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WO2005023868A1 - Serine protease, et nucleotides codant cette serine protease - Google Patents

Serine protease, et nucleotides codant cette serine protease Download PDF

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Publication number
WO2005023868A1
WO2005023868A1 PCT/CN2003/000756 CN0300756W WO2005023868A1 WO 2005023868 A1 WO2005023868 A1 WO 2005023868A1 CN 0300756 W CN0300756 W CN 0300756W WO 2005023868 A1 WO2005023868 A1 WO 2005023868A1
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WO
WIPO (PCT)
Prior art keywords
protein
amino acid
seq
sequence
polynucleotide
Prior art date
Application number
PCT/CN2003/000756
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English (en)
Chinese (zh)
Inventor
Yuancong Zhou
Yunlong Hu
Qian Jin
Hong Zhu
Yiqing Wang
Wenhua Huang
Junjie Gu
Original Assignee
Shanghai Chuangeng Bio-Tech. Co., Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shanghai Chuangeng Bio-Tech. Co., Ltd. filed Critical Shanghai Chuangeng Bio-Tech. Co., Ltd.
Priority to PCT/CN2003/000756 priority Critical patent/WO2005023868A1/fr
Priority to AU2003264312A priority patent/AU2003264312A1/en
Publication of WO2005023868A1 publication Critical patent/WO2005023868A1/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)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482—Serine endopeptidases (3.4.21)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention belongs to the field of biotechnology and medicine. Specifically, the present invention relates to a novel serine protease (SC protein for short) with antitumor function and a polynucleotide encoding an SC protein. The invention also relates to a method for preparing and using the polynucleotide and protein, and a pharmaceutical composition containing the SC protein. Background technique
  • Serine proteolytic enzyme also known as serine protease is one of the proteolytic enzymes, and its main function is to hydrolyze proteins.
  • serine proteolytic enzymes There are many types of serine proteolytic enzymes that have been discovered so far. However, as one of the proteolytic enzymes, serine proteolytic enzymes have various uses, such as hydrolyzing protease activity, and some serine proteolytic enzymes also have BAEE activity and NGF. Activity, as well as tumor suppressive activity.
  • the object of the present invention is to provide a new SC protein and its fragments, analogs and derivatives.
  • Another object of the invention is to provide polynucleotides encoding these proteins.
  • Another object of the present invention is to provide a method for producing these proteins and uses of the protein and coding sequences.
  • an isolated SC protein comprising: a protein having the amino acid sequence of SEQ ID NO: 2, or a conservatively mutated protein thereof, or an active fragment thereof, or an active derivative thereof.
  • the protein is selected from the group consisting of:
  • the amino acid sequence of SEQ ID NO: 2 is formed by substitution, deletion or addition of one or more (such as 1-10, preferably 1-8) amino acid residues, and has a serine proteolytic function Polypeptides derived from (a). Is a protein having the amino acid sequence of SEQ H NO: 2.
  • an isolated polynucleotide which encodes the SC protein described above.
  • the polynucleotide polynucleotide encodes a protein having the amino acid sequence shown in SEQ ID NO: 2. More preferably, the polynucleotide contains the sequence from positions 1 to 723 in SEQ ID NO: 1.
  • a vector which contains the above-mentioned polynucleotide encoding an SC protein. And a host cell transformed or transduced by the vector or a host cell directly transformed or transduced by the above polynucleotide
  • a method for preparing a protein includes steps:
  • Figure 2 shows the gene sequence of the SC protein and its encoded protein sequences (SEQ ID NOs: 1 and 2).
  • Figure 3 shows the construction of the eukaryotic expression vector yeast GS115 expressing the SC protein. detailed description
  • isolated refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment).
  • polynucleotides and proteins in the natural state in a living cell are not separated and purified, but the same polynucleotides or proteins such as from the natural state and other substances present In the separation, it is separation and purification.
  • an isolated SC polypeptide protein is substantially free of other proteins, lipids, carbohydrates, or other substances with which it is naturally associated. Those skilled in the art can use standard protein purification techniques (especially
  • the protein of the present invention may be a recombinant protein, a natural protein, or a synthetic protein, and preferably a recombinant protein.
  • the protein of the present invention may be a naturally purified product, or a chemically synthesized product, or it may be produced from a prokaryotic or eukaryotic host (for example, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant technology.
  • a prokaryotic or eukaryotic host for example, bacteria, yeast, higher plants, insects, and mammalian cells
  • the protein of the invention may be glycosylated, or it may be non-glycosylated.
  • the proteins of the invention may also include or exclude initial methionine residues.
  • the invention also includes fragments, derivatives and analogs of SC proteins.
  • fragment refers to a protein that retains substantially the same biological function or activity as the natural SC protein of the invention.
  • a protein fragment, derivative or analog of the present invention may be (i) a protein having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a protein with a substituent group in one or more amino acid residues, or (iii) a mature protein with another compound (such as a compound that extends the half-life of a protein, such as Polyethylene glycol), or (iv) a protein formed by fusing additional amino acid sequences to this protein sequence (e.g., a leader sequence or a secreted sequence or a sequence or zymogen sequence used to purify this protein, or Formation of a fusion protein with an
  • the invention also provides analogs of SC proteins.
  • the difference between these analogs and the natural SC protein may be a difference in the amino acid sequence, a difference in the modified form that does not affect the sequence, or both.
  • These proteins include natural or induced genetic variants. Induced variants can be obtained by a variety of techniques, such as random mutagenesis by radiation or exposure to mutagens, or by site-directed mutagenesis or other known molecular biology techniques.
  • Analogs also include analogs with residues different from natural L-amino acids (e.g., D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (e.g., 3, Y-amino acids). It should be understood that the protein of the present invention is not limited to the representative protein exemplified above.
  • Modified (usually unchanged primary structure) forms include: Chemically derived forms of proteins in vivo or in vitro such as acetylated or carboxylated. Modifications also include glycosylation, such as those produced by glycosylation modification during protein synthesis and processing or further processing steps. This modification can be accomplished by exposing the protein to an enzyme that undergoes glycosylation, such as mammalian glycosylation or deglycosylation. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are proteins that have been modified to improve their proteolytic properties or to optimize their lytic properties.
