WO2022143186A1 - 一种制备丝氨酸蛋白酶的方法 - Google Patents

一种制备丝氨酸蛋白酶的方法 Download PDF

Info

Publication number
WO2022143186A1
WO2022143186A1 PCT/CN2021/138653 CN2021138653W WO2022143186A1 WO 2022143186 A1 WO2022143186 A1 WO 2022143186A1 CN 2021138653 W CN2021138653 W CN 2021138653W WO 2022143186 A1 WO2022143186 A1 WO 2022143186A1
Authority
WO
WIPO (PCT)
Prior art keywords
serine protease
inhibitor
expression vector
plasminogen activator
seq
Prior art date
Application number
PCT/CN2021/138653
Other languages
English (en)
French (fr)
Inventor
王丁力
Original Assignee
佛山汉腾生物科技有限公司
广州汉腾生物科技有限公司
佛山普津生物技术有限公司
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 佛山汉腾生物科技有限公司, 广州汉腾生物科技有限公司, 佛山普津生物技术有限公司 filed Critical 佛山汉腾生物科技有限公司
Publication of WO2022143186A1 publication Critical patent/WO2022143186A1/zh

Links

Images

Classifications

    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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
    • 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
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • 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
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8114—Kunitz type inhibitors
    • C07K14/8117—Bovine/basic pancreatic trypsin inhibitor (BPTI, aprotinin)
    • 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
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8121—Serpins
    • 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
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8121—Serpins
    • C07K14/8125—Alpha-1-antitrypsin
    • 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
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8121—Serpins
    • C07K14/8132—Plasminogen activator inhibitors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6456—Plasminogen activators
    • C12N9/6459—Plasminogen activators t-plasminogen activator (3.4.21.68), i.e. tPA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21068—Tissue plasminogen activator (3.4.21.68), i.e. tPA
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Definitions

