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WO2009039704A1 - Procédé de fabrication de kallikréine 1 humaine - Google Patents

Procédé de fabrication de kallikréine 1 humaine Download PDF

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Publication number
WO2009039704A1
WO2009039704A1 PCT/CN2007/070800 CN2007070800W WO2009039704A1 WO 2009039704 A1 WO2009039704 A1 WO 2009039704A1 CN 2007070800 W CN2007070800 W CN 2007070800W WO 2009039704 A1 WO2009039704 A1 WO 2009039704A1
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WIPO (PCT)
Prior art keywords
rhkl
polynucleotide
hkl
chromatography
protein
Prior art date
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Ceased
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PCT/CN2007/070800
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English (en)
Chinese (zh)
Inventor
Xiudong Huang
Peixin Chen
Shusheng Wang
Yaoguo Chen
Jun Wang
Xuegong Pan
Zhifang Cao
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Shanghai Wanxing Biopharmaceuticals Co Ltd
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Shanghai Wanxing Biopharmaceuticals Co Ltd
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Priority to PCT/CN2007/070800 priority Critical patent/WO2009039704A1/fr
Publication of WO2009039704A1 publication Critical patent/WO2009039704A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6445—Kallikreins (3.4.21.34; 3.4.21.35)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the present invention is in the field of genetic engineering, and more particularly, the present invention relates to a polynucleotide encoding human kallikrein 1 and a method for large-scale expression of recombinant human kallikrein 1. Background technique
  • hKl belongs to the peptidase class S1 family of serine proteases (or kallikrein subfamily). It specifically hydrolyzes peptide bonds between Met-Lys or Arg-Ser amino acid residues in low molecular weight or high molecular weight kininogen molecules, releasing Lys-bradykinin or vasodilating hormone, respectively. (Kallidin). The kinins released by these hydrolysis will regulate the body's blood pressure, electrolyte balance, inflammation and cell proliferation.
  • Chinese Patent No. CN02116783.4 discloses that hK1 can be used for the preparation of a medicament for the treatment and prevention of cerebral infarction.
  • human kallikrein-1 there are two main methods for preparing human kallikrein-1: One method is to directly extract hK or hKl components from human urine or animal tissues, and the preparation cost is high and the quality of raw materials cannot be guaranteed. Another method is to express human kallikrein 1 in microorganisms or cells such as E. coli or yeast using genetic recombination techniques. However, the current gene recombination studies on human kallikrein 1 are mainly based on the expression of Pro-hKl or Pr-ro-hKl or Met-hKl, and when the direct secretion of hKl protein is secreted, the signal peptide sequence is found. Can not be removed smoothly. See, for example, Angermann, A.
  • rhK1 Protein Expr & Purif. 1998, 12: 361-370.
  • the existing recombinant production of rhK1 has the following disadvantages: (a) low expression yield (200 ⁇ g/L ⁇ 30 mg/L); (b) recombinant rhK1 is unglycosylated or heterogeneous in degree of glycosylation or signal The peptide can not be naturally excised, requires additional protease digestion, and the product is not uniform. The molecular weight distribution range is large (usually 27 ⁇ 40kDa); (c) Due to low product yield, heterogeneity, complicated purification process and low effective yield.
  • Another object of the present invention is to provide a large-scale acquisition of recombinant human kallikrein by a methanol yeast expression system.
  • -1 Recombinant Human Kallikrein 1, rhKl
  • a vector comprising the polynucleotide.
  • a host cell comprising the vector or the polynucleotide of the host cell in which the polynucleotide is integrated is provided.
  • the host cell is a yeast. More preferably, the host cell is methanol yeast. In another preferred embodiment, the expression level of recombinant human kallikrein l (rhKl) expressed by the host cell is 1. 3 ⁇
  • the specific activity of the expressed rhK1 is not less than 5. 2 AU/mg.
  • a method of preparing human kallikrein 1, hK1 comprising the steps of:
  • the whole salt medium is used for the fermentation in the step (a), and the pH of the induction phase is 6. 0 ⁇ 0. 5 (preferably 60 ⁇ 0.2), and the temperature is 25. ⁇ 5 ° C, induction time 65 ⁇ 15 hours (preferably, induction time is 65 ⁇ 10 hours, more preferably, induction time is 65 ⁇ 7 hours).
  • purification is carried out in step (b) using a method selected from the group consisting of hydrophobic chromatography, anion exchange chromatography and gel filtration chromatography.
  • the step (b) comprises: sequentially performing hydrophobic chromatography, Ni 2+ and Cu 2+ chelate chromatography, and anion exchange chromatography to obtain purified hK1.
  • the purified hK1 has a purity greater than 95%, more preferably greater than 98%, and most preferably greater than 99%.
  • the hydrophobic chromatography is performed using a Phenyl Sepharose 6 FF (HS) filler, and the Ni 2+ and Cu 2+ chelate chromatography is performed using a Chelating Sepharose FF filler, the anion exchange chromatography. Q Sepharose FF packing was used.
  • step (ii) chromatographically extracting the hK1 eluate obtained in step (i) with M 2+ -Chelating Sepharose FF, using 20 mM PB, pH 7.0 eluent to hKl; and/or upper Cu 2+ -Chelating Sepharose FF layer Analysis, elution of hKl with 20 mM PB, 10 mM imidazole, pH 7.0;
  • the activity yield of hK1 after purification is higher than 22%.
