB10 top agarose were mixed at 37 ° C in advance. Inoculated on a warmed TB10 bot tom agar plate). This was incubated at 37 e C— ⁇ .
  • probe 1 was used for hybridization in the same manner as in primary screening.
  • Phage DNA was prepared from 16 of the obtained phage clones, and the obtained phage DNA was digested with restriction enzyme EcoRI, followed by agarose gel electrophoresis to confirm the insert. Most phage clones had one or two inserts with a size of 0.5-2 kb. Furthermore, Southern hybridization using probe 2 was performed, and a 1.2-1.3 kb fragment reported in the literature (JF Kaumeyer et al. Nucl. Acids Res. 14 (20), p 7839-7850, 1986) was reported. 6 claw with insert CDNA was excised from the DNA and subcloned into the EcoRI site of pUC19. Restriction enzyme mapping was performed, and the nucleotide sequence was confirmed using the obtained plasmid DNA.
  • sequence was confirmed by the dideoxy method using [a-32p] dCTP. Specifically, Sequenase ver.2 (Pharmacia) and 7-DEAZA Sequenceing kit ver.2 (Takara Shuzo) were used according to the protocol.
  • Synthesized DNA (Fig. 8) of the 5'-end and 3'-end regions of the UTI gene corresponding to the above modification was converted to a DNA synthesizer (Applied Biosystems Japan, model 392).
  • the synthetic DNA was designed so that both ends become the cohesive ends of each restriction enzyme after annealing.
  • the synthetic DNA corresponding to the C-terminal coding region has a sequence in which 6 bases corresponding to 2-amino acid at the C-terminal have been deleted and 21 bases in the 3 'untranslated region have been deleted from an operational point of view. I made it.
  • DNA containing UTIcDNA obtained by digestion with Bbd at base number position 504 of UT Ic DNA contained in plasmid Ji7 of subcloning strain No. 7 to restore blunt ends, and further digesting with EcoRl.
  • the fragment was subcloned into the Smal-EcoRI region of pUC19 ( ⁇ / Bbe ERI, Fig. 9). Based on this / Bb-ERI, each synthetic DNA was incorporated.
  • # 7 / BbeI-ERI was digested with BamHI and EcoRl, and a DNA fragment containing UTicDNA was recovered. This was digested with Sau3AI at the base number 686 of UT I cDNA to recover a Sau3A EcoRI fragment.
  • # 7 / BbeI-ER [was digested with Aval and EcoRl at the base number 645 of UTI cDNA to recover the vector portion. The obtained DNA fragments and the synthesized DNA were ligated to obtain a clone pUTI-N in which the N-terminal coding region was modified (FIG. 10).
  • ⁇ / BbeI-ERI was digested with Pst [and Ecol? The DNA fragment containing the latter half of the DNA was recovered. This was digested with Hhal at base number 1089 of UT I cDNA to recover a Pstl-Hhal fragment.
  • # 7 / Bbeto ER I was digested with Pstl and Smal at base number 1146 of UTI cDNA to recover the vector portion. The obtained both DNA fragments and the synthetic DNA were ligated to obtain a clone ⁇ -C in which the C-terminal coding region was modified (FIG. 11).
  • pUTI-N was digested with Pstl to recover a DNA fragment between the PsU site derived from PUC19 and the Pstl site at the base number 877 of the UTI cDNA. This was ligated to the Pstl site of pUTI-C to create a clone pUTIN-C in which the N-terminal and C-terminal coding regions were modified (FIG. 12).
  • This plasmid was digested with Aor51HI-SmaI to obtain 3 ' UTI cDNA from which the poly A sequence of the translation region has been removed can be obtained.
  • PA0807N Japanese Unexamined Patent Publication No. 2-104290 (EP-A-0344459) which is an expression plasmid of yeast belonging to the genus Pichia has an EcoRI site in the closing site.
  • plasmid pUTIN-C carrying cassette UTI cDNA is digested with SmaI and treated with CIP (calf intestine alkaline phosphatase: Takara Shuzo Co., Ltd.). (Takara Shuzo Co., Ltd.), and the resulting cells were transformed into E. coli HB101 by a conventional method to produce plasmid PUT I NC / ER I (FIG. 13).
  • UTIcDNA from which the poly A sequence of the 3 'untranslated region has been removed can be obtained.
  • this DNA fragment has the 5 'end corresponding to the N-terminal of native UTI, and can be directly ligated to the SUC2 signal.