  • the invention relates to the field of molecular biology, in particular to a method for preparing serine protease.
  • Serine proteases are enzymes that cleave peptide bonds in proteins with serine as the nucleophilic amino acid in the active site of the enzyme. They are ubiquitous in eukaryotes and prokaryotes. Serine proteases can be divided into two broad categories according to their structure: trypsin-like (chymotrypsin-like) or subtilisin-like proteases. Among them trypsin-like serine proteases cleave peptide bonds after positively charged amino acids (lysine or arginine). This specificity is determined by residues (usually negatively charged aspartic or glutamic acids) located at the bottom of the enzyme's substrate-binding pocket.
  • Trypsin-like serine proteases play an important role in maintaining the homeostasis of organisms, such as the control of blood coagulation, fibrinolysis, kinase-kallikrein, and the complement system. Under normal circumstances, the body's own endogenous protease inhibitors can protect the potential damage caused by the protease. If the concentration of protease inhibitors decreases, the balance between protease and anti-protease is destroyed. Activation of excess proteases can lead to the development of various diseases, such as pancreatitis or disseminated intravascular coagulation. Trypsin-like serine proteases can activate various diseases, and their inhibitors are usually effective drugs for the treatment or prevention of thrombosis, inflammatory diseases, autoimmunity and related diseases.
  • Mammalian cells are commonly used to express recombinant proteins in bioengineering, and some trypsin-like serine proteases (such as various coagulation factors and thrombolytic enzymes) used for clinical treatment often have the problem of low expression in mammalian cells, which increases the The production cost of the drug.
  • trypsin-like serine proteases such as various coagulation factors and thrombolytic enzymes
  • the reason for this is that the activity of the overexpressed trypsin-like serine protease causes cytotoxicity. Even protease precursors with low or inactive expression may be partially or slightly activated in advance during the expression process. When the cytotoxicity exceeds a certain limit, it will cause a certain screening of the cell population expressing trypsin-like serine proteases. Pressure, resulting in the final screened cell lines are almost all low expression. Even if there are occasional high-expressing cell lines, they cannot exist stably for a long time and are difficult to use for large-scale commercial production.
  • the present invention provides expression vectors that co-express a serine protease and a serine protease inhibitor.
  • the serine protease is a trypsin-like serine protease, or may be referred to as a trypsin-like protease.
  • the expression vector is a constitutive expression vector.
  • the expression vector is expressed without induction of an inducer.
  • the present invention adopts the constitutive expression vector to co-express the serine protease and the serine protease inhibitor, which can realize the constitutive expression of the target protein without induction, thereby avoiding the inhibition of cell growth or metabolism caused by the addition of an additional inducer, thereby affecting the post-translation of the protein retouch.
  • the expression vector provided by the present invention has high safety and is more conducive to subsequent purification of the target protein and then use as a medicine.
  • the expression vector is a pXC17.4 vector, a pXC18.4 vector, or a ligation vector of pXC17.4 and pXC18.4.
  • the serine protease is a tissue plasminogen activator (tPA).
  • tPA tissue plasminogen activator
  • the serine protease has the amino acid sequence set forth in SEQ ID NO:1.
  • the serpin is selected from the group consisting of alpha-antitrypsin, bovine trypsin inhibitor, plasminogen activator inhibitor-1, plasminogen activator inhibitor-2, plasminogen activator inhibitor At least one of zymogen activator inhibitor-3, neuro-derived serine protease inhibitor and glial cell-derived connexin.
  • the serpin is selected from the group consisting of alpha-antitrypsin, bovine trypsin inhibitor, plasminogen activator inhibitor-2, plasminogen activator inhibitor-3, and neurogenic at least one of the sex serine protease inhibitors.
  • the serpin is selected from the group consisting of bovine trypsin inhibitor, plasminogen activator inhibitor-2, plasminogen activator inhibitor-3, and neuroderived serpin at least one of.
  • the serine protease inhibitor is selected from at least one of bovine trypsin inhibitor and neuroderived serine protease inhibitor.
  • the alpha-antitrypsin has the amino acid sequence set forth in SEQ ID NO:2.
  • the bovine trypsin inhibitor has the amino acid sequence set forth in SEQ ID NO:3.
  • the plasminogen activator inhibitor-1 has the amino acid sequence shown in SEQ ID NO:4.
  • the plasminogen activator inhibitor-2 has the amino acid sequence shown in SEQ ID NO:5.
  • the plasminogen activator inhibitor-3 has the amino acid sequence shown in SEQ ID NO:6.
  • the neuroderived serine protease inhibitor has the amino acid sequence set forth in SEQ ID NO:7.
  • the glial cell-derived connexin has the amino acid sequence set forth in SEQ ID NO:8.
  • the expression vector for co-expressing a serine protease and a serine protease inhibitor is prepared by a method comprising the steps of: inserting an expression vector encoding a nucleotide sequence encoding the serine protease (tPA) and inserting an expression vector encoding a serine protease (tPA)
  • the expression vector of the nucleotide sequence of the serine protease inhibitor (SPI) is connected to form a double expression cassette expression vector.
  • the expression vector for co-expressing serine protease and serine protease inhibitor is prepared by a method comprising the following steps: inserting the nucleotide sequence encoding the tPA into the pXC17.4 expression vector to obtain pXC17.4. 4-tPA; insert the nucleotide sequence encoding the SPI into the pXC18.4 expression vector to obtain pXC18.4-SPI; the pXC17.4-tPA and pXC18.4-SPI are digested with NotI and PvuI, Ligated into a double expression cassette expression vector pXC-tPA-SPI expressing both tPA and SPI.
  • the present invention provides a host cell into which the expression vector is exogenously transferred.
  • the host cells are mammalian cells, such as cells from mammals such as rats, mice, hamsters, guinea pigs, monkeys, humans, and the like.
  • the present invention uses mammalian cells as host cells, which can achieve the constitutive type of target protein without induction. expression, thereby avoiding the inhibition of cell growth or metabolism caused by the addition of additional inducers, which in turn affects the post-translational modification of the protein.
  • the embodiment provided by the present invention has high safety and is more conducive to subsequent purification of the target protein and then use as a medicine.
  • the host cell is a Chinese hamster ovary cell (Chinese hamster ovary cells, CHO).
  • the present invention provides a non-covalent complex of a serine protease and a serine protease inhibitor.
  • the non-covalent complex formed by the serine protease and the serine protease inhibitor provided by the present invention is loose in structure, and the formation of the non-covalent complex is reversible.
  • the serine protease is a trypsin-like serine protease, or may be referred to as a trypsin-like protease.
  • the serine protease is a tissue plasminogen activator.
  • the serine protease has the amino acid sequence set forth in SEQ ID NO:1.
  • the present invention has found through a lot of practice that if an inhibitor with too strong inhibitory effect is used, it will form a covalent complex with the serine protease, and if the inhibitory effect is too weak, it is not enough to reduce the cytotoxicity caused by the protease.
  • the serpin is selected from the group consisting of alpha-antitrypsin, bovine trypsin inhibitor, plasminogen activator inhibitor-1, plasminogen activator inhibitor-2, plasminogen activator inhibitor At least one of zymogen activator inhibitor-3, neuro-derived serine protease inhibitor and glial cell-derived connexin.