  • the hK1 produced in the step (b) has the following characteristics: the molecular weight range is 28 to 32 kDa, all of the molecules are N-type sugar chain modifications of methanol yeast, and the sugars account for 8 of the entire glycoprotein. 8 ⁇ 1. 3 ⁇ The 2, the isoelectric point (pi) of the protein is 4. 2 ⁇ 0. 3.
  • the use of the polynucleotide or the protein encoded thereby for the preparation of a medicament for preventing or treating cerebral infarction is provided.
  • the medicament is further for: increasing cerebral blood flow in a mammal, reducing cerebral vascular resistance in a mammal, increasing blood flow in the neck and vertebral arteries, reducing vascular resistance, and increasing blood flow in the femoral artery.
  • Figure 1 shows SDS-PAGE electrophoresis of reduced (R) and non-reduced (NR) induced expression in six high-resistant Zeocin clones screened on 24-well plates in vitro.
  • Figure 2 shows the SDS-PAGE reduction electrophoresis pattern of rhKl produced in the 30L fermentation induction phase.
  • Figure 3 shows the SDS-PAGE electrophoresis of Phenyl-S-printed harose FF chromatography (A) and Superdex 75 molecular sieve chromatography (B).
  • FIG. 4 3D Scheyl-Sepharose FF (HS), Chelating-Sepharose FF and Q_SepharoseFF chromatograms of reduction (R) and non-reduction (NR) SDS-PAGE of each target peak.
  • Figure 5 shows the results of isoelectric point measurement of rhKl.
  • Fig. 6 is a mass spectrum of different degree of glycosylation r-hKl purified by gel filtration chromatography, wherein r-hKl-A is a mass spectrum of rhKl with low molecular weight, and r-hKl-B is high molecular weight during electrophoresis. The mass spectrum of r_hKl.
  • Figure 7 shows the SDS-PAGE electrophoresis (A) and mass spectrogram (B) of PNGaseF removal of N-glycosylation of two glycosylated rhKl.
  • the molecular weight standard of each protein used in SDS-PAGE electrophoresis in the picture is LMW Marker Kit (Amersham Pharmacia Biotech), and the molecular weight of each band is from upper (large) to lower (small): 97. OkDa, 66. OkDa, 45. 0 kDa, 30. 0 kDa, 20. 1 kDa, 14. 4 kDa.
  • hKl human Kallikrein
  • hKl human Kallikrein
  • the expression of rhK1 in the host cell, especially methanol yeast can obtain very high protein expression.
  • the expression product has a good glycosylation modification mode, high specific activity, uniform product and easy purification.
  • the present invention also provides a method of expressing the rhK1, which is simple in process, easy to control expression conditions, and low in cost. The present invention has been completed on this basis.
  • the present invention separates and obtains the polynucleotide from the human kidney cDNA library, and performs recombinant expression, directly obtaining a high expression amount of high activity protein secreted in the form of hKl, and the product is uniform, the purification process is simple, and the effective yield is high,
  • the expression yield of rhKl can reach 2. 5mg/ml fermentation broth in 64 hours after fermentation induction; it has similar efficacy compared with the protein directly isolated from urine, but overcomes the animal tissue or human
  • the direct extraction of hKl components in urine is costly and the quality of the raw materials cannot be guaranteed very well.
  • isolated means that the substance is separated from its original environment (if it is a natural substance, the original environment is the natural environment).
  • the polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotide or polypeptide is separated and purified, such as from other substances existing in the natural state. .
  • kallikrein refers to an alcohol-insoluble protease that hydrolyzes kininogens in the body to release kinins, which causes a decrease in blood pressure, including, but not limited to, urokininogenase, urinary kallikrein, and pancreatic Peptidase, pancreatic kallikrein, tissue kallikrein.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or artificially synthesized DNA.
  • DNA can be single-stranded or double-stranded.
  • the DNA can be a coding strand or a non-coding strand.
  • the polynucleotide of the present invention is preferably provided in an isolated form, more preferably purified to homogeneity.
  • the polynucleotide of the present invention or a fragment thereof can usually be obtained by a PCR amplification method, a recombinant method or a synthetic method.
  • primers can be designed in accordance with the disclosed nucleotide sequences, particularly open reading frame sequences, and can be prepared using commercially available cDNA libraries or conventional methods known to those skilled in the art.
  • the library is used as a template to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then the amplified fragments are spliced together in the correct order.
  • the polynucleotide can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it to a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • synthetic sequences can be used to synthesize related sequences, especially when the fragment length is short.
  • a long sequence of fragments can be obtained by first synthesizing a plurality of small fragments and then connecting them.
  • polynucleotide sequence can be obtained completely by chemical synthesis. This sequence can then be introduced into various existing DNA molecules (e.g., vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
  • a method of amplifying DNA/RNA by PCR technique (Saiki et al., Science 1985; 230: 1350-1354) is preferably used to obtain the gene of the present invention.
  • the RACE method can be preferably used.
  • primers for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein, and can be synthesized by a conventional method.
  • the amplified DNA/RNA fragment can be isolated and purified by a conventional method such as by gel electrophoresis.
  • the invention also relates to vectors comprising the polynucleotides of the invention, and host cells genetically engineered with the vectors of the invention or the polynucleotide sequences of the invention, and methods for producing rhKl by recombinant techniques.