  • this DNA fragment has the Bori A sequence in the 3 ′ untranslated region removed.
  • a synthetic SUC2 signal gene pUTIN-C / ERI, prepared to have an EcoRI junction site at the 5 'end, was obtained by digestion with A0r51HI and EcoRI. After mixing and ligating the obtained DNA fragment of about 0.4 kb and the three DNA fragments of EcoRI digested pAO807N, the competent cell E.c01iHB1 01 was transformed. Plasmid DNA was prepared from the resulting transformant by a conventional method, treated with restriction enzymes and treated with AOX1 promoter and SUC2. The nucleotide sequence of the signal • UTI gene connecting region and the UTI gene • ⁇ 1 terminator one connecting region was confirmed, and the plasmid, which was an expression vector for Pichia yeast, was named ⁇ 310. Figure 14).
  • the obtained transformant was inoculated into 5 ml of YNB medium (0.7% yeast nitrogen base / o Amino Acids, 2% dextrose) and cultured at 30 ° C for 2 days. This was inoculated 10 times into 5 ml 2 x YP-23 ⁇ 4 glycerol medium (2% yeast extract, 4ept eptone, 2% glycerol), and cultured at 30 ° C for 2 days to grow the cells. This was centrifuged (2000 rpm, 5 min. At room temperature), and the obtained cells were treated with 5 ml of YP-2% methanol medium (1% Yeast Extract, 2% Peptone,
  • the cells were suspended in 2% methanol) and further cultured at 30 days for 5 days.
  • Knitz-type domain 1 is a polypeptide obtained by adding the N-terminal peptide of UTI consisting of 21 amino acids to the N-terminus of the original Knitz-type domain 1, that is, A It was expressed as polybeptide consisting of 1a1 to Arg77 (hereinafter referred to as N-terminal peptide-added Knitz type domain 1).
  • Each Knitz-type domain expression plasmid was prepared as follows. (1) Construction of expression plasmid
  • the synthetic DNA of the N-terminal coding region of Kunitz type domain 2 had a structure having a blunt end at the 5 ′ and an Ap I junction end at the 3 ′ to be linked to the SUC2 signal.
  • the restriction enzyme SphI site was created in the sequence without any amino acid change due to the single base substitution, C in base number 902 (see FIG. 6) of the UTI cDNA was changed to A. This newly provided SphI site is effective for re-linking Kunitz-type domain 1 and Kunitz-type domain 2 and modifying the N-terminal code region of Knitz-type domain 2 .
  • N-terminal peptide-added Kunitz-type domain 1 (N-terminal peptide-added Kunitz-type domain 1: base numbers 66 1 to 891 of UTI cDNA; see FIGS. 3 to 7) )
  • the pHH310 constructed in Example 1 (2) was digested with restriction enzymes EcoRI and PstI, and a fragment containing (SUC2 signal + Knitz type domain 1 structural gene with N-terminal peptide added) was cut out. .
  • This fragment was ligated to the EcoRI site of pUC19 together with the synthetic DNA corresponding to the C-terminal coding region of Kunitz-type domain 1 shown in FIG.
  • a plasmid pHH305 carrying the nitz-type domain 1 gene unit was obtained (Fig. 19 ).
  • the gene unit obtained by digesting this pHH305 with EcoRI was inserted into the EcoRI site of pA ⁇ 807N to create an N-terminal peptide-added Kunitz-type domain 1 expression plasmid pHH313 (Fig. 20).
  • the pUTIN-CZERI constructed in Example 1 (2) was digested with restriction enzymes ApaI and EcoRI to obtain a DNA fragment of the Kunitz type 2 domain gene.
  • This fragment and the synthetic DNA and synthetic SUC2 signal gene corresponding to the N-terminal coding region of Knitz-type domain 2 shown in Fig. 10 were ligated to the EcoRI site of pUC19, and secreted Kunitz A plasmid pHH306 carrying the type domain 2 gene unit (see FIG. 18) was obtained (FIG. 21).
  • the gene unit obtained by digesting this pHH306 with EcoRI was inserted into the EcoRI site of PAO807N to prepare a Kunitz type domain 2 expression plasmid pHH314 (FIG. 22).