  • the serpin is selected from the group consisting of alpha-antitrypsin, bovine trypsin inhibitor, plasminogen activator inhibitor-2, plasminogen activator inhibitor-3, and neurogenic at least one of the sex serine protease inhibitors.
  • the serpin is selected from the group consisting of bovine trypsin inhibitor, plasminogen activator inhibitor-2, plasminogen activator inhibitor-3, and neuroderived serpin at least one of.
  • the serpin is selected from at least one of bovine trypsin inhibitor and neuroderived serpin.
  • the alpha-antitrypsin has the amino acid sequence set forth in SEQ ID NO:2.
  • the bovine trypsin inhibitor has the amino acid sequence set forth in SEQ ID NO:3.
  • the plasminogen activator inhibitor-1 has the amino acid sequence shown in SEQ ID NO:4.
  • the plasminogen activator inhibitor-2 has the amino acid sequence shown in SEQ ID NO:5.
  • the plasminogen activator inhibitor-3 has the amino acid sequence shown in SEQ ID NO:6.
  • the neuroderived serine protease inhibitor has the amino acid sequence set forth in SEQ ID NO:7.
  • the glial cell-derived connexin has the amino acid sequence set forth in SEQ ID NO:8.
  • the present invention provides a method for preparing a non-covalent complex of the serine protease and the serine protease inhibitor.
  • the preparation method comprises: transfecting the expression vector provided in the first aspect of the present invention into a host cell for expression.
  • the preparation method comprises: using the host cell provided by the second aspect of the present invention for expression.
  • the preparation method includes the following specific steps: firstly screening the host cells transformed into the expression vector, and then culturing the screened cells to express the target protein.
  • the preparation method includes the following specific steps: performing pressurized screening on the CHO K1 cells transferred into the expression vector with methionine sulfoxide imide, and then culturing the screened cells to express the target protein .
  • the present invention provides the application of the expression vector, the host cell, the non-covalent complex or the preparation method of the non-covalent complex in the preparation of serine protease.
  • the present invention provides a method for preparing serine protease.
  • the serine protease on the basis of expressing protein through the expression vector or host cell, obtaining the non-covalent complex, or preparing the non-covalent complex by the preparation method, the serine protease can be further obtained by a purification process.
  • the main purpose of the purification is to remove serine protease inhibitors.
  • protein expression can be performed through the expression vector or the host cell of the present invention, and then the serine protease inhibitor in the expression product can be removed to obtain a serine protease.
  • the non-covalent complex can be prepared by the non-covalent complex or the method, and then the serine protease inhibitor is removed to obtain a serine protease.
  • the purification described in the present invention can adopt protein purification methods known in the art, such as molecular size exclusion, ion exchange, and the like.
  • a suitable serine protease inhibitor is selected to form a loose reversible non-covalent complex with the serine protease (the inhibitor with too strong inhibitory effect will form with the serine protease).
  • the inhibitory effect is too weak, it is not enough to reduce the cytotoxicity caused by the protease), and subsequent purification of the serine protease can be considered by removing the serine protease inhibitor through a downstream purification process.
  • the serine protease inhibitor By inhibiting the partial activity of the overexpressed serine protease by the serine protease inhibitor, the cytotoxicity of the serine protease to the expressing cell line is reduced, and the tolerance of the expressing cell line to the same expression amount of serine protease is improved.
  • Co-expression of serine protease inhibitors can increase the expression level of serine protease in the same host cell under the condition of unchanged receptivity.
  • Figure 1 is a schematic diagram of the detection results of SDS-PAGE in the supernatant of cell pool culture
  • lane 1 tPA
  • lane 2 tPA+AAT
  • lane 3 tPA+BPTI
  • lane 4 tPA+PAI-1
  • lane 5 tPA+PAI-2
  • lane 6 tPA+PAI-3
  • lane 7 tPA+PI-12
  • lane 8 tPA+PI-7
  • M stands for marker.
  • Figure 2 is a schematic diagram of the results of SDS-PAGE detection of the cell pool fed batch culture supernatant
  • lane 1 tPA
  • lane 2 tPA+AAT
  • lane 3 tPA+BPTI
  • lane 4 tPA+PAI-2
  • lane 5 tPA+PAI-3
  • lane 6 tPA+PI-12
  • lane 7 0.2 ⁇ g Actilyse (reference)
  • lane 8 0.5 ⁇ g Actilyse (reference)
  • lane 9 1.0 ⁇ g Actilyse (reference)
  • M represents marker.
  • Tissue plasminogen activator is one of the earliest recombinant proteins produced by Chinese hamster ovary cells (CHO), and is commonly used to dissolve thrombus in clinic. Relative to its high dose (50mg/cup), its expression is rarely more than 500mg/L.
  • amino acids in the amino acid sequence can be conservatively substituted without changing the activity or function of the protein, see Table 1 below:
  • Residues conservative substitution Residues conservative substitution Ala Ser Leu Ile; Val Arg Lys Lys Arg; Gln Asn Gln; His Met Leu; Ile Asp Glu Phe Met; Leu; Tyr Gln Asn Ser Thr; Gly Cys Ser Thr Ser; Val Glu Asp Trp Tyr Gly Pro Tyr Trp; Phe His Asn; Gln Val Ile; Leu Ile Leu; Val
  • tPA (Uniprot entry: P00750) was synthesized by the whole gene.
  • the codons used were optimized with the preferred codons of CHO cells.
  • the 5' and 3' restriction sites were HindIII and EcoRI, respectively, and the amino acids were as shown in SEQ ID NO. : 1 (36-562 are mature peptides).
  • SPI serine protease inhibitor
  • the sequence of tPA was inserted into the pXC17.4 expression vector to construct an expression plasmid pXC17.4-tPA expressing tPA alone, as a control; the sequence of SPI was inserted into the pXC18.4 expression vector and pXC18.4-SPI.
  • the pXC17.4-tPA and pXC18.4-SPI were digested with NotI and PvuI to form a double expression cassette plasmid pXC-tPA+SPI expressing both tPA and SPI, and SPI was SEQ ID NO:2 ⁇ SEQ ID NO:8 any of the .
  • pXC17.4-tPA and seven pXC-tPA+SPI plasmids were transfected into suspension serum-free acclimated CHO K1 cells by electroporation. After transfection, the transfected cells were pressurized and screened with CD CHO medium containing 25 ⁇ M MSX, and the medium was changed every 3-4 days until the cell viability recovered to more than 90%, and MSX was removed. SDS-PAGE was used to detect the protein in the culture supernatant. There may be some SPIs whose inhibitory effect is too strong and form an irreversible covalent complex with a larger molecular weight with serine proteases, which leads to the loss of application value of the target protein and should be excluded. inhibitor.
  • the screened cell pool was inoculated into a 250 ml Erlenmeyer flask containing 60 ml Dynamis medium at about 0.5 ⁇ 10 6 cells/ml, and the culture conditions were: 37° C., 140 RPM, 5% CO 2 , and 85% humidity. From the 3rd day, the feed medium was fed daily with 3% (v/v) Cell Boost 7a and 0.3% (v/v) Cell Boost 7b, and the glucose was controlled at a concentration of 5-8 g/L. 11 days. The supernatant was harvested by centrifugation at 2000 rmp for 10 min, filtered through a 0.22 ⁇ m filter, and stored at 2-8 °C.
  • Example 3 100 uL of the culture supernatant obtained in Example 3 was taken, diluted 10 times with PBS, and then 20 uL of the sample was taken and analyzed by reducing SDS-PAGE.
  • Serine protease broad-spectrum chromogenic substrate S-2288 is a chemically synthesized small peptide with a chromophore (pNA) at one end; serine protease can catalyze the dissociation of pNA; free pNA can be detected by spectrophotometer or microplate reader detection to determine the activity of the corresponding substance.
  • pNA chromophore
  • Dilution Buffer 0.05M Tris-HCl, 0.15M NaCl, 1mg/ml BSA, 0.01% Tween 80, pH 7.4.
  • Reaction Buffer 0.05M Tris-HCl, 0.15M NaCl, 1mg/ml BSA, 0.01% Tween 80, pH 7.4, 0.5mM S-2288.
  • tPA is set to 100% tPA 0.624 100% tPA+AAT 0.638 102% tPA+BPTI 1.582 253% tPA+PAI-2 0.969 155% tPA+PAI-3 0.981 157% tPA+PI-12 >2.000 >320.5%