  • the polynucleotide sequence of the present invention can be used to express or produce rhK1 by conventional recombinant DNA technology (Science, 1984; 224: 1431). Generally, the following steps are carried out: (1) using a polynucleotide of the present invention, or transforming or transducing a suitable host cell with a recombinant expression vector containing the polynucleotide; (2) culturing in a suitable medium Host cells; (3). Isolation and purification of proteins from culture media or cells.
  • the polynucleotide sequence can be inserted into a recombinant expression vector.
  • recombinant expression vector refers to Bacterial plasmids, yeast plasmids or other vectors well known in the art. An important feature of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and a translational control element.
  • the recombinant expression vector is a vector suitable for expression in yeast or E. coli; and most preferred is a vector suitable for expression in methanol yeast.
  • Vectors comprising appropriate DNA sequences and appropriate promoters or control sequences can be used to transform appropriate host cells to enable expression of the protein.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • the host cell is selected from the group consisting of: yeast or Escherichia coli; more preferably, the host cell is methanol yeast.
  • the polynucleotide of the present invention is introduced into a methanol yeast through a suitable vector, and the obtained recombinant methanol yeast is particularly suitable for expressing hK1, and the expression amount is high, and the rhK1 has a good glycosylation pattern and is excellent in biological activity.
  • Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated by the CaCl 2 method, and the procedures used are well known in the art.
  • Another method is to use MgCl 2 .
  • Conversion can also be carried out by electroporation if desired.
  • the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, and the like.
  • the obtained transformants are cultured to express the polypeptide encoded by the polynucleotide of the present invention.
  • the expressed protein is purified by hydrophobic chromatography, anion exchange chromatography, or gel filtration chromatography in sequence; or, hydrophobic chromatography, Ni 2+ and Cu 2+ are sequentially used. Chelating chromatography, and anion exchange chromatography. The present inventors have found that a high purity protein can be obtained by the above purification method, and the biological activity of the protein can be well retained.
  • the present inventors obtained the gene sequence corresponding to the hKl mature protein by direct PCR amplification from the human kidney cDNA library through the synthesized primers.
  • the hK1 gene fragment obtained by PCR of the present invention is inserted into the pPICZ ⁇ ⁇ vector to construct a pPICZ a -hK1 expression vector, which is linearized and transformed into a methanol yeast host strain X33, and finally realized.
  • the expression of hK1 is secreted in methanol yeast. It was confirmed by measurement that the N-terminus of the recombinant product was completely identical to the naturally extracted hK1, and also the methanol yeast glycosylation modification mode. This is the recombinant human kallikrein-1 (rhKl).
  • the present invention provides the use of the polynucleotide of the present invention or a protein encoded thereby for the preparation of a medicament for preventing or treating cerebral infarction.
  • the inventors found through animal experiments that rhKl can significantly reduce the area of cerebral infarction area and the weight of cerebral infarction area, which shows that it has a good therapeutic effect on cerebral infarction. Further animal studies have also found that rhKl can significantly increase cerebral blood flow in mammals and significantly reduce cerebral vascular resistance. Moreover, rhKl can significantly increase the blood flow in the neck and vertebral arteries, reduce vascular resistance, and exhibit significant vasodilatation; femoral artery flow also increases to varying degrees.
  • rhKl The selectivity of rhKl to cerebral blood vessels is about 3 times stronger than that of peripheral blood vessels.
  • Intravenous administration of rhKl has a tendency to lower blood pressure, especially in diastolic blood pressure, but has no significant effect on mammalian heart rate.
  • the invention produces the protein drugs traditionally produced by the biological extraction method through modern bioengineering technology, which is a concentrated expression of technological progress in the field of biopharmaceuticals.
  • the quality of the product can be fundamentally controlled to minimize contamination of the product by bacterial viruses of human and animal origin. Therefore, the advantages of the present invention for producing rhKl in methanol yeast are:
  • the production process is simple: the secretory expression of methanol yeast, using conventional hydrophobic, chelated, anion exchange chromatography media, High-purity rhKl (95%) can be purified from the fermentation supernatant on a large scale with low production costs.
  • hKl which is routinely extracted from human urine, uses a self-crosslinking high-cost affinity chromatography medium, and its product uniformity is not high.
  • the stability study of rhKl's water-needle formulation is underway. It is preliminarily judged that the variety can be used in the form of water injection, which not only reduces the production components, but also makes the clinical use more convenient.
  • the specific activity of the product is high: the expression of methanol yeast is secreted, rhKl has glycosylation modification, and the specific activity is high (5.2 AU/mg).
  • the specific activity of the biological extract from urine is only 3 ⁇ 4AU/mg, and the difference between batches is obvious.
  • the quality controllability of the product is good: The genetic engineering product has little dependence on the environment, has few external pollution sources, and can be well controlled, and the product quality is controllable. When extracted from the urine, the quality control of raw materials (urine) will be greatly affected by the seasons. At the same time, in practice, it is impossible to ensure that the urine of healthy people is collected, and the quality control is difficult.
  • the average total activity unit is calculated according to the specific activity of 5. 2 AU / mg, the specific activity unit is calculated according to the specific activity of 5. 2 AU / mg.
  • the efficacy is better than or at least equivalent to extracting from the pancreas or human urine:
  • Pharmacodynamic studies conducted in animals that are officially carried out in animals have shown that the efficacy is comparable when using the same dose.
  • the acute, chronic and reproductive toxicity tests commissioned by the School of Pharmacy of the Second Military Medical University showed that rhKl is safe and non-toxic.