  • glycosylation such as those produced by glycosylation modification during protein synthesis and processing or further processing steps. This modification can be accomplished by exposing the protein to an enzyme that undergoes glycosylation, such as mammalian glycosylation or deglycosylation. Modified forms also include sequences having phosphorylated amino
  • SC conservatively mutated protein means that there are up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 compared to the amino acid sequence of SEQ ID NO: 2. Amino acids are replaced by similar or similar amino acid residues to form proteins. These conservatively mutated proteins are best produced by amino acid substitutions according to Table 1. Table 1
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA, or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be coding or non-coding.
  • the coding region sequence encoding the mature protein may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant.
  • degenerate variant refers in the present invention to a nucleic acid sequence that encodes a protein having SEQ ID NO: 2, but which differs from the coding region sequence shown in SEQ ID NO: 1.
  • polynucleotide encoding a protein may include a polynucleotide encoding the protein, and may also include a polynucleotide encoding additional coding and / or non-coding sequences.
  • the present invention also relates to a polynucleotide that hybridizes to the sequence described above and has at least '50%, 'preferably at least' 70%, and more preferably at least 80% homology between the two sequences.
  • the invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the invention under stringent conditions.
  • “strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 X SSC, 0.1% SDS, 60 ° C ; or (2) during hybridization Added denaturing agents, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42 ° C, etc .; or (3) the homology between only two sequences is at least 90% Above, it is more preferable that the hybridization occurs at 95% or more.
  • the protein encoded by the hybridizable polynucleotide has the same biological function and activity as the mature protein shown in SEQ ID NO: 2.
  • nucleic acid fragment that hybridizes to the sequence described above.
  • a "nucleic acid fragment” contains at least 15 nucleotides in length, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides.
  • Nucleic acid fragments can be used in nucleic acid amplification techniques, such as PCR, to identify and / or isolate polynucleotides encoding SC proteins.
  • the protein and polynucleotide in the present invention are preferably provided in an isolated form, and are more preferably purified to homogeneity.
  • the full-length SC nucleotide sequence or a fragment thereof of the present invention can usually be obtained by a PCR amplification method, a recombinant method, or a synthetic method.
  • primers can be designed according to the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequences, and a commercially available cDNA library or cDNA prepared according to conventional methods known to those skilled in the art
  • the library is used as a template and amplified to obtain the relevant sequence.
  • Relevant sequences can also be obtained directly by RT-PCR. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then stitch the amplified fragments together in the correct order.
  • a DNA sequence encoding a protein (or a fragment thereof, or a derivative thereof) of the present invention can be obtained completely through chemical synthesis.
  • This DNA sequence can then be introduced into a variety of existing DNA molecules (or such as vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequence of the invention by chemical synthesis. -.
  • a method for amplifying DNA / RNA using PCR technology is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-cDNA terminal rapid amplification method
  • the primers for PCR may be appropriately selected based on the sequence information of the present invention disclosed herein
  • the amplified DNA / RNA fragments can be separated and purified by conventional methods such as by gel electrophoresis.
  • the present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell genetically engineered using the vector of the present invention or the 'SC protein coding sequence, and a method for producing the protein of the present invention by recombinant technology.
  • polynucleotide sequence of the present invention can be used to express or produce recombinant SC proteins. Generally there are the following steps:
  • the SC protein polynucleotide sequence can be inserted into a recombinant expression vector.
  • group g expression vector refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art.
  • Vectors suitable for use in the present invention include, but are not limited to: T7-based expression vectors expressed in bacteria; pMSXND expressed in mammalian cells Expression vectors and baculovirus-derived vectors expressed in insect cells. In short, any plasmid and vector can be used as long as it can be replicated and stabilized in the host.
  • An important feature of expression vectors is that they usually contain origins of replication, promoters, marker genes and translation control elements.
  • Methods known to those skilled in the art can be used to construct expression vectors containing SC protein-encoding DNA sequences and appropriate transcriptional / translational control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombinant technology.
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to guide niRNA synthesis. Representative examples of these promoters are: E.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. .
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • GFP fluorescent protein
  • tetracycline or ampicillin resistance for E. coli.
  • the obtained transformants can be cultured by a conventional method to express the protein encoded by the gene of the present invention.
  • the medium used in the culture may be selected from various conventional mediums. Host cell Cultivate under growing conditions. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • the present invention also provides a pharmaceutical composition containing a safe and effective amount of the SC protein of the present invention and a pharmaceutically acceptable carrier or excipient.
  • Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by using a normal saline solution or an aqueous solution containing glucose and other adjuvants by a conventional method.
  • Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods.
  • Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions.
  • the active ingredient is administered in a therapeutically effective amount, for example, from about 0.1 ng / kg body weight to about 10 mg / kg body weight per day.
  • the protein of the present invention can be used with other therapeutic agents.
  • the downstream primers were: 5 'TTTCTCCTCT T (G / A) A (G / C) (C / T) (A / T) TTTCAAA 3 * (SEQ ID NO: 4).
  • the primer synthesis was entrusted to Shanghai Biotech Bioengineering Company.
  • T-carrier carrier pBluescript- SK (purchased from Stratagene) lg cut with EcoRI, equal volume phenol: chloroform treatment, ethanol precipitation, dissolved in 50 ⁇ 1 system, containing lOxPCR buffer 5 ⁇ , 100m mol / L dNTP2 L, Taq enzyme 1 unit. Incubate at 70 ° C for 2h. Treat with equal volume of phenol: chloroform. After ethanol precipitation, dissolve in 10 ⁇ l sterilized water.
  • carrier pBluescript- SK purchased from Stratagene
  • the sequence of the protein encoded by the SC protein cDNA shares 54% homology with another protein encoded by Ziffl / briciAs bimastus. Therefore, this is a new unreported SC protein gene whose molecular weight is 29.375KD as determined by mass spectrometry.