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Mycology (AREA)
  • Cell Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

提供了共表达丝氨酸蛋白酶和丝氨酸蛋白酶抑制剂的表达载体以及宿主细胞，利用所述表达载体或宿主细胞制备丝氨酸蛋白酶的方法，通过由丝氨酸蛋白酶抑制剂抑制过量表达的丝氨酸蛋白酶的部分活性，减少丝氨酸蛋白酶对表达细胞株造成的细胞毒性，提高表达细胞株对同等表达量的丝氨酸蛋白酶的耐受性。

Description

一种制备丝氨酸蛋白酶的方法 技术领域
本发明涉及分子生物学领域，具体涉及一种制备丝氨酸蛋白酶的方法。
背景技术
丝氨酸蛋白酶(或称丝氨酸内肽酶)是一种能在蛋白质中裂解肽键的酶，其中丝氨酸作为酶活性部位的亲核氨基酸。它们普遍存在于真核生物和原核生物中。丝氨酸蛋白酶根据其结构可分为两大类：类胰蛋白酶(类糜蛋白酶)或类枯草杆菌蛋白酶。其中胰蛋白酶样丝氨酸蛋白酶在带正电的氨基酸(赖氨酸或精氨酸)后裂解肽键。这种特异性是由位于酶的底物结合口袋底部的残基(通常是带负电的天冬氨酸或谷氨酸)决定的。
胰蛋白酶样丝氨酸蛋白酶对维持生物体的内环境稳定起着重要作用，如控制血液凝聚、血纤蛋白溶解、激酶-激肽释放酶和补体系统。正常情况下，人体自身的内源性蛋白酶抑制剂能保护保护蛋白酶所致的潜在的破坏作用，若蛋白酶抑制剂浓度下降，则破坏了蛋白酶和抗蛋白酶之间的平衡。过量蛋白酶的活化会导致各种疾病的发展，如胰腺炎或弥散性血管内凝血疾病等。胰蛋白酶样丝氨酸蛋白酶能使各种疾病激活，而其抑制剂通常是治疗或预防血栓形成、炎症疾病、自身免疫及相关疾病的有效药物。
生物工程中常用哺乳动物细胞表达重组蛋白，而某些用于临床治疗的胰蛋白酶样丝氨酸蛋白酶(如各类凝血因子和溶栓酶等)常存在哺乳动物细胞内表达量低的问题，进而增加药物的生产成本。其原因在于过量表达的胰蛋白酶样丝氨酸蛋白酶的活性会引起细胞毒性。即使是表达活性较低或无活性的蛋白酶前体，也可能在表达过程中有部分或少量被提前激活，细胞毒性超过一定限度时，会对表达胰蛋白酶样丝氨酸蛋白酶的细胞群体造成一定的筛选压力，导致最终筛选得到的细胞株几乎都是低表达的。即使偶有高表达细胞株，也无法长期稳定存在，难以用于大规模商业化生产。
发明内容
第一方面，本发明提供共表达丝氨酸蛋白酶和丝氨酸蛋白酶抑制剂的表达载体。
在一些实施方式中，所述丝氨酸蛋白酶为胰蛋白酶样丝氨酸蛋白酶，或可称为类胰蛋白酶。
在一些实施方式中，所述表达载体为组成型表达载体。优选地，所述表达载体的表达无需诱导剂的诱导。本发明采用组成型表达载体共表达丝氨酸蛋白酶和丝氨酸蛋白酶抑制剂，可以无需诱导而实现目标蛋白的组成型表达，从而避免了额外添加诱导剂导致的细胞生长或代谢抑制，进而影响蛋白的翻译后修饰。且本发明提供的表达载体安全性高，更利于目标蛋白后续的纯化继而作为药物使用。
在一些实施方式中，所述表达载体为pXC17.4载体、pXC18.4载体或pXC17.4和pXC18.4的连接载体。
在一些实施方式中，所述丝氨酸蛋白酶为组织型纤溶酶原激活剂(tissue plasminogen activator,tPA)。
在一些实施方式中，所述丝氨酸蛋白酶具有如SEQ ID NO:1所示的氨基酸序列。
在一些实施方式中，所述丝氨酸蛋白酶抑制剂选自α-抗胰蛋白酶、牛胰蛋白酶抑制剂、纤溶酶原激活剂抑制剂-1、纤溶酶原激活剂抑制剂-2、纤溶酶原激活剂抑制剂-3、神经源性丝氨酸蛋白酶抑制剂和胶质细胞源性连接蛋白中的至少一种。
在一些实施方式中，所述丝氨酸蛋白酶抑制剂选自α-抗胰蛋白酶、牛胰蛋白酶抑制剂、纤溶酶原激活剂抑制剂-2、纤溶酶原激活剂抑制剂-3和神经源性丝氨酸蛋白酶抑制剂中的至少一种。
在一些实施方式中，所述丝氨酸蛋白酶抑制剂选自牛胰蛋白酶抑制剂、纤溶酶原激活剂抑制剂-2、纤溶酶原激活剂抑制剂-3和神经源性丝氨酸蛋白酶抑制剂中的至少一种。
在一些实施方式中，所述丝氨酸蛋白酶抑制剂选自牛胰蛋白酶抑制剂和神经 源性丝氨酸蛋白酶抑制剂中的至少一种。
在一些实施方式中，所述α-抗胰蛋白酶具有如SEQ ID NO:2所示的氨基酸序列。
在一些实施方式中，所述牛胰蛋白酶抑制剂具有如SEQ ID NO:3所示的氨基酸序列。
在一些实施方式中，所述纤溶酶原激活剂抑制剂-1具有如SEQ ID NO:4所示的氨基酸序列。
在一些实施方式中，所述纤溶酶原激活剂抑制剂-2具有如SEQ ID NO:5所示的氨基酸序列。
在一些实施方式中，所述纤溶酶原激活剂抑制剂-3具有如SEQ ID NO:6所示的氨基酸序列。
在一些实施方式中，所述神经源性丝氨酸蛋白酶抑制剂具有如SEQ ID NO:7所示的氨基酸序列。
在一些实施方式中，所述胶质细胞源性连接蛋白具有如SEQ ID NO:8所示的氨基酸序列。
在一些实施方式中，所述共表达丝氨酸蛋白酶和丝氨酸蛋白酶抑制剂的表达载体采用包括如下步骤的方法制备得到：将插入编码所述丝氨酸蛋白酶(tPA)的核苷酸序列的表达载体和插入编码所述丝氨酸蛋白酶抑制剂(SPI)的核苷酸序列的表达载体，连接成双表达框表达载体。
在一些实施方式中，所述共表达丝氨酸蛋白酶和丝氨酸蛋白酶抑制剂的表达载体采用包括如下步骤的方法制备得到：将编码所述tPA的核苷酸序列插入pXC17.4表达载体中，得到pXC17.4-tPA；将编码所述SPI的核苷酸序列插入pXC18.4表达载体，得到pXC18.4-SPI；所述将pXC17.4-tPA和pXC18.4-SPI用NotI和PvuI进行酶切，连接成同时表达tPA和SPI的双表达框表达载体pXC-tPA-SPI。
第二方面，本发明提供外源转入了所述表达载体的宿主细胞。
在一些实施方式中，所述宿主细胞为哺乳动物细胞，如来自于大鼠、小鼠、仓鼠、豚鼠、猴、人等哺乳动物的细胞。相对于酵母细胞需要加入醇氧化酶启动 子进行诱导表达，以及细菌细胞(如大肠杆菌)需要加入乳糖进行诱导表达，本发明采用哺乳动物细胞作为宿主细胞，可以无需诱导而实现目标蛋白的组成型表达，从而避免了额外添加诱导剂导致的细胞生长或代谢抑制，进而影响蛋白的翻译后修饰。本发明提供的实施方式安全性高，更利于目标蛋白后续的纯化继而作为药物使用。