  • the invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are merely illustrative of the invention and are not intended to limit the scope of the invention.
  • Kal5 5'-primer, SEQ ID NO: 3
  • Kal3 3' - Primer, SEQ ID NO: 4
  • Kal5 5.
  • Kal3 5. -cat gcg gcc get tag gag ttc tec get atg gtg tec tc_3.;
  • the hKl gene was obtained by PCR using Panomics' human kidney cDNA library (P/N: 7202, L/N: P0260602) as a template.
  • the primer Kal5 can add the DNA restriction endonuclease Xhol site (CTCGAG) at the 5'-end of the hK1 gene, and the codon AAA AGA corresponding to the recognition sequence Lys-Arg of the Kex2 protease, which will ensure the expression of the inserted hK1 gene during secretion.
  • CTCGAG DNA restriction endonuclease Xhol site
  • the ⁇ _signal peptide can be successfully excised to obtain the hKl protein molecule with the natural ⁇ -terminal sequence; the primer Kal3 is added to the 3'-end stop codon TAA and the DNA restriction endonuclease Notl site (GCGGCCGC).
  • PCR amplification was carried out using DNA polymerase (Pyrobest), and the system was as follows:
  • Kal5 (5, -primer) 2 ⁇ 1
  • Kal3 (3' - Primer) 2 ⁇ 1
  • PCR conditions Denaturation at 94 °C for 3 minutes, followed by circulation at 94 °C, 30 seconds / 55 °C, 30 seconds / 72 °C, 1 minute. After the end of PCR, 2 ⁇ l of the amplified product was electrophoresed on 1.5% agarose, and a length of about 730 bp was obtained, indicating that the PCR was successful.
  • the PCR product was purified by a column recovery kit (Sangon), and the PCR product recovered by the column was double-cleaved with restriction enzymes Xhol and Notl, and the hK1 gene was recovered by gelation.
  • the pPICZ a A vector (Invitrogen) was double-cut with Xhol and Notl, and a large fragment of the vector was recovered as a vector for inserting the gene of interest, and the recovered hK1 gene fragment was ligated with the pPICZ a vector as follows:
  • the reaction was carried out at 22 °C for about 1 hour.
  • the above-mentioned ligation product 5 ⁇ 1 was mixed with NovaBlue (Novagen) competent 100 ⁇ 1 prepared by CaCl 2 method, 4 ° C, 30 min, 42 ° C, heat. Shock for 90 seconds, put on ice for 3 to 5 minutes, then add 500 ⁇ ⁇ LB medium, incubate at 37 ° C for 200 minutes at 45 rpm, and take 100 ⁇ l of bacteria solution coated with LZ (LB+Ze 0C) In 25 ⁇ g/ml) + l. 5% agarose plates, overnight culture at 37 ° C, positive clones that were successfully transformed were grown.
  • NovaBlue Novagen competent 100 ⁇ 1 prepared by CaCl 2 method, 4 ° C, 30 min, 42 ° C, heat. Shock for 90 seconds, put on ice for 3 to 5 minutes, then add 500 ⁇ ⁇ LB medium, incubate at 37 ° C for 200 minutes at 45 r
  • the host cell competent cells of methanol yeast X-33 (purchased from Invitrogen) were prepared according to the method of Invitrogen ⁇ methanolica Expression Kit.
  • the plasmid expression vector was linearized by Sacl digestion, and the protein was removed by phenol-chloroform extraction.
  • the linearized vector after ion water dissolution was mixed with X33 competent cells, electrotransformed, and plated on a plate containing Zeocin 500 ug/ml YPD (1% yeast extract, 2% polypeptone, 2% glucose), 30 Incubate at °C for 2 to 3 days until a yeast single colony grows.
  • the recombinants were first screened by increasing the resistance of the Zeocin antibiotics, and 100 recombinants were picked and seeded in a 24-well plate of 400 ⁇ l YPD (Zeocin 600 ⁇ g/ml) medium. Incubate overnight at 30 °C on a shaker, and screen out the high-resistant Zeocin wells (6 eligible clones) and select them for transfer to the tubes. Add 2 ml of YPD medium to continue the proliferation, and collect them 24 hours later. The cells were induced to express at 30 °C with methanol containing 0.5% BMMY medium, and methanol was added for 24 hours.
  • the final concentration in the bacterial solution was 0.5%, and the induced state was maintained for 48 hours.
  • the supernatant of the bacterial liquid was collected by centrifugation, and the comparison was made before and after the induction by SDS-PAGE electrophoresis, and it was found that there was a 30 kDa protein strip in the fermentation supernatant after the induction. Bring it out. Since it is difficult to accurately judge the expression yield by electrophoresis observation of the protein band type change, the pre-induction supernatant was used as a blank control, and the chromogenic substrate S-2266 was used to accurately determine the rhKl activity unit yield in the induced fermentation supernatant.
  • Figure 1 is a SDS-PAGE electrophoresis map of reduced (R) and non-reduced (NR) induced expression of six high anti-Zeocin clones screened in 24-well plates in a test tube; Lane 0 is the fermentation before induction of expression. Supernatants; Lanes 1 to 6 were 48-hour results for cloning induction of high anti-Zeocin resistance (500 ⁇ g/ml).
  • the fermentation temperature of the initial cell proliferation stage is 30. 0 ⁇ 0. 5 ° C, pH 5. 00 ⁇ 0. 3, the initial rotation speed is 300 rpm, the ventilation is 0. 5wm, the dissolved oxygen (DO) value is 100%, and the PTM1 is added. .