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Abstract

L'invention concerne une protéine antinéoplasique (SC), l'acide nucléique qui la code, et un procédé relatif à la production de la protéine en question. L'invention concerne également des procédés relatifs à l'utilisation de cette construction à acide nucléique pour le traitement de maladies (tumeurs, etc.). L'invention concerne en outre une composition pharmaceutique renfermant ladite protéine SC.
PCT/CN2003/000756 2003-09-05 2003-09-05 Serine protease, et nucleotides codant cette serine protease WO2005023868A1 (fr)

Priority Applications (2)

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PCT/CN2003/000756 WO2005023868A1 (fr) 2003-09-05 2003-09-05 Serine protease, et nucleotides codant cette serine protease
AU2003264312A AU2003264312A1 (en) 2003-09-05 2003-09-05 Serine protease and polynucleotides which encode the serine protease

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PCT/CN2003/000756 WO2005023868A1 (fr) 2003-09-05 2003-09-05 Serine protease, et nucleotides codant cette serine protease

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1135358A (zh) * 1994-12-26 1996-11-13 东菱药品工业株式会社 治疗再狭窄和动脉硬化的药物
EP0814164A2 (fr) * 1996-06-21 1997-12-29 Mogam Biotechnology Research Institute Nouvel ADNc de sérine protéase, ayant une action fibrinolytique directe
CN1322834A (zh) * 2000-05-09 2001-11-21 上海博德基因开发有限公司 一种新的多肽——人atp依赖的丝氨酸蛋白水解酶9.2和编码这种多肽的多核苷酸
WO2001087346A2 (fr) * 2000-05-17 2001-11-22 Toximed Gmbh Cellules dendritiques chargees de substances toxiques

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1135358A (zh) * 1994-12-26 1996-11-13 东菱药品工业株式会社 治疗再狭窄和动脉硬化的药物
EP0814164A2 (fr) * 1996-06-21 1997-12-29 Mogam Biotechnology Research Institute Nouvel ADNc de sérine protéase, ayant une action fibrinolytique directe
CN1322834A (zh) * 2000-05-09 2001-11-21 上海博德基因开发有限公司 一种新的多肽——人atp依赖的丝氨酸蛋白水解酶9.2和编码这种多肽的多核苷酸
WO2001087346A2 (fr) * 2000-05-17 2001-11-22 Toximed Gmbh Cellules dendritiques chargees de substances toxiques

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