在一些实施方式中，所述宿主细胞为中国仓鼠卵巢细胞(Chinese hamster ovary cells，CHO)。
第三方面，本发明提供丝氨酸蛋白酶与丝氨酸蛋白酶抑制剂形成的非共价复合物。本发明提供的所述丝氨酸蛋白酶与所述丝氨酸蛋白酶抑制剂形成的非共价复合物结构松散，且该非共价复合物的形成可逆。
在一些实施方式中，所述丝氨酸蛋白酶为胰蛋白酶样丝氨酸蛋白酶，或可称为类胰蛋白酶。
在一些实施方式中，所述丝氨酸蛋白酶为组织型纤溶酶原激活剂。
在一些实施方式中，所述丝氨酸蛋白酶具有如SEQ ID NO:1所示的氨基酸序列。
本发明通过大量实践发现，如果采用抑制作用过强的抑制剂会与丝氨酸蛋白酶形成共价复合物，抑制作用过弱则不足以减弱蛋白酶所导致的细胞毒性。
在一些实施方式中，所述丝氨酸蛋白酶抑制剂选自α-抗胰蛋白酶、牛胰蛋白酶抑制剂、纤溶酶原激活剂抑制剂-1、纤溶酶原激活剂抑制剂-2、纤溶酶原激活剂抑制剂-3、神经源性丝氨酸蛋白酶抑制剂和胶质细胞源性连接蛋白中的至少一种。
在一些实施方式中，所述丝氨酸蛋白酶抑制剂选自α-抗胰蛋白酶、牛胰蛋白酶抑制剂、纤溶酶原激活剂抑制剂-2、纤溶酶原激活剂抑制剂-3和神经源性丝氨酸蛋白酶抑制剂中的至少一种。
在一些实施方式中，所述丝氨酸蛋白酶抑制剂选自牛胰蛋白酶抑制剂、纤溶酶原激活剂抑制剂-2、纤溶酶原激活剂抑制剂-3和神经源性丝氨酸蛋白酶抑制剂中的至少一种。
在一些实施方式中，所述丝氨酸蛋白酶抑制剂选自牛胰蛋白酶抑制剂和神经源性丝氨酸蛋白酶抑制剂中的至少一种。
在一些实施方式中，所述α-抗胰蛋白酶具有如SEQ ID NO:2所示的氨基酸序列。
在一些实施方式中，所述牛胰蛋白酶抑制剂具有如SEQ ID NO:3所示的氨基酸序列。
在一些实施方式中，所述纤溶酶原激活剂抑制剂-1具有如SEQ ID NO:4所示的氨基酸序列。
在一些实施方式中，所述纤溶酶原激活剂抑制剂-2具有如SEQ ID NO:5所示的氨基酸序列。
在一些实施方式中，所述纤溶酶原激活剂抑制剂-3具有如SEQ ID NO:6所示的氨基酸序列。
在一些实施方式中，所述神经源性丝氨酸蛋白酶抑制剂具有如SEQ ID NO:7所示的氨基酸序列。
在一些实施方式中，所述胶质细胞源性连接蛋白具有如SEQ ID NO:8所示的氨基酸序列。
第四方面，本发明提供所述丝氨酸蛋白酶与所述丝氨酸蛋白酶抑制剂的非共价复合物的制备方法。
在一些实施方式中，所述制备方法包括：将本发明第一方面提供的表达载体转染至宿主细胞中进行表达。
在一些实施方式中，所述制备方法包括：利用本发明第二方面提供的宿主细胞进行表达。
在一些实施方式中，所述制备方法包括如下具体步骤：先对转入所述表达载体的宿主细胞进行隆筛选，然后对筛选后的细胞进行培养，表达目标蛋白。
在一些实施方式中，所述制备方法包括如下具体步骤：对转入所述表达载体的CHO K1细胞用蛋氨酸亚砜酰亚胺进行加压筛选，然后对筛选后的细胞进行培养，表达目标蛋白。
第五方面，本发明提供所述表达载体、所述宿主细胞、所述非共价复合物或所述非共价复合物的制备方法在制备丝氨酸蛋白酶中的应用。
第六方面，本发明提供一种制备丝氨酸蛋白酶的方法。
本发明在通过所述表达载体或宿主细胞进行蛋白质表达、获得所述非共价复合物或者通过所述制备方法制备得到所述非共价复合物的基础上，可以进一步采用纯化工艺获得丝氨酸蛋白酶，所述纯化的主要目的是去除丝氨酸蛋白酶抑制剂。
在一些实施方式中，可以通过本发明所述表达载体或所述宿主细胞进行蛋白质表达，然后去除表达产物中的丝氨酸蛋白酶抑制剂，从而得到丝氨酸蛋白酶。
在一些实施方式中，可以通过所述非共价复合物或所述方法制备得到所述非共价复合物，然后去除其中的丝氨酸蛋白酶抑制剂，从而得到丝氨酸蛋白酶。
本发明所述纯化可以采用分子排阻、离子交换等本领域已知的蛋白纯化方式。
本发明通过共表达丝氨酸蛋白酶和相应的丝氨酸蛋白酶抑制剂，选择合适的丝氨酸蛋白酶抑制剂使之与丝氨酸蛋白酶形成松散的可逆的非共价复合物(抑制作用过强的抑制剂会与丝氨酸蛋白酶形成共价复合物，抑制作用过弱则不足以减弱蛋白酶所导致的细胞毒性)，后续可以考虑通过下游纯化工艺去除丝氨酸蛋白酶抑制剂来提纯丝氨酸蛋白酶。通过由丝氨酸蛋白酶抑制剂抑制过量表达的丝氨酸蛋白酶的部分活性，减少丝氨酸蛋白酶对表达细胞株造成的细胞毒性，提高表达细胞株对同等表达量的丝氨酸蛋白酶的耐受性，换言之，在细胞毒性耐受度不变的情况下，共表达丝氨酸蛋白酶抑制剂可提高丝氨酸蛋白酶在同一宿主细胞中的表达量。
附图说明
图1为细胞池培养上清SDS-PAGE检测结果示意图；
其中，泳道1：tPA,泳道2：tPA+AAT,泳道3：tPA+BPTI,泳道4：tPA+PAI-1；泳道5：tPA+PAI-2,泳道6：tPA+PAI-3,泳道7：tPA+PI-12,泳道8：tPA+PI-7；M代表marker。
图2为细胞池补料批培养上清SDS-PAGE检测结果示意图；
其中，泳道1：tPA,泳道2：tPA+AAT,泳道3：tPA+BPTI,泳道4：tPA+PAI-2；泳道5：tPA+PAI-3,泳道6：tPA+PI-12,泳道7：0.2μg Actilyse(参考品),泳道8：0.5μg Actilyse(参考品)，泳道9：1.0μg Actilyse(参考品)；M代表marker。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。
组织型纤溶酶原激活剂(tissue plasminogen activator,tPA)是最早的用中国仓鼠卵巢细胞(CHO)生产的重组蛋白之一，临床上常用于溶解血栓。相对于其高剂量(50mg/支),其表达量一般鲜有超过500mg/L的。
根据本发明的技术方案，在不改变蛋白的活性或功能的情况下，可以对氨基酸序列中的某些氨基酸进行保守取代，参见下表1：
表1
残基 保守性替换 残基 保守性替换
Ala Ser Leu Ile；Val
Arg Lys Lys Arg；Gln
Asn Gln；His Met Leu；Ile