  • DO dissolved oxygen
  • the dissolved oxygen value is decreasing, but the DO value should be maintained at no less than 20%.
  • the carbon source is consumed, the dissolved oxygen value rises rapidly, and the wet weight of the bacteria reaches about 100g/L. Enter the rate-limiting growth phase. During the first 2 hours of this phase, add 50% glycerol solution at a rate of 240 ml/hour and maintain a D0 value of not less than 20%.
  • the feed rate was increased to 360 ml/hour.
  • the D0 value is maintained at a level of not less than 20% by adjusting the stirring speed, air flow rate, and tank pressure ( ⁇ 0.8 bar).
  • the feeding is stopped and the value of D0 rises.
  • the increase in the value of D0 indicates that the carbon source in the medium is depleted, and the carbon source and the inducer methanol may be fed, which enters the critical stage of the fermentation to induce the expression phase: the pH is controlled to 6. 0 ⁇ 0. 2 with ammonia water. , start adding methanol.
  • the initial addition amount of methanol was controlled at 30 ml/hr, and the amount of methanol added was slowly increased, and the feed rate was set to 60 ml/hr after about 2 hours. After 4 hours of induction, the methanol feed rate was set to 120 ml/hr. At this time, methanol induction was successful, and the cells could use methanol as a carbon source at a normal high speed. Maintain D0 value not less than 20%, temperature 30 ° C, pH value of 6.0 ⁇ 0.2, perform fermentation expression, take samples every 8 hours during the whole fermentation period, determine activity (Table 1) and SDS- PAGE electrophoresis, as shown in Figure 2.
  • Figure 2 shows the SDS-PAGE reduction electrophoresis of rhK1 produced during the 30 L fermentation induction phase: samples were taken every 8 hours at the induction period, and the numbers below each lane indicate the number of hours induced by methanol. After the end of the fermentation process, the supernatant was collected by centrifugation, and the expression yield was measured by SDS-PAGE electrophoresis and S-2266 chromogenic substrate method.
  • the purification process consists of three steps: hydrophobic, anion exchange and gel filtration chromatography.
  • Hydrophobic chromatography selects Phenyl Sepharose FF
  • anion exchange medium is Q_S printed harose FF
  • gel filtration chromatography uses Superdex75. The specific process is as follows:
  • the above treated fermentation broth was applied to a Phenyl-S-printed harose FF column with a column type of Index 70/500, a bed volume of 500 ml, an equilibration buffer of 20 mM PB, 1. 0 M (NH 4 ) 2 S0 4 , pH 6 0 ⁇
  • the elution buffer is 20 mM PB, 0. 7M (NH 4 ) 2 S0 4 , pH 6. 0, 20 mM PB, 0. 4M (NH 4 ) 2 S0 4 , pH 6. 0 and 20 mM PB, pH 6. 0 , collect each elution peak.
  • the target protein rhKl was mainly in the elution peak of 20 mM PB, pH 6.0 (Fig. 3A).
  • Figure 3A is an SDS-PAGE electrophoresis of Phenyl_S-printed harose FF chromatography.
  • the Phenyl-S-printed Harose FF chromatography medium was equilibrated with 20 mM PB, 1.0 M (NH 4 ) 2 S0 4 , pH 6.0 buffer; wherein: Lane 1 was 200 mM PB, pH 6.0 and 3M (NH 4 ) 2 S0 4
  • the mother liquor is added to the fermentation broth, and the final concentration in the fermentation broth is 20 mM PB, 1.0 M (NH 4 ) 2 S0 4 , pH 6.0, and then filtered using a membrane of 0. 45 ⁇ m.
  • Lane 2 is the effluent; Lane 3 is 20 mM PB, 0.7 M (NH 4 ) 2 S0 4 , elution peak at pH 6.0; Lane 4 is 20 mM PB, 0. 35M (NH 4 ) 2 S0 4 , pH 6 0 0 eluted peaks, wherein the molecular weight of the larger and slightly lower hKl each accounted for about 50%; Lane 5 was 20 mM PB, pH 6.0 elution peak. 3.
  • the hydrophobic chromatography elution main peak collection solution was adjusted to pH 7.5 with 1.0 M NaOH, diluted conductance 8.
  • the respective elution peaks were collected in a volume of 200 ml, an equilibration buffer of 20 mM PB, a pH of 7.5, an elution buffer of 20 mM PB, 0.2 M NaCl, pH 7.5 and 20 mM PB, 0.5 M NaCl, pH 7.5.
  • the target protein rhKl is mainly in the elution peak of 20 mM PB, 0.5 M NaCl, pH 7.5.
  • the balance and elution buffer of the Superdex 75 molecular sieve chromatography are 20 mM PB, 0.15 M NaCl, pH 7.5, and the elution peaks appearing at a volume of 0.5 V column are collected in stages, and are divided into 1, 2, 3, Sections 4, 5 and 6 have the largest molecular weight and a small amount.
  • the actual molecular weight measured by mass spectrometry r-hKl-B is 32871. 16Da, the fourth segment is the main peak of hKl, the largest amount, and the mass spectrometry r-hKl-A determines its The actual molecular weight is 28975. 79 Dalton.