  • the obtained transformant was inoculated into 5 ml of YNB medium and cultured at 30 ° C. for 2 days. This was inoculated in a 5 ml 2 YP-2P glycerol medium at 10%, cultured at 30 ° C. for 1 day, added with methanol to a final concentration of 2%, and further cultured at 30 at 3 days.
  • the culture solution was centrifuged (100 rpm, 5 minutes, 4 times), and the obtained culture supernatant was examined for its inhibitory activity against tribsine and human neutrophil elastase according to the methods described in Reference Examples 2 and 3.
  • the culture supernatant of the Kunitz-type domain 2 expression strain shows remarkable trypsin inhibitory activity and slightly It showed elastase inhibitory activity.
  • Each expression system created in the present invention is a prototype, and if further improvement is required in the future, the N-terminal peptide-added Knitz-type domain 1 C-terminal coding region was used to replace synthetic DNA.
  • the Pst I site can be used, and in the case of the Knitz-type domain 2, the same can be done using the ScaI site for the C-terminal code region and the newly provided SphI site for the N-terminal code region. it can.
  • modified DNA was used for pH H305 in which the Kunitz-type domain 1 gene was added as a cassette and pHH306 in which the Kunitz-type domain 2 gene was added as a cassette. It can be used to create a Pichia yeast expression system.
  • Example 3 UTI (hereinafter referred to as ntntact-rUTI) prepared by genetic recombination in Example 1 and Kunitz type domain 2 (hereinafter, r) of UTI prepared by genetic recombination in Example 2 D2) from Pichia yeast culture supernatant
  • I ntact - r UT I produce yeast culture supernatant, 1 0 k D cut of concentrated approximately 1 0-fold with an ultrafiltration membrane (F iltron OMEGA 10K MINISETTE 75 SQ FT), 50mM Tris, 20mM CaCl 2 containing Dialysis was performed using the solution (PH8.0).
  • the culture supernatant of the rD2 producing yeast was adjusted to pH 8.0 with ⁇ -NaOH after adding CaCl 2 to a final concentration of 20 m.
  • Urine-derived natural UTI was prepared according to the method described in JP-A-5-9200. Also, natural UT
  • I-derived domain 2 is reported by Hochstrasser et al. CHoppe-Seyler's Z. Physiol, chem.
  • Intact-rUTI and rD2 were subjected to SDS-PAGE according to the method of Laemmli et al.
  • the three clones of Intact-rUTI show similar migration patterns, and no difference was observed in migration patterns between reducing and non-reducing conditions. won. The same was true for the two clones of rD2.
  • Intact-r UTI showed a main band at 21.3 kD, a broad minor band around 43 kD and a sharp minor band at 19.4 kD.
  • a single band was shown at 7.31 (0), and the Kunitz type domain 2 derived from native UTI had the same migration position.
  • Fig. 23 shows the HPLC-GPC pattern of Intact-rUTI
  • Fig. 24 shows the HP LC-GPC pattern of rD2.
  • N-terminal amino acid sequence analysis was performed on the three bands observed in SDS-PAGE of the Intact-rUTI clone and the 7.3 kD band of rD2. Table 1 shows the results.
  • Ki values of purified Intact-rUTI and rD2 with respect to pepsin trypsin and human neutrophil elastase were measured.
  • the reaction buffer was a buffer (pH 8.0) consisting of 0.1 M Tris. LOm CaCh and 0.1% Triton X-100 (hereinafter referred to as buffer A), and the substrate was S-2222 (tryptic). S-2484 (manufactured by kabi) was used for neutrophil elastase.
  • the reaction conditions are as follows.
  • Ushitoripushin were diluted 20mM C a C 1 2, with 0.1% tri-ton X- 1 0 0-containing solution (pH 3. 0) 6. 3 X 1 0_ ⁇ ⁇ .
  • the UT I sample 8 0 // 1 buffer was varying concentrations diluted 1 0_ 8 to 1 0- 9 M in ⁇ addition, further buffer A 220 ju 1 or 3 1 0 ⁇ Added one. After reacting at 25 ° C for 10 minutes, 1201 or 30a1 of 5 mM dissolved in distilled water was added to each reaction solution, and the absorbance at 405 nm was measured over time.
  • Neutrophils Heras evening over Ze is, 20mM C a C l 2, 0. 1 OmM acetate buffer containing 1% Tri ton X- 1 0 0 5 x 1 0 in (pH 4. 5, hereinafter referred to as buffer B) It was diluted with an 8 M.