Asp Glu Phe Met；Leu；Tyr
Gln Asn Ser Thr；Gly
Cys Ser Thr Ser；Val
Glu Asp Trp Tyr
Gly Pro Tyr Trp；Phe
His Asn；Gln Val Ile；Leu
Ile Leu；Val    
此外，因为碱基的简并性，在不改变多核苷酸序列的活性或功能的情况下，可以对多核苷酸序列的碱基进行取代，参见下表2：
表2
Figure PCTCN2021138653-appb-000001
实施例1：质粒构建
全基因合成tPA(Uniprot entry:P00750)的DNA序列，所用密码子以CHO细胞的偏好密码子进行优化，5’和3’的限制性酶切位点分别为HindIII和EcoRI，氨基酸如SEQ ID NO:1所示(36-562为成熟肽)。
全基因合成丝氨酸蛋白酶抑制剂(Serine protease inhibitor,SPI),所用密码子以CHO细胞的偏好密码子进行优化，5’和3’的限制性酶切位点分别为HindIII和EcoRI。具体而言：α-抗胰蛋白酶(AAT,Uniprot entry:P01009)的序列如SEQ ID NO:2所示；牛胰蛋白酶抑制剂(BPTI,Uniprot entry:P00974)的序列如SEQ ID NO:3所示；纤溶酶原激活剂抑制剂-1(PAI-1,Uniprot entry:P05121)的序列如SEQ ID NO:4所示；纤溶酶原激活剂抑制剂-2(PAI-2,Uniprot entry:P05120)的序列如SEQ ID NO:5所示；纤溶酶原激活剂抑制剂-3(PAI-3,Uniprot entry:P05154)的序列如SEQ ID NO:6所示；神经源性丝氨酸蛋白酶抑制剂(PI-12,Uniprot entry:Q99574)的序列如SEQ ID NO:7所示；胶质细胞源性连接蛋白(PI-7，Uniprot entry:P07093)的序列如SEQ ID NO:8所示。
上述氨基酸序列具体如表3所示。
表3：氨基酸序列
Figure PCTCN2021138653-appb-000002
Figure PCTCN2021138653-appb-000003
将tPA的序列插入至pXC17.4表达载体中，构建单独表达tPA的表达质粒pXC17.4-tPA，作为对照；将SPI的序列插入pXC18.4表达载体，并pXC18.4-SPI。 将pXC17.4-tPA和pXC18.4-SPI用NotI和PvuI酶切连接成一个同时表达tPA和SPI的双表达框质粒pXC-tPA+SPI，SPI为SEQ ID NO:2～SEQ ID NO:8中的任意一个。
实施例2：细胞池构建
将pXC17.4-tPA和7个pXC-tPA+SPI质粒通过电穿孔转染至经悬浮无血清驯化的CHO K1细胞中。转染后用含25μM MSX的CD CHO培养基对转染后的细胞进行加压筛选，每3-4天更换一次培养基，直至细胞活率恢复至90％以上，撤去MSX。用SDS-PAGE对培养上清中蛋白进行检测，可能存在某些SPI的抑制作用过强而与丝氨酸蛋白酶形成分子量更大的不可逆的共价复合物，导致目标蛋白失去应用价值，应排除此类抑制物。
结果如图1所示，在本实施例中，排除了PAI-1(泳道4)和PI-7(泳道8)。
实施例3：细胞池补料批培养
将筛选后的细胞池以约0.5×10 6cell/ml接种至含60ml Dynamis培养基的250ml三角摇瓶中，培养条件：37℃，140RPM,5％CO 2，85％湿度。从第3天起，每天流加补料培养基3％(v/v)Cell Boost 7a和0.3％(v/v)Cell Boost 7b，并将葡萄糖控制在5-8g/L的浓度，培养第11天。2000rmp离心10min收获上清，再经0.22μm滤膜过滤后保存于2-8℃。
实施例4：表达量检测
取实施例3所得的100uL培养上清，用PBS稀释10倍后，取20uL样品用还原性于SDS-PAGE分析。
由图2结果可知，共表达SPI的细胞池所表达的tPA表达水平要明显高于单独表达tPA的细胞池。
实施例5：活性分析
用底物S-2288对培养上清进行活性检测。丝氨酸蛋白酶广谱发色底物S-2288为化学合成的小肽，其一端为发色基团(pNA)；丝氨酸蛋白酶可将pNA催化解离下来；游离pNA可通过分光光度计或酶标仪检测，从而确定相应物质的活性。
稀释Buffer:0.05M Tris-HCl,0.15M NaCl,1mg/ml BSA,0.01％Tween 80,pH 7.4。
反应Buffer:0.05M Tris-HCl,0.15M NaCl,1mg/ml BSA,0.01％Tween 80,pH 7.4,0.5mM S-2288。
取1mg/mL的Actilyse(参考品),用稀释Buffer稀释成1.0、0.5、0.25、0.125、0.0625、0.03125、0.015625mg/mL的工作浓度用于制备标准曲线。细胞池补料批培养上清用稀释buffer 1:1稀释后作为待测样品。取5μL参考品工作液或待测样品，与95μL反应buffer在96孔板混合后，迅速用分光光度计检测检测5分钟内406nm处的吸收值，以工作浓度为横坐标，吸光值为纵坐标拟合标准曲线，计算细胞池补料批培养上清中的tPA含量。检测结果如表4所示。
表4：细胞池培养上清表达量检测(S-2288法)
细胞池 表达量(g/L) tPA设为100％
tPA 0.624 100％
tPA+AAT 0.638 102％
tPA+BPTI 1.582 253％
tPA+PAI-2 0.969 155％
tPA+PAI-3 0.981 157％
tPA+PI-12 ＞2.000 ＞320.5％
由表4结果可知，除tPA+AAT外，其余细胞池均有50％以上tPA表达量提升。其中，tPA+PI-12细胞池的表达量超出检测上限，可能与其双链tPA的比例高于其他细胞池有关。
虽然，上文中已经用一般性说明、具体实施方式及试验，对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。