  • Method II Hydrophobic chromatography, Ni 2+ and Cu 2+ chelate chromatography, and anion exchange chromatography
  • the molecular sieve step is limited for large-scale production, the same result can be obtained according to the method, which is particularly advantageous for scale-up production of the process scale, wherein the second and third steps are adjusted and completely stabilized in the fermentation conditions.
  • the step can be omitted, if the two steps are carried out in sequence, the product purity is higher, and the fermentation broth can be used for different conditions, even the crude extract of biological tissues, and the hKl or hKl analog in the urine of human or animal. You can purify the extraction by following the steps below.
  • Phenyl Sepharose 6FF (HS) filler was used.
  • the equilibration buffer was 20 mM PB, 1.0 M Na 2 SO 4 , pH 7.0, and the elution buffer was 20 mM PB, 0.58 M Na 2 S0 4 , pH 7.0, 20 mM PB, 0.23 M Na 2 SO 4 , pH 7. 0, water was injected into the column, and 20 mM PB, 0.23 M Na 2 SO 4 , pH 7.0 eluate was collected.
  • the Chelating Sepharose FF filler was used.
  • Hang M 2+ , equilibration buffer is 20 mM PB, 0.5 M NaCl, pH 7.0
  • elution buffer is 20 mM PB, pH 7.0, 50 mM EDTANa 3 ⁇ 4lM NaCl, pH 8.0, collect 20 mM PB, pH 7.0 eluate .
  • the third step Cu 2+ metal chelate chromatography
  • the Chelating Sepharose FF filler was used. Cu 2+ was suspended, the equilibration buffer was 20 mM PB, 0.15 M NaCl, pH 7.0, and the elution buffer was 20 mM PB, pH 7.0 and 20 mM PB, 10 mM imidazole, pH 7.0, 50 mM hydrazine, ⁇ NaCl, pH 8 .0, 20 mM PB, 10 mM imidazole, pH 7.0 eluate was collected.
  • the equilibration buffer was 20 mM PB, pH 7.0, and the elution buffer was 20 mM PB, 0.15 M NaCl, pH 7.0 20 mM PB, 0.25 M NaCl, pH 7.0, and collected 20 mM PB, 0.25 M NaCl, pH 7.0. Deliquoring.
  • the purified rhKl was subjected to protein purity (SDS-PAGE electrophoresis, RP-HPLC), and the concentration of rhKl having a purity of not less than 98% was accurately determined by the Lorry method.
  • the sample to be tested is diluted with a 20 mM Tris-Cl, pH 8.0 buffer to a series of multiples, such as 100, 200, 300, etc., based on the estimated concentration of the sample to be tested.
  • the temperature of the solution in each measuring tube was kept constant at 37 °C for 5 minutes in a water bath, and then 40 ⁇ l of the chromogenic substrate S-2266 was added, mixed, accurately timed, and reacted in a water bath at 37 ° C for 15 minutes.
  • reaction sample was added with 40 ⁇ l of 50% acetic acid solution to terminate the reaction, and the spectrophotometer was adjusted to zero by the control, and the absorption value ⁇ 4 ° 5 was measured at a wavelength of 405 nm, and the value of ⁇ 4 ° 5 should be 0.1. Between ⁇ 0. 2, when not in this range, increase or decrease the dilution factor and repeat.
  • the determination of hKl activity by S-2266 chromogenic substrate is the basis of the whole research work.
  • This method can be used to accurately and accurately quantify hKl activity in the sample (fermentation solution, stock solution, purified intermediate sample, etc.). 5 ⁇ g/ml (equivalent to 0. 017 ⁇ ) ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇
  • the specific activity of rhKl can be obtained, and the actual result is about 5. 2 AU/mg.
  • the rhKl protein was collected by gel filtration chromatography and the molecular weight was low and the molecular weight was high.
  • the two groups were entrusted to the Proteomics Research Center of the Institute of Biochemistry and Cell Research, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. N-terminal sequence analysis showed that although there were differences in molecular weight between the two, there was no significant difference in the in vitro activity of the S-2266 chromogenic substrate method, and the N-terminal sequences of their polypeptide chains were completely identical.
  • the first 15 amino acid residues at the N-terminus are: I le-Val-Gly-Gly-Trp-Glu-Cys-Glu-Gln-His-Ser-Gln-Pro-Trp-Gln.
  • the mass spectrum of each peptide was determined by trypsin digestion of rhKl.
  • the 12-amino acid residue at the carboxyl end was determined by mass spectrometry to be -Val-Lys-Trp-I le-Glu-Asp-Thr-I le-Ala-Glu. -Asn-Ser. This indicates that we use the methanol yeast system to secrete rhK1, but there is no N-terminal heterogeneity described by previous researchers. Electrophoresis shows that the molecular heterogeneity is caused by different degrees of glycosylation, but the sugar can be separated by purification. RhKl protein molecules with different degrees of basement.
  • the isoelectric point of rhKl protein was determined by isoelectric focusing method.
  • the pi standard protein used was GE Pharma Life Science's Broad pi Kit pH 3 ⁇ 10.
  • the pi of high glycosylation and low glycosylation modified rhK1 protein was not significantly different.
  • the next step is Amylglucosidase (3. 50), Trypsin inhibitor (4. 55), ⁇ -lactoglobulin a ( ⁇ -Lactoglobul in a) (5. 20), cattle.
  • Bovine carbonic anhydrase b (5. 85), Human carbonic anhydrase b (6.55), Acidoglobin (acid band) (6.85), Alkali Myoglobin (basic band) (7. 35), Lenti l lectin, acidic (8. 15), Lenti l lectin (middle) (8. 45) , Lenti lectin, basic (8. 65), Trypsinogen (9. 30).