  • the diluted neutrophils Heras evening Ichize solution 20 1 a UT I sample 8 0 pi 1 diluted to various concentrations of 1 0 one 5 ⁇ 1 0- beta Micromax with buffer A was added, further buffer A 241 or 2901 was added. 25 of this mixture. Reaction for 10 minutes at C After addition, add 100- 21 or 50 ⁇ 1 of S-2484 dissolved in 25% DMS- ⁇ -distilled water at 4 mM to each reaction solution, and measure the absorbance at 405 nm over time. Was measured.
  • Table 2 shows the results obtained using the Dixon block
  • Table 3 shows the results obtained using the Easson-Stedman block.
  • a modified UTI in which a part of the amino acid sequence of Kunitz type domain 1 of UTI was modified was prepared.
  • the modification was performed by replacing the region containing the active site of Kunitz type domain 1 (Met-Gly-Met-Thr-Ser) with (Ile-Ala-Phe-Phe-Pro).
  • the substitution was performed by the site direct mutagenesis method using a U.S.E. mutagenesis kit (manufactured by Pharmacia). Except where noted, the operation was performed according to the protocol attached to the kit.
  • the Kunitz type domain 1 gene contained on PHH305 created in Example 2 is inserted in the direction opposite to the transcription direction of the lacZ gene of pUC19. Therefore, the mutagenic primer was designed to be on the same chain as the selection primer of the kit.
  • FIG. 25 shows the sequence of the mutage nic primer. The 5 'end of the prepared mutagenic primer was phosphorylated with T4 kinase, and site-directed mutagenesis was performed according to the kit's lip. Note that Sspl / Stul selection primer (manufactured by Pharmacia) was used as the selection primer. The nucleotide sequence of the obtained clone was confirmed, and the plasmid carrying the Kunitz type domain 1 gene into which the desired modification was introduced was designated pT-325.
  • PTK325 was digested with EcoRII and PstI to recover an N-terminal peptide-added modified Kunitz type domain 1 gene fragment lacking the 3 'region.
  • PHH306 was digested with Sphl and EcoRI to recover a Kunitz-type domain 2 gene fragment lacking the 5 'region.
  • a synthetic DNA corresponding to the region between the Sphl sites on Kunitz type domain 2 was prepared from the Pst 1 site on Kunitz type domain 1.
  • PTK332 carrying the modified UT [gene of interest (Epl-UTI gene).
  • Epl-U expression plasmid pHH334 was constructed by linking the secreted modified UTI gene unit obtained by digesting PTK332 with EcoRI to the EcoR site of the expression plasmid PA0807N (Fig. 26).
  • Epl-UTI expression plasmid PHH334 was digested with a restriction enzyme Stul, and Pichia yeast strain GTS115 was transformed in the same manner as in Examples 1 and 2.
  • the obtained transformant was inoculated into 5 ml YNB medium and cultured at 30 ° C. for 2 days. This is inoculated into 5 ml of 2 x YP-2% glycerol medium at 10%, cultured at 30 ° C for 2 days, then methanol is added to the medium to a final concentration of 4%, and further cultured at 30 ° C for 5 days did.
  • the culture supernatant obtained by centrifuging the culture solution was measured for trypsin inhibitory activity and human neutrophil elastase inhibitory activity according to the methods described in Reference Examples 2 and 3.
  • trypsin inhibitory activity while exhibiting remarkable trypsin inhibitory activity, the enhancement of human neutrophil elastase inhibitory activity was remarkable as compared with the unmodified [ntact-rUT] shown in Example 2.
  • the eluted fraction is collected as 5 OmM Tris, 2 OmM C a C It was dialyzed against a 12-containing solution (pH 8.0) and concentrated with Centri-Reb 10 to obtain a purified product.
  • the UTI titer of the purified product was 2160/1, and the recovery relative to the column loading titer was 50.1%.
  • the SDS electrophoresis pattern of the purified product showed a main band at 20.9 kD and a minor band at 17.1 kD.
  • 20.9 kD is a complete molecular form in terms of amino acid sequence, and 17.1 kD has 22 amino acids at the N-terminus deleted, and it is considered that sugar chains are added to all molecules.
  • Ep1-UTI enzymes trypsin (human, human), plasmin, and neutrophil elastase] were measured according to the following method.
  • the buffer used in the reaction system was Buffer A.
  • the substrate used was S-2222 (manufactured by Kabi) for trypsin, S-2484 (manufactured by Kabi) for neutrophil elastase, and S-2251 (No. 1 for plasmin). (Manufactured by Chemicals).
  • the reaction conditions are as follows.
  • Ushitoribushin is 20mM C a C 12, 0. 1 % Tri ton X- 1 0 0, with ⁇ ⁇ 3. 0 6. 3 x 1 0 - diluted 9 Micromax.
  • buffer solution ⁇ in 1 0_ 8 ⁇ 0- 8 M a UT I sample 8 0 ⁇ 1 made various concentrations diluted in addition, further buffer A plus 220 ⁇ 1 or 3 1 0/1 .
  • 1201 or 301-1 of S-2222 dissolved in distilled water to 5 mM was added to each reaction solution, and the absorbance at 405 nm was measured over time.
  • Human Toribushin were diluted 20 mM C a C 12, 0. 1% Tri ton X- 1 0 0-containing solution ( ⁇ 3. 0) at 1 X 1 0 _ 7 ⁇ .
  • a UT I sample 8 0 mu. 1 made various concentrations diluted in buffer ⁇ to 1 0_ 8 ⁇ 1 0- 8 M was added Further, Buffer A was added to 220 u ⁇ or 3101. These were treated in the same manner as in the case of cytrypsin, and the absorbance was measured.
  • Neutrophil elastase was diluted to 5 ⁇ 10 8 M in buffer B.
  • Plasmin was diluted to 8 ⁇ 10 8 M in buffer B.
  • S-2251 dissolved in distilled water at 5 mM or 2.5 mM was added to each reaction solution, and the absorbance at 405 nm was measured over time. .
  • Table 4 shows the Ki values of purified Ep1-1UTI together with the values of Intact-rUTI and native UTI similarly measured.
  • the values marked with * were obtained by the secondary plot of Lineweaver-Burk, and the other values were obtained by the Dixon plot.
  • Intact—rUT I and Ep 1—UT I had almost the same values, whereas neutrophil elastase, whose inhibitory activity is attributed to K unitz-type domain 1, had Knitz-type domain 1
  • the Ep 1 -UT I modified at the active center had an order of magnitude lower than that of the native UTI and a 4-order-one Ki value lower than that of Intact-rUT I, indicating an increase in inhibitory capacity.
  • Chloramine T (manufactured by Nacalai Tesque) solution which was serially diluted 3-fold up to 67 M, was added at 120/1, and allowed to react at room temperature for 10 minutes.Then, 10 OmM dissolved in water was added to each reaction solution. The reaction was stopped by adding L-methionine (Nacalai Tesque, Inc.) ⁇ B neutrophils Elastica dissolved in evening Ichize.
  • FIG. 27 shows the effects of Ep1-UTI oxidized by chloramine T and native UTI on human neutrophil elastase inhibition.
  • the residual inhibition rate in Fig. 27 is the inhibition rate of chloramine T non-oxidized inhibitor when the inhibitory rate is defined as the ratio of the decrease in the initial reaction rate when the inhibitor is added to the initial reaction rate when the inhibitor is not added ().
  • the ratio (%) of the inhibition rate of the chloramine T oxidation inhibitor with respect to is shown.
  • FIG. 27 when the concentration of the reacted chloramine T was increased, neutrophil elastase inhibitory activity was reduced in the urinary native UTI due to chloramine T oxidation, whereas Ep1-1UT In I, the inhibitory activity did not decrease.
  • Ep 1—UTI is a methionine at position 36 from the N-terminus of urine-derived natural UTI (see Figure 5) and is position 38 from the N-terminus of urine-derived natural UTI (see Figure 5).
  • the methionine is replaced by phenylalanine, and this substitution is expected to improve oxidation resistance.
  • Chloramine T is known to oxidize methionine to methionine sulfoxide in neutral or weakly acidic conditions, but as shown in Figure 27, the neutrophil elastase inhibitory activity of Ep1-UTI It was confirmed that compared to UTI, it was not affected by chloramine T oxidation. It is known that neutrophils release active oxygen together with elastase in the field of inflammation, but EP 1 -UTI is expected to work stably even in such a field. You.
  • Example 5 Preparation of N-terminal 21 amino acid deletion modified UTI (hereinafter referred to as Ep 1 -d 21) and its Pichia yeast expression system
  • PTK332 obtained in Example 4 was digested with Ec052I and EcoRI, and added to the N-terminal side of the EcoRI fragment of the vector DNA and the Knitz-type domain 1. An Eco 52 I-EcoRI fragment of the UTI gene lacking the 5 'terminal region encoding the amino acid region was recovered.
  • the synthetic DNA shown in FIG. 15 was used for the SUC2 signal.
  • the region from the 5 'end which encodes the 21 amino acid region added to the N-terminal side of the Knitz-type domain 1 to the Ec052 I site, originally contains the corresponding synthetic DNA. It was prepared by eliminating the vull site ( Figure 28). By linking these four members, a plasmid pHH336 carrying the Ep1-d21 gene was prepared (FIG. 29).
  • Ep1-d21 gene obtained by digesting pHH336 obtained in (1) with EcoRI was ligated to the EcoRI site of PAO 807 N, and the expression plasmid PHH339 was ligated. Created ( Figure 30).
  • Ep1-d21 expression plasmid pHH339 was digested with restriction enzyme StuI, and Pichia yeast strain GTS115 was transformed in the same manner as in Examples 1 and 2.
  • the obtained transformant was inoculated into 5mlYNB medium and cultured for 2 days at 30 e C. This 2 days after culture in 10% inoculated 30 e C to 2xYP-2% glycerol medium of 5 m 1, methanol was added to the medium to a final concentration of 4%, and further cultured for 5 days at 30 e C .
  • the obtained culture supernatant showed remarkable human neutrophil elastase inhibitory activity as compared with the unmodified Intact-rUTI obtained in Example 1.
  • the UTI titer of the culture supernatant after concentration was 23 uZm 1.
  • the eluted fraction was dialyzed against distilled water and concentrated with Centriprep 10 to obtain a purified product.
  • the UTI titer of the purified product was 244 4 / m1.
  • the SDS electrophoresis pattern of the purified product showed a main band at 17.7 kD. It was considered that a sugar chain was added.
  • Ki values of various purified Ep 1 -d 21 enzymes [trypsin (pishi, human), human plasmin, and human neutrophil elastase] were determined.
  • the Ki value was determined according to the following method.
  • Citribulin contains 2 OmM Ca C 12, 0.1% Triton X—100 Diluted 9 M - 6. 3 x 1 0 with a solution (pH 3. 0). To the Ushitoripushin liquid 2 0 1 Buffer E p 1 and various concentrations diluted 1 0 one 8 ⁇ 1 0- beta Micromax in A - d 2 1 sample 8 0 1 was added, further buffer A 2 2 0 1 Or 3 1 0 ⁇ 1 added. After reacting at 25 for 10 minutes, add S-222, dissolved in distilled water to 5 mM, to each reaction solution, add 121 or 30/1, and measure the absorbance at 405 nm over time. It was measured.
  • Human Toribushin is 2 0 mM C a C 1 2 , 1 X 1 0 with 0.1% tri-ton X- 1 0 0-containing solution (pH 3. 0) - were diluted into 7 M.
  • Neutrophils Heras evening Ichize was diluted to 5 XI CT e M with buffer B.
  • a medium favorable diluted elastase solution 2 0 [/ 1, buffer 1 0 5-1 0 A - diluted to various concentrations in 9 M E pl - d 2 1 sample 8 0 1 was added, Buffer A was added to 240 41 or 2901. After reacting at 25 for 10 minutes, add 100 / zl or 50/1 of S-2484 dissolved in 4 mM 25% DMS-Z distilled water to each reaction solution, and add 4 The absorbance at 05 nm was measured over time.
  • Plasmin was diluted to 8 ⁇ 10-8 M in buffer B.
  • the Burasumin liquid 4 0 ⁇ 1 diluted buffer A at 1 (T 5 ⁇ l 0_ 8 E p 1 was diluted to various concentrations Micromax - d 2 1 test body 8 0 1 was added, further buffer A 1 8 After adding 10 minutes at 25, the reaction mixture was added with S-2251 dissolved in distilled water at 5 mM or 2.5 mM, and added to each reaction solution. The absorbance at nm was measured over time.
  • Table 5 shows the Ki values of purified Ep 1 -d 21 for various enzymes [trypsin (human, human), blasmin, and human neutrophil elastase] measured under the above measurement conditions. The values are shown together with the Ki values of Intact-rUTI, natural UTI and Ep1-UTI.
  • the values marked * 1 were determined by Easson-Stedman plots, the values marked * 2 were determined by the Lineweaver-Burk secondary plot, and the other values were determined by Dixon plots.
  • Enzyme inhibitor constant ( ⁇ i: ⁇ )
  • the Ki value of UTI (Intact-rUTEpl—iUTI, Ep1-d21) created by genetic recombination was the same as that of native UT. I ranged from 1.6 to 1.6 times that of I, but with regard to neutrophil elastase inhibition, Ep 1— 1/25 of UTI in native UTI and 1 in Ep1-d21 in Ep1-d21. The Ki value was remarkably reduced to 39, and a clear increase in inhibitory capacity was observed.
  • Ep 1-d21 and natural UTI each diluted to 5 M with 5 OmM phosphoric acid buffer solution (pH 7.0) and natural UTI each 25], and the same buffer continuously from 2 OmM to 1.28 M 5 Chloramin T (manufactured by Nacalai Tesque, Inc.) was added in a concentration of 25 ⁇ 1 and reacted at room temperature for 20 minutes. Then, after dilution 9. 4 X 1 0- 8 M in each reaction to 20 Omm of L one Mechionin aqueous 50 i 1 added to stop the reaction buffer A, to the 40 1, buffer A110 u ⁇ , and buffer B at 2.
  • Table 6 shows the treatment concentrations of chloramine T in which human neutrophil elastase activity remains at 80% or more in Ep1-d21 and native UTI. Natural UTIs are lost at relatively low concentrations.
  • Ep 1—d21 uses low concentrations of chloramin T, which inactivates natural UTIs as well as Ep1-1 UTIs. Oxidation did not deactivate at all, and an apparent increase in oxidation resistance was observed.
  • the culture supernatant of the Ep1-UTI expression strain obtained in Example 4 (4) and the culture supernatant of the Ep1-d21 expression strain obtained in Example 5 (3) were The expression level of UTI was measured by a sandwich ELISA using an anti-rUTI antibody (Reference Example 1).
  • Germogenic enzyme VECTOR LA BORATOR IES
  • peroxydase color-developing kit 0 Sumitomo Bei-Client
  • Example 6 Ep1-UTI having the N-terminal peptide of UTI consisting of 21 amino acids was expressed in a larger amount by genetic engineering compared to Ep1-d21 not having it. It was suggested that To confirm this, flask cultivation (high-density flask cultivation) was performed with increased bacterial cell density during methanol induction, and the expression levels were compared.
  • Example 4 (4) obtained in Ep 1-UT I expressing strain and Examples 5 (3) E obtained in p 1 - a d 21 expression strain was inoculated into ⁇ medium 2m 1 30 e C For two days. This was inoculated into 100 ml of 3 XYP—2% glycerol medium (3% yeast extract, 6% eptone, 2% glycerol) in 1 ml portions, and cultured at 30 ° C for 2 days. The turbidity at O nm (OD ⁇ o.) was measured.
  • each OD eo After harvesting from the culture, each OD eo .
  • the suspension was suspended in 2XYP-4% methanol medium (2% Yeast Extract. 43 ⁇ 4 Peptone, 43 ⁇ 4 methanol) so that the value was about 250. Place 40ml of these in a 300ml Erlenmeyer flask with baffle.
  • the cells were cultured at 150 rpm for 72 hours at C. The turbidity was measured every 24 hours, and the culture supernatant for measuring the expression level was collected and stored frozen in TC (TC).
  • TC TC
  • ODeoo ODgoo expression level ODeoo expression level ODfioo expression level Modified UTI (mg / L) (mg / L) (mg / L)
  • Pichia yeast as a host in an expression system is that the density of cells can be increased under optimally controlled culture conditions.
  • the E1-UTI expression strain having a 21 amino acid UTI N-terminal peptide was transformed into an Ep1-d21 expression strain without it.
  • the remarkably superior expression level suggests that the expression level can be further increased by examining the culture conditions in a controllable culture tank.
  • the heron anti-blood immunized with natural UTI was subjected to ammonium sulfate decay, and Ig fractions were collected. This is adsorbed to an affinity column in which natural UTI is immobilized on FMP-activated Cell mouth Fine (manufactured by Seikagaku Kogyo Co., Ltd.). After washing, the column was eluted with 0.1 M glycine hydrochloride (pH 2.5). In addition, if necessary, biotinylation was performed according to a conventional method.
  • the reaction was stopped by mixing 1 ml of 20% acetic acid with the reaction solution, and the absorbance at 405 nm was measured.
  • UTI was diluted with the above reaction buffer, and tribcine was diluted with 20 mM CaCl 2 , 1.5 mg / ml Gelatin, pH 3.0.

Abstract

L'invention concerne un procédé de production d'un inhibiteur humain de la trypsine urinaire et des domaines de celui-ci en utilisant une levure du genre Pichia comme cellule hôte; un système d'expression associé; un nouveau polypeptide ayant au moins la séquence des acides aminés représentée par la formule I de la description; un ADN codant pour cette séquence; et un vecteur d'expression contenant cet ADN; une cellule transformée contenant ce vecteur; et un procédé pour produire le polypeptide. Le procédé de l'invention permet de produire l'inhibiteur humain de la trypsine urinaire et des domaines de celui-ci à grande échelle et d'une manière efficace. L'utilisation du système d'expression facilite la préparation de variantes ou de dérivés de l'inhibiteur humain de la trypsine urinaire et permet de mettre au point de nouveaux médicaments du type inhibiteurs de protéases. L'invention concerne également un nouveau polypeptide qui est meilleur comme inhibiteur de l'élastase que l'inhibiteur humain de la trypsine urinaire naturel, et qui peut être produit à grande échelle.
PCT/JP1995/001449 1994-07-21 1995-07-21 Procede de production d'un inhibiteur de la trypsine urinaire et des domaines de celui-ci, nouveau polypeptide associe et procede de production de ce polypeptide WO1996003503A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0965597A4 (fr) * 1996-12-27 2003-01-08 Mochida Pharm Co Ltd Medicaments diriges vers la membrane cellulaire
US6583108B1 (en) 1996-03-11 2003-06-24 Bayer Corporation Human bikunin
US11725043B2 (en) 2020-03-05 2023-08-15 DiaMedica USA Inc. Ulinastatin polypeptides

Citations (3)

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Publication number Priority date Publication date Assignee Title
JPS6344899A (ja) * 1986-08-12 1988-02-25 リサーチ・コーポレーション・テクノロジーズ・インコーポレーテッド 酵母による分泌型タンパク質の生産方法
JPH0584083A (ja) * 1990-11-13 1993-04-06 Mochida Pharmaceut Co Ltd 新規ポリペプチド、それをコードする新規ｄｎａ、新規ポリペプチドの製造方法、新規医薬組成物、および新規酵素阻害方法
JPH06315386A (ja) * 1993-05-01 1994-11-15 Mochida Pharmaceut Co Ltd Ｄｎａ断片およびそれを含むベクター、該ベクターによって形質転換された形質転換体、該ベクターを用いる蛋白質の産生方法

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JPS6344899A (ja) * 1986-08-12 1988-02-25 リサーチ・コーポレーション・テクノロジーズ・インコーポレーテッド 酵母による分泌型タンパク質の生産方法
JPH0584083A (ja) * 1990-11-13 1993-04-06 Mochida Pharmaceut Co Ltd 新規ポリペプチド、それをコードする新規ｄｎａ、新規ポリペプチドの製造方法、新規医薬組成物、および新規酵素阻害方法
JPH06315386A (ja) * 1993-05-01 1994-11-15 Mochida Pharmaceut Co Ltd Ｄｎａ断片およびそれを含むベクター、該ベクターによって形質転換された形質転換体、該ベクターを用いる蛋白質の産生方法

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Title
NUCLEIC ACIDS RESEARCH, 1986, Vol. 14, No. 20, KAUMEYER J.F., "The mRNA for a Proteinase Inhibitor Related to the HI-30 Domain of Inter-alpha-Trypsin Inhibitor Also Encodes alpha-1-Microglobulin (Protein HC)", pages 7839-7850. *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6583108B1 (en) 1996-03-11 2003-06-24 Bayer Corporation Human bikunin
US7019123B2 (en) 1996-03-11 2006-03-28 Bayer Corporation Human bikunin
US7452859B2 (en) 1996-03-11 2008-11-18 Aerovance, Inc. Human bikunin
EP0965597A4 (fr) * 1996-12-27 2003-01-08 Mochida Pharm Co Ltd Medicaments diriges vers la membrane cellulaire
US11725043B2 (en) 2020-03-05 2023-08-15 DiaMedica USA Inc. Ulinastatin polypeptides

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