Claims (10)

  1. 共表达丝氨酸蛋白酶和丝氨酸蛋白酶抑制剂的表达载体，其特征在于，所述丝氨酸蛋白酶为胰蛋白酶样丝氨酸蛋白酶；
    优选地，所述表达载体为组成型表达载体；
    更优选地，所述表达载体的表达无需诱导剂的诱导。
  2. 根据权利要求1所述的表达载体，其特征在于，所述丝氨酸蛋白酶为组织型纤溶酶原激活剂；
    优选地，所述丝氨酸蛋白酶具有如SEQ ID NO:1所示的氨基酸序列。
  3. 根据权利要求1或2所述的表达载体，其特征在于，所述丝氨酸蛋白酶抑制剂选自α-抗胰蛋白酶、牛胰蛋白酶抑制剂、纤溶酶原激活剂抑制剂-1、纤溶酶原激活剂抑制剂-2、纤溶酶原激活剂抑制剂-3、神经源性丝氨酸蛋白酶抑制剂和胶质细胞源性连接蛋白中的至少一种；
    优选地，所述丝氨酸蛋白酶抑制剂选自α-抗胰蛋白酶、牛胰蛋白酶抑制剂、纤溶酶原激活剂抑制剂-2、纤溶酶原激活剂抑制剂-3和神经源性丝氨酸蛋白酶抑制剂中的至少一种；
    优选地，所述丝氨酸蛋白酶抑制剂选自牛胰蛋白酶抑制剂、纤溶酶原激活剂抑制剂-2、纤溶酶原激活剂抑制剂-3和神经源性丝氨酸蛋白酶抑制剂中的至少一种；
    优选地，所述丝氨酸蛋白酶抑制剂选自牛胰蛋白酶抑制剂和神经源性丝氨酸蛋白酶抑制剂中的至少一种。
    更优选地，所述α-抗胰蛋白酶具有如SEQ ID NO:2所示的氨基酸序列；和/或，所述牛胰蛋白酶抑制剂具有如SEQ ID NO:3所示的氨基酸序列；和/或，所述纤溶酶原激活剂抑制剂-1具有如SEQ ID NO:4所示的氨基酸序列；和/或，所述纤溶酶原激活剂抑制剂-2具有如SEQ ID NO:5所示的氨基酸序列；和/或，所述纤溶酶原激活剂抑制剂-3具有如SEQ ID NO:6所示的氨基酸序列；和/或，所述神经源性丝氨酸蛋白酶抑制剂具有如SEQ ID NO:7所示的氨基酸序列；和/或，所述胶质细胞源性连接蛋白具有如SEQ ID NO:8所示的氨基酸序列。
  4. 根据权利要求1～3任意一项所述的表达载体，其特征在于，所述共表达丝氨酸蛋白酶和丝氨酸蛋白酶抑制剂的表达载体采用包括如下步骤的方法制备得到：将插入编码所述丝氨酸蛋白酶的核苷酸序列的表达载体和插入编码所述丝氨酸蛋白酶抑制剂的核苷酸序列的表达载体，连接成双表达框表达载体；
    优选地，所述共表达丝氨酸蛋白酶和丝氨酸蛋白酶抑制剂的表达载体采用包括如下步骤的方法制备得到：将编码所述tPA的核苷酸序列插入pXC17.4表达载体中，得到pXC17.4-tPA；将编码所述SPI的核苷酸序列插入pXC18.4表达载体，得到pXC18.4-SPI；所述将pXC17.4-tPA和pXC18.4-SPI用NotI和PvuI进行酶切，连接成同时表达tPA和SPI的双表达框表达载体pXC-tPA-SPI。
  5. 外源转入了权利要求1～4任意一项所述表达载体的宿主细胞；
    优选地，所述宿主细胞为哺乳动物细胞，优选为来自大鼠、小鼠、仓鼠、豚鼠、猴或人的细胞；
    更优选地，所述宿主细胞为中国仓鼠卵巢细胞，优选为CHO K1细胞。
  6. 丝氨酸蛋白酶与丝氨酸蛋白酶抑制剂形成的非共价复合物，其特征在于，所述丝氨酸蛋白酶为胰蛋白酶样丝氨酸蛋白酶，优选所述非共价复合物的形成可逆；
    优选地，所述丝氨酸蛋白酶为组织型纤溶酶原激活剂。
  7. 根据权利要求6所述的非共价复合物，其特征在于，所述丝氨酸蛋白酶抑制剂选自α-抗胰蛋白酶、牛胰蛋白酶抑制剂、纤溶酶原激活剂抑制剂-1、纤溶酶原激活剂抑制剂-2、纤溶酶原激活剂抑制剂-3、神经源性丝氨酸蛋白酶抑制剂和胶质细胞源性连接蛋白中的至少一种；
    优选地，所述丝氨酸蛋白酶抑制剂选自α-抗胰蛋白酶、牛胰蛋白酶抑制剂、纤溶酶原激活剂抑制剂-2、纤溶酶原激活剂抑制剂-3和神经源性丝氨酸蛋白酶抑制剂中的至少一种；
    优选地，所述丝氨酸蛋白酶抑制剂选自牛胰蛋白酶抑制剂、纤溶酶原激活剂抑制剂-2、纤溶酶原激活剂抑制剂-3和神经源性丝氨酸蛋白酶抑制剂中的至少一种；
    优选地，所述丝氨酸蛋白酶抑制剂选自牛胰蛋白酶抑制剂和神经源性丝氨酸蛋白酶抑制剂中的至少一种。
  8. 制备权利要求6或7所述非共价复合物的方法；
    优选地，所述方法包括：将权利要求1～4任意一项所述表达载体转染至宿主细胞中进行表达，或者利用权利要求5所述宿主细胞进行表达；
    更优选地，所述方法包括：对转入所述表达载体的宿主细胞进行筛选，然后对筛选后的细胞进行培养，表达目标蛋白；
    更优选地，所述方法包括：对转入所述表达载体的CHO K1细胞用蛋氨酸亚砜酰亚胺进行加压筛选，然后对筛选后的细胞进行培养，表达目标蛋白。
  9. 权利要求1～4任意一项所述表达载体、权利要求5所述宿主细胞、权利要求6或7所述非共价复合物或权利要求8所述方法在制备丝氨酸蛋白酶中的应用。
  10. 一种制备丝氨酸蛋白酶的方法，其特征在于，包括如下步骤：通过权利要求1～4任意一项所述表达载体或权利要求5所述宿主细胞进行蛋白质表达，然后去除表达产物中的丝氨酸蛋白酶抑制剂；
    或者，获得权利要求6或7所述非共价复合物或通过权利要求8所述方法制备得到所述非共价复合物，然后去除其中的丝氨酸蛋白酶抑制剂。
PCT/CN2021/138653 2020-12-30 2021-12-16 一种制备丝氨酸蛋白酶的方法 WO2022143186A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202011625925.7A CN114686519A (zh) 2020-12-30 2020-12-30 一种制备丝氨酸蛋白酶的方法
CN202011625925.7 2020-12-30