  • Lane r-hKl-A is a protein molecule with a low degree of glycosylation
  • lane r-hKl-B is a protein molecule with a high degree of glycosylation. 2 ⁇ The pi is about 4. 2 ⁇ 0.3.
  • the molecular weight calculated from the amino acid sequence should be 26405. 74Dalton, while the actually purified rhKl has a high molecular weight and a slightly lower molecular weight.
  • the two kinds can be seen by SDS-PAGE electrophoresis (Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 7A). More precisely, the molecular weight of the purified rhKl protein is determined by mass spectrometry, according to Fig. 6 (for coagulation).
  • the mass spectrum of rhKl with different glycosylation degree was purified by gel filtration chromatography.
  • r-hKl-A is the low molecular weight of r-hKl, which is the highest in the whole secreted protein.
  • r-hKl-B is SDS-PAGE. The molecular weight of the higher part of the electrophoresis is lower.
  • the mass spectral molecular weight of rhKl is 28975. 79Dalton and 31871. 16Dalton, which is a net increase of 2570. 05 ⁇ 5465. 42Dalton, which is based on the theoretical calculation of the amino acid sequence theory. Yeast secreted expression results in post-translational modification of the protein.
  • RhKl was treated with PNGase F protease (New England BioLabs) according to the standard procedure in the manual. SDS-PAGE electrophoresis showed that the rhK1 molecule became smaller after removal of the N-glycan (Fig. 7A: where r_hKl_A and r_hKl_B were not subjected to desugating At the time of treatment, De-rhKl is treated with PNGaseF, and rhKl with high glycosylation and low glycosylation modification can be seen. The molecular weights are both small and uniform in size, and there are a small amount of rhKl that has not been cleaved.
  • PNGase F protease New England BioLabs
  • Test drug The r_hKl stock solution of the present invention with the batch number of 20060218 has a purity of 98%, a protein concentration of 4. 8 mg/ml, and an active concentration of 25 PNAU/ml. Dilute to the desired concentration with physiological saline before use.
  • Positive control drug Nimodipine injection (Shandong Xinhua Pharmaceutical Co., Ltd., product batch number 0411022), containing 10 mg of nimodipine per 50 ml injection.
  • Solvent Control 0. 9% sodium chloride injection (Shanghai Baite Medical Products Co., Ltd., product batch number A6B11307).
  • Reagents Red tetrazolium (TTC) (Shanghai Shenggong Bioengineering Co., Ltd., batch number 2803B19), prepared in a concentration of 2% with PBS buffer before use.
  • TTC Red tetrazolium
  • the external jugular vein was separated and threaded two times for use.
  • the distal end of the heart is ligated, and the proximal end of the artery is clamped. Cut the mouth obliquely and insert a 45-degree angle into the venous tube filled with liquid or saline.
  • the proximal end of the catheter is ligated to remove the arterial clip. A slight re-examination was successful, and was administered at 0.1 ml/100 g body weight 30 min after the cerebral infarction model was caused.
  • Another thread is placed under the catheter, the end of the catheter is ligated, the catheter is pulled out, and the layers are sutured layer by layer. Return to the cage.
  • the room temperature is strictly controlled at 24 ⁇ 25 °C. After 24 h, the head was decapitated and the area of the infarct area and the weight of the infarct area were measured.
  • the experiment was divided into three groups (model group, positive control group, test group), and about 15 male SD rats in each group.
  • the model group was modeled and given an equal volume of physiological saline 0.1 ml/lOOg.
  • the positive drug control group was given nimodipine injection 0.5 mg / kg.
  • the test group was given r-hKl 30 X 10 PNAU/kg, 0.1 ml/100 g.
  • the positive control drug nimodipine significantly reduced the area of cerebral infarction and the weight of the cerebral infarction area ( ⁇ ⁇ 0.05) compared with the model group.
  • the test drug r-hKl significantly reduced the area of cerebral infarction and the weight of cerebral infarction (P ⁇ 0.01).
  • the experimental results show that: similar to nimodipine, r-hKl has a good therapeutic effect on acute ischemic cerebral infarction.
  • Example 6 Comparison of the effects of hKl and rhKl extracted from human urine on cerebral blood flow in Beagle dogs
  • hKl batch number: 20060218
  • Ureklin for injection trade name Kailikang ®, purified from human urine, referred to as uK, batch number 31051201
  • Beagle Dog provided by Weiguang Experimental Animal Center, Fuyang City, Anhui province, License No. SCXK ( ⁇ ) 06-001. Feeding Observed for more than two weeks.
  • BVF cone arterial blood flow
  • the above various analog signals are input to the MP-100 data acquisition system via the body-6300 multi-channel physiological recorder, and continuous real-time A/D conversion, data acquisition (sampling frequency 300 Hz), and digital signals are stored in the computer. Data analysis was performed using Acqknowledge v.3.5.7 software.
  • the measured values of each parameter are recorded as pre-dose values.
  • the infusion pump was used for constant-vein intravenous administration, and the volume was 30 mL/mouse, and the speed was 1 mL/min.
  • the test drug rhKl was used for constant-vein intravenous administration, and the volume was 30 mL/mouse, and the speed was 1 mL/min.
  • mice were divided into 5 groups, each group of six dogs, saline control group, 3 groups given the test drug rhKl, doses of 1.25X10- 3, 2.5X10- 3, 5.0 X 10- 3 AU / kg, positive drug Kallikrein in administration group (uK), a dose of 2.5X10- 3 AU / kg, 5, 10, 20, 30, 40, 50, 60, 90, 120 minutes.
  • Intravenous administration of rhKl significantly increased cerebral blood flow, and the mean flow during the doses of 1.25 X 10- 3 , 2.5X 10- 3 , 5X 10 - 3 AU/kg increased by 4.7%, 13.4%, 20.9%, respectively, compared with before administration. ; can significantly reduce cerebral vascular resistance, decreased by 5.1%, 12.2%, 19.2%.
  • Equal doses of rhKl and uK increased cerebral blood flow (13.4% vs 9.9%) and decreased cerebrovascular resistance (12. 2% vs 11.8%).
  • Intravenous rhKl can significantly increase the internal carotid and vertebral artery blood flow, reduced vascular resistance, showed significant vasodilatation, 1.25X10- 3, 2.5X10- 3, 5 X 10- 3 AU / kg administered during carotid average
  • the flow rate increased by 5.1%, 13.6%, and 21.6%, respectively.
  • the vertebral artery flow increased by 2.6%, 12.8%, and 18.1%.
  • the femoral artery flow also increased to different degrees, increasing by 3.5%, 4.7%, and 6.3%, respectively.
  • the selectivity to cerebral blood vessels is about 3 times stronger than that of peripheral blood vessels.
  • Intravenous rhKl tends to lower blood pressure, diastolic blood pressure decreased more obvious especially after 5X10- 3 AU / kg administered diastolic blood pressure decreased significantly, the maximum decrease of about 6 mmHg, soon after drug discontinuation. Intravenous administration of rhKl had no significant effect on heart rate in anesthetized dogs. The intensity of action of rhKl is comparable to that of uK.

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Abstract

L'invention porte sur un polynucléotide isolé codant pour une kallikréine 1 humaine, sur un procédé de fabrication de la kallikréine 1 codée, sur un vecteur comportant le polynucléotide et sur une cellule hôte comprenant le vecteur ou une cellule hôte dans laquelle le polynucléotide est intégré. Le polynucléotide peut s'exprimer fortement dans la cellule hôte. Et la pureté de la kallikréine 1 humaine après purification est d'au moins 98 %.
PCT/CN2007/070800 2007-09-27 2007-09-27 Procédé de fabrication de kallikréine 1 humaine Ceased WO2009039704A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130315891A1 (en) * 2012-05-25 2013-11-28 Matthew Charles Formulations of human tissue kallikrein-1 for parenteral delivery and related methods
US9364521B2 (en) 2012-06-04 2016-06-14 Diamedica Inc. Human tissue kallikrein 1 glycosylation isoforms
CN113073092A (zh) * 2021-04-15 2021-07-06 宁波瑞林生物科技有限公司 重组人组织型激肽释放酶及其制备方法
WO2023088272A1 (fr) * 2021-11-16 2023-05-25 江苏众红生物工程创药研究院有限公司 Kallikréine i sous-glycosylée, son modificateur polyéthylène glycol et son utilisation pharmaceutique
US11857608B2 (en) 2017-03-09 2024-01-02 Diamedica Inc. Dosage forms of tissue kallikrein 1

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CN101092598A (zh) * 2006-06-19 2007-12-26 上海万兴生物制药有限公司 甲醇酵母生产人激肽释放酶－1

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CN1384199A (zh) * 2001-02-20 2002-12-11 深圳市人民医院 一种表达人胰腺组织激肽释放酶基因的重组表达载体及重组人胰腺组织激肽释放酶的制备
CN1403156A (zh) * 2002-05-13 2003-03-19 广东天普生化医药股份有限公司 人尿激肽原酶在制备治疗和预防脑梗塞药物中的应用
CN101092598A (zh) * 2006-06-19 2007-12-26 上海万兴生物制药有限公司 甲醇酵母生产人激肽释放酶－1

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130315891A1 (en) * 2012-05-25 2013-11-28 Matthew Charles Formulations of human tissue kallikrein-1 for parenteral delivery and related methods
US20160000704A1 (en) * 2012-05-25 2016-01-07 Diamedica Inc. Formulations of human tissue kallikrein-1 for parenteral delivery and related methods
US9616015B2 (en) * 2012-05-25 2017-04-11 Diamedica Inc. Formulations of human tissue kallikrein-1 for parenteral delivery and related methods
US9364521B2 (en) 2012-06-04 2016-06-14 Diamedica Inc. Human tissue kallikrein 1 glycosylation isoforms
US9839678B2 (en) 2012-06-04 2017-12-12 Diamedica Inc. Human tissue kallikrein 1 glycosylation isoforms
US11857608B2 (en) 2017-03-09 2024-01-02 Diamedica Inc. Dosage forms of tissue kallikrein 1
US12329805B2 (en) 2017-03-09 2025-06-17 Diamedica Inc. Dosage forms of tissue kallikrein 1
CN113073092A (zh) * 2021-04-15 2021-07-06 宁波瑞林生物科技有限公司 重组人组织型激肽释放酶及其制备方法
WO2023088272A1 (fr) * 2021-11-16 2023-05-25 江苏众红生物工程创药研究院有限公司 Kallikréine i sous-glycosylée, son modificateur polyéthylène glycol et son utilisation pharmaceutique

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