Publications (1)

Publication Number Publication Date
WO2022143186A1 true WO2022143186A1 (zh) 2022-07-07

Family

ID=82133749

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/138653 WO2022143186A1 (zh) 2020-12-30 2021-12-16 一种制备丝氨酸蛋白酶的方法

Country Status (2)

Country Link
CN (1) CN114686519A (zh)
WO (1) WO2022143186A1 (zh)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5648254A (en) * 1988-01-15 1997-07-15 Zymogenetics, Inc. Co-expression in eukaryotic cells
CN102388135A (zh) * 2009-03-03 2012-03-21 B.R.A.I.N.生物技术研究和信息网络公司 用于创伤调理和皮肤护理的新型蛋白酶
IN201947051091A (zh) * 2017-05-31 2019-12-20 B.R.A.I.N. Biotechnology Research And Information Network Ag

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5648254A (en) * 1988-01-15 1997-07-15 Zymogenetics, Inc. Co-expression in eukaryotic cells
CN102388135A (zh) * 2009-03-03 2012-03-21 B.R.A.I.N.生物技术研究和信息网络公司 用于创伤调理和皮肤护理的新型蛋白酶
IN201947051091A (zh) * 2017-05-31 2019-12-20 B.R.A.I.N. Biotechnology Research And Information Network Ag
CN110770340A (zh) * 2017-05-31 2020-02-07 B.R.A.I.N.生物技术研究和信息网络公司 在宿主细胞中丝氨酸蛋白酶的表达的优化

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
SØREN E. DEGN, THIEL STEFFEN, JENSENIUS JENS C.: "Recombinant expression of the autocatalytic complement protease MASP-1 is crucially dependent on co-expression with its inhibitor, C1 inhibitor", PROTEIN EXPRESSION AND PURIFICATION, ACADEMIC PRESS, SAN DIEGO, CA., vol. 88, no. 2, 1 April 2013 (2013-04-01), SAN DIEGO, CA. , pages 173 - 182, XP055402258, ISSN: 1046-5928, DOI: 10.1016/j.pep.2013.01.002 *
TSUZUKI SATOSHI, MURAI NOBUHITO, MIYAKE YUKA, INOUYE KUNIYO, HIRAYASU HIROFUMI, IWANAGA TOSHIHIKO, FUSHIKI TOHRU: "Evidence for the occurrence of membrane-type serine protease 1/matriptase on the basolateral sides of enterocytes", BIOCHEMICAL JOURNAL, PUBLISHED BY PORTLAND PRESS ON BEHALF OF THE BIOCHEMICAL SOCIETY., GB, vol. 388, no. PART 2, 1 June 2005 (2005-06-01), GB , pages 679 - 687, XP008103674, ISSN: 0264-6021, DOI: 10.1042/BJ20041639 *

Also Published As

Publication number Publication date
CN114686519A (zh) 2022-07-01

Similar Documents

Publication Publication Date Title
KR0148575B1 (ko) 효소원 또는 피브린 특이성을 갖는 조직 플라스미노겐 활성제
FI88932C (fi) Framstaellning av funktionellt maenskligt urokinasprotein
RU2564131C2 (ru) Варианты плазминогена и плазмина
EP2661493B1 (en) Plasminogen and plasmin variants
JP2529816B2 (ja) 新規なヒト組織プラスミノゲン活性化因子突然変異体
JP5096759B2 (ja) セリンエンドペプチダーゼの安定化調製物、その製造および使用
JP6000353B2 (ja) プラスミノーゲンおよびプラスミンの変異体
EP0703788A1 (en) A stabilised phenylalanine ammonia lyase
JPS5942321A (ja) ヒト組織プラスミノ−ゲン活性化因子
Guyot et al. Proteolytic susceptibility of the serine protease inhibitor trappin-2 (pre-elafin): evidence for tryptase-mediated generation of elafin
JP2916228B2 (ja) 遺伝子操作した組み換えアプロチニン変異型、均質に処理されたアプロチニン変異型の微生物調製の方法およびその治療学的使用
Pourmotabbed et al. Characteristics of 92 kDa type IV collagenase/gelatinase produced by granulocytic leukemia cells: structure, expression of cDNA in E. coli and enzymatic properties
EP0398859B1 (en) Novel 92-kDa type IV collagenase
WO2022143186A1 (zh) 一种制备丝氨酸蛋白酶的方法
JPH07143878A (ja) 改善された繊維素溶解性及びトロンビン阻害作用を有する２官能性ウロキナーゼ変異体
Goodfellow et al. Characterization of a maize root proteinase
JPH08231595A (ja) 繊維素溶解性のトロンビン阻害性質を有するキメラたん白質
JP2001513992A (ja) 新規の組織特異的カルパイン、その製造およびその使用
JPH08231594A (ja) 繊維素溶解性の血液凝固阻害性質を有するたん白質
Inokuchi et al. Isolation of pepsinogen A from gastric mucosa of bullfrog, Rana catesbeiana
Hori et al. Purification and characterization of myonase from X-chromosome linked muscular dystrophic mouse skeletal muscle
TWI252234B (en) Novel chromogenic substrates and their use in assaying carboxypeptidase activity
US20110200541A1 (en) Recombinant preparation of bromelain inhibitors and bromelain inhibitor precursor
GB2300190A (en) Elastase inhibitor extracted from the leech, Hirudo nipponia
CA3211023A1 (en) Uricase activator and uric acid measurement reagent

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21913927

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21913927

Country of ref document: EP

Kind code of ref document